Metabolic Labeling and Chemoselective Ligation

Metabolic labeling refers to methods in which chemical detection- or affinity-tags can be added to biomolecules in vivo using the endogenous synthesis and modification machinery of living cells. When analogs of molecular building blocks (e.g., amino acids) can be designed to contain specifically targetable tags that do not interfere with the metabolic machinery of the cell, they provide a mechanism for metabolic labeling. The strategy makes possible a number of powerful experimental approaches for the investigation of cellular pathways.

Page Contents:

• Metabolic Labeling and Staudinger Ligation
• Bioorthogonal Labeling Compounds
• Glycosylation Labeling Reagents
• References

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Staudinger Ligation Reaction Chemistry

Metabolic Labeling and Staudinger Ligation

The Staudinger ligation (azide-phosphine) chemistry is one of several crosslinking techniques that are amenable to in vivo metabolic labeling applications. Because the azide component of the chemoselective reaction pair is so small, it can be supplied to living cells in the form of bioorthogonal molecules that substitute for the building blocks cells use to synthesize proteins or other macromolecules.

Staudinger chemoselective ligation as a strategy for metabolic labeling. Top left. Phosphine activation of proteins is easily accomplished with reactive, phosphine-containing chemical modification reagents; alternatively, fluorescent dyes and affinity tags such as biotin are available ready-made in phosphine-activated form. Top right. The tiny azide tag can be added to biomolecules by in vivo incorporation of azide-containing derivatives of metabolic building blocks (amino acids, sugars, etc.); alternatively, proteins or other molecules can be modified in vitro with reactive azide-containing reagents. Bottom. When combined, phosphine-activated compounds conjugate with high specificity to azide-tagged molecules, resulting in stable covalent attachment of "A" and "B" molecules.

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Bioconjugate Techniques,
2nd Edition (2008)
Greg T. Hermanson,
Academic Press, Inc.,
1202 pages.

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Azide-Phosphine Reagent Selection Guide

Metabolic Labeling Reagents

Chemoselective Ligation Crosslinker Pairs

Bioorthogonal Labeling Compounds

In practical terms, whether or not a particular labeling chemistry can be used in metabolic labeling depends upon its chemoselectivity (reaction specificity) and metabolic compatibility (i.e., production of bioorthogonal derivatives through metabolism).

In this chemoselective ligation strategy, one component of the reaction pair is supplied as a substitute (an analog) of a naturally occurring molecule that is required for catabolism of the target macromolecules. This is the meaning of "bioorthogonal" – that the biological function of the molecule is unaffected by the reactive group it contains. In other words, the reactive group is "invisible" to the biological system.

The azide group in the Staudinger reaction pair has this bioorthogonal property. When supplied to cells, synthetic azide-containing analogs of amino acids or sugars can be incorporated during protein synthesis or post-translational glycosylation using cellular metabolic or regulatory machinery. Thus, the relevant chemoselective reactive group is added in vivo by metabolic labeling. Alternatively, bioorthogonal derivatives can be incorporated into specific non-protein targets using in vitro enzymatic reactions.

Once target molecules are labeled (tagged) with the bioorthogonal group (azide), they can be chemoselectively conjugated or tagged by reaction with the desired phosphine-activated reagent (biotin, fluor, etc.) using the Staudinger reaction (see figure below). In this way, chemoselective ligation using bioorthogonal derivatives blends the simplicity of metabolically encoded tags with specific labeling and the versatility of small-molecule probes.

Photoreactive amino acids and SILAC (stable isotope labeling using amino acids in cell culture) are two other types of bioorthogonal reagents for metabolic labeling. However, these reagents do not comprise chemoselective ligation pairs; instead, they enable conjugation and detection by other means.

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Azide-Tagged Sugars
(bioorthogonal sugars for post-translational glycosylation)

Photoreactive Amino Acids
(bioorthogonal amino acids for UV-activatable crosslinking)

SILAC Reagents and Kits
(stable isotope, bioorthogonal labeling using amino acids in cell culture)

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Azido-Sugar Bioorthogonal Labeling Reagents

Phosphine-PEG3-Biotin Labeling Reagent

DyLight Fluorescent Phosphine Labeling Reagents

Glycosylation Labeling Reagents

Metabolic labeling with bioorthogonal monosaccharides (sugars) that are used by cells to glycosylate proteins and other cell constituents provides for a variety of experimental approaches. The effects of drugs or other treatment conditions on total or sugar-specific glycosylation can be measured. When combination with protein-specific purification or antibody-detection, the glycosylation patterns for specific glycoproteins can be investigated.

Three azido sugar reagents are available for metabolic labeling with the Staudinger ligation (azide-phosphine) chemistry. As illustrated in the figure above, the azide tag of incorporated sugars can be chemoselectively targeted with phosphine-activated compounds for a variety of purposes (e.g., detection or affinity purification).

Bioorthogonal azido sugars for metabolic labeling of glycoproteins.

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Azido-Sugar Bioorthogonal Labeling Reagents

References

1. Agard, N., et al. (2006). A comparative study of bioorthogonal reactions with azides. ACS Chemical Biology 1(10):644-648.
2. Prescher, J.A. and Bertozzi, C.R. (2005). Chemistry in living systems. Nature Chem. Bio. 1(1):13-21.
3. Varki, A., et al. (2008). Essentials of Glycobiology. Second Edition. Cold Spring Harbor Press: Cold Spring Harbor, NY.
4. Saxon, E. and Bertozzi, C. (2000). Cell surface engineering by a modified Staudinger reaction. Science 287:2007-10.
5. Berlett, B. and Stadtman, E. (1997). Protein oxidation in aging, disease, and oxidative stress. JBC 272(33):20313-16.
6. Nessen, M.A., et al. (2009). Selective enrichment of azide-containing peptides from complex mixtures. J Proteome Res 8(7):3702-11.