Before the emergence of bioorthogonal chemistry, fluorescent protein labeling was the most popular and extensive method at the protein level. The gene-edited protein, combined with the green fluorescent protein or its variant to track the structure and function of the labeled object. However, this method has an obvious drawback: the marker proteins are often bulky, which can easily affect the labeled biomolecules and alter the experimental results.

Bertozzi's bioorthogonal chemistry approach is much more convenient. Two small molecules interact in a living cell environment to form a covalent bond of a single type of reaction. Then, the combination acts like a "bridge": one of the two small molecules is integrated into the target sugar molecule by a metabolic marker, while the other binds to a chemical marker. In this process, the integration of small molecules and targets takes advantage of existing biochemical reactions in the organism without affecting the reactions themselves. In 2005, Bertozzi's team detailed the experimental strategy in a paper published in Nature Chemical Biology.

Fig. 1 The bioorthogonal chemical reporter strategy. (Prescher, 2005)

The initial bioorthogonal chemistry mainly referred to coupling reactions. In 2000, Bertozzi's team developed the Staudinger ligation, namely the azide-phosphine conjugation, based on the Staudinger reduction, and applied it to the chemical modification of cell surface. Since then, a variety of bioorthogonal reactions developed rapidly.

In 2002, organic chemists Karl Barry Sharpless (USA) and Morten P. Meldal (Denmark) together with their teams independently reported a copper-catalyzed azide-alkyne cycloaddition (CuAAC). The reactions followed the selectivity principle of click chemistry and had the advantages of high yield and wide applications, which was very suitable for bioorthogonal chemistry. The two small functional groups used in the CuAAC reaction were easy to bind to target molecules while not affecting natural biochemical reactions.

Despite the obvious features, the CuAAC reaction had a defect in that copper catalysts reacting in cells will produce toxic reactive oxygen species (ROS). Therefore, Bertozzi's team found that an organic compound called cyclooctyne could react with azide to perform a strain-promoted [3+2] azide-alkyne cycloaddition (SPAAC) under physiological conditions. In a few months, the team built the reactive molecule and selectively modified it first on proteins and then on cells. The results were satisfactory and non-toxic. Later in 2004, the team published a paper in the Journal of the American Chemical Society demonstrating the application potential of copper-free click chemical. Subsequently, the team further improved the cyclooctyne reagent for faster reaction kinetics, and successfully imaged the membrane-associated glycans in developing living zebrafish to identify and track the expression patterns of glycans.

While Bertozzi's team was making leaps and bounds in bioorthogonal chemistry, other teams were actively exploring new reaction strategies. In 2008, Joseph Fox's team at the University of Delaware published a paper in the Journal of the American Chemical Society, first proposed the cycloaddition reaction of S-tetrazine and trans-Cyclooctene (TCO) without catalyst under physiological conditions. The tetrazine ligation has been one of the fastest known bioorthogonal reactions so far. Fox said, "with the most powerful TCO, the reaction rate exceeds 3,300,000 M^-1 S^-1".

In 2010, Chen's team from the School of Chemistry and Molecular Engineering at Peking University, first put forward the concept of bioorthogonal cleavage reaction. Before that, bioorthogonal reactions often followed the bond reaction, but Chen's team broke the bonds in bioorthogonal reactions to activate proteins and even deliver drugs.

As a result, Chen's team developed a unique protein in which a key residue at the active site was "caged" by a protective functional group. The bioorthogonal cleavage reaction was then used to "uncaging", removing the protection and activating key residue. The team achieved in situ deprotection of protein side chains (conversion of proc-lysine to natural lysine) in living cells. The advantage of this strategy was that unnatural amino acids were directly inserted into the catalytic active site of the target protein enzyme, making it completely "off". In the process of activation, a small number of proteins in the "on" state were enough to study its function and related biological functions. In 2019, in the JACS, the team proposed another bioorthogonal cleavage reaction of "dual-substituted propargyl (dsPra) or propargyloxycarbonyl (dsProc)/copper complexes", extending from terminal uncaging to intramolecular cleavage. Based on this, the team designed and prepared controlled-release antibody-drug conjugates (cleavable ADCs) based on amino and phenol hydroxyl groups, which successfully achieved selective killing of cancer cells.

Summary

Bioorthogonal chemistry was innovatively pioneered by Bertozzi and over the last 20 years, scientists have been expanding its implications and implications. Although bioorthogonal reactions have more or fewer problems in terms of efficiency, rate, substrate stability, accessibility, biological compatibility, mutual orthogonality, and ease of operation, the technique has grown independently and now become one of the core fields of biochemistry from the supplement to the coupling reaction. Bioorthogonal chemistry not only promotes the development of glycobiology but also the potential scope of drug targeting is likely to be further expanded in the future. For example:

• In situ synthesis of pharmaceutical agents: Bioorthogonal chemistry may facilitate the assembly of drugs from smaller precursors. By creating drugs in the time needed, they are more effective and less toxic and the range of drug interventions could also be expanded.

• Glycan labeling: Scientists have used folic acid ligands to generate lipid nanoparticles containing azide-labeled galactosamine. Tumor membranes incorporated azide-functionalized dibenzocyclooctyne, triggering an immune response when tumor cells were exposed to human sera.

• Click to Release: This method uses bioorthogonal chemistry to control the timing and location of drug release to produce drugs with selective toxicity to target cells.