Chemoselective Ligation Reaction Chemistry
Introduction to Chemoselective Ligation
Chemoselective ligation involves using unique pairs of mutually specific reactive chemical groups to accomplish molecular conjugation. Examples of this chemistry include hydrazide-aldehyde condensation, click chemistry (azide-alkyne) and Staudinger ligation (azide-phosphine). Of these reaction types, the Staudinger chemistry has the best efficiency and compatibility for live-cell labeling and mass spectrometry (MS) applications for biological research.
Unlike typical crosslinking methods used in biological research, this reaction chemistry depends upon a pair of unique reactive groups that are specific to one another and also foreign to biological systems. Because phosphines and azides do not occur in cells, they are "invisible" to biological processes (called bioorthogonal) and react only with each other (chemoselective), resulting in minimal background and few artifacts.
In addition, the azide component of the reaction pair is so small and bioorthogonal that it can be supplied to living cells as tagged substitutes of the building blocks cells use to synthesize proteins or other macromolecules.
Bioconjugate Techniques, 3rd Edition
Bioconjugate Techniques, 3rd Edition (2013) by Greg T. Hermanson is a major update to a book that is widely recognized as the definitive reference guide to the field of bioconjugation.
Bioconjugate Techniques is a complete textbook and protocols-manual for life scientists wishing to learn and master the biomolecular crosslinking, labeling and immobilization techniques that form the basis of many laboratory applications. The book is also an exhaustive and robust reference for researchers looking to develop novel conjugation strategies for entirely new applications. It also contains an extensive introduction to the field of bioconjugation that covers all of the major applications of the technology used in diverse scientific disciplines as well as containing tips for designing the optimal bioconjugate for any purpose.
Azide-Phosphine Reaction Chemistry
The Staudinger reaction occurs between a methyl ester phosphine (P3) and an azide (N3) to produce an aza-ylide intermediate that is trapped to form a stable covalent bond. This crosslinking chemistry, invented in the 1900s by polymer chemist and Nobel Laureate (1953) Hermann Staudinger, has only recently been applied to biological systems as a bioconjugation technique (Saxon and Bertozzi, 2000). The chemical biology application is now known as Staudinger ligation.
Staudinger chemistry (and most other varieties of chemoselective ligation) can be used to modify and conjugate two purified biomolecules. For example, a purified antibody could be modified (labeled) using NHS-Phosphine while an enzyme reporter could be modified using NHS-Azide; subsequently, when combined, the two proteins would crosslink together to form an antibody-enzyme conjugate.
Because the azide group is extremely small, amino acids, sugars and other building blocks for metabolism can be synthesized to contain azide groups and then supplied to cells as substitutes of their natural counterparts. In this manner, the selectable azide tag can be incorporated into molecules of interest by the cellular metabolic machinery, allowing the underlying metabolic activity to be detected and measured via phosphine-activated reagents. The figure below illustrates this type of application.
Azide-Alkyne Click Chemistry
Staudinger ligation requires fewer harmful additives than other chemoselective ligation chemistries that have been developed for use with biological samples. Although azide-alkyne ("click") chemistry uses the same azide component as the azide-phosphine (Staudinger) chemistry, it requires special copper-containing reaction buffers that have damaging effects on cellular components.
- Agard, N., et al. (2006). A comparative study of bioorthogonal reactions with azides. ACS Chemical Biology 1(10):644-648.
- Prescher, J.A. and Bertozzi, C.R. (2005). Chemistry in living systems. Nature Chem. Bio. 1(1):13-21.
- Varki, A., et al. (2008). Essentials of Glycobiology. Second Edition. Cold Spring Harbor Press: Cold Spring Harbor, NY.
- Saxon, E. and Bertozzi, C. (2000). Cell surface engineering by a modified Staudinger reaction. Science 287:2007-10.
- Berlett, B. and Stadtman, E. (1997). Protein oxidation in aging, disease, and oxidative stress. JBC 272(33):20313-16.
- Nessen, M.A., et al. (2009). Selective enrichment of azide-containing peptides from complex mixtures. J Proteome Res 8(7):3702-11.
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