Sneaking A Look At Sugars—Inside Cells

Bioorthogonal Chemistry: Carbohydrate labeling strategy has come a long way in 10 years

Stu Borman

Cell surface glycans light up in this microscopy image of the head of a zebrafish embryo labeled using bioorthogonal chemistry. Credit: Courtesy Of Carolyn Bertozzi

Cell surface glycans light up in this microscopy image of the head of a zebrafish embryo labeled using bioorthogonal chemistry.
Credit: Courtesy Of Carolyn Bertozzi

In 2004, Carolyn R. Bertozzi of the University of California, Berkeley, and coworkers took a key step toward imaging carbohydrates to study their roles in live animals. They visualized cell surface glycans by injecting into mice a functionalized metabolic sugar substrate followed by a peptide probe. The sugar and probe reacted inside the mice, creating a complex bound to the cell surface that could be detected using a fluorescent antibody after the cells were removed from the animals (Nature 2004, DOI: 10.1038/nature02791).

The work was an advance in bioorthogonal chemistry, a field dedicated to studying chemical reactions that are carried out in living cells but that don’t interfere with natural life processes. Bioorthogonal chemistry, a name Bertozzi coined, has come far since its beginnings in 2000, when her group developed the Staudinger ligation, the addition of a peptide-bound phosphine to an azide to form an amide. This was the first reaction used to label cell surface ­glycans.

A number of improved bioorthogonal reactions have since been developed by Bertozzi’s group and others. The chemistry is now carried out completely inside living animals, reagents for the reactions are available commercially, and applications are growing.

Staudinger ligation worked well, except that it was so slow that the reagents sometimes cleared from an animal’s body before they could react. Bertozzi and her coworkers noted that a copper-catalyzed ­azide-alkyne addition, a so-called click reaction, was orders of ­magnitude faster than Staudinger ligation and would be excellent for bioorthogonal chemistry, except that the copper catalyst is toxic to cells. So in 2007 they redesigned the click reaction by using a difluorinated cyclooctyne as the alkyne reagent. The compound’s strained ring and difluoro group prime it for reaction, eliminating the need for copper. Other groups later developed similar reactions using cyclooctynes with attached phenyl rings.

With copper-free click chemistry in hand, the obvious next step was to go whole hog into live animals. Bertozzi and coworkers made that step in 2008 by introducing azide-derivatized N-acetylgalactosamine and difluorinated cyclo­octyne into zebrafish embryos. The technique made it possible to detect glycan biosynthesis in specific locations during zebrafish development.

Two other groups later developed tetrazine ligation, the ­cycloaddition of tetrazine and strained alkene derivatives, which proceeds at fast rates that dwarf those of all other bioorthogonal transformations. Tetrazine ligation has been used for antibody-based imaging of cancer cells in live mice. Yet another team ­developed photoclick chemistry, in which light-induced cycloadditions make it possible to carry out bioorthogonal chemistry experiments with ­enhanced spatial and time precision.

Bioorthogonal chemistry has enabled widespread applications in biological imaging, bioconjugation, polymer and dendrimer synthesis, and protein engineering. For example, biotech companies have carried out research in which bioorthogonal functional groups are added to selected sites on recombinant antibodies, allowing drugs to be incorporated specifically at those sites. Preclinical studies suggest that the site-specific conjugates have better therapeutic properties and could be manufactured more easily than previous antibody-drug conjugates.

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In a 2004 study, Bertozzi and coworkers visualized cell surface glycans by injecting into mice an azide-functionalized sugar (above arrow) followed by a peptide probe bound to a phosphine (below arrow). The sugar and phosphine probe react, creating a complex that can be detected in isolated cells with a fluorescent antibody.

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