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Chemoselective Ligation Reaction Chemistry

Chemoselective ligation refers to the use of mutually specific reactive chemical groups to accomplish molecular conjugation. Staudinger chemistry, based on the specific crosslinking reaction between azide- and phosphine-labeled molecules, is one of the more promising and versatile examples of chemoselective strategies for applications such as metabolic labeling.

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Metabolic Labeling

Chemistry of Crosslinking

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.

Overview of chemoselective ligation for biomolecular conjugation.
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, Greg Hermanson
Bioconjugate Techniques,
2nd Edition (2008)
Greg T. Hermanson,
Academic Press, Inc.,
1202 pages.

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.

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Azide-Phosphine Reagent Selection Guide
Staudinger ligation reaction chemistry.
Staudinger ligation reaction scheme (azide-phosphine conjugation). Phosphine-activated proteins or labeling reagents react with azide-labeled target molecules to form aza-ylide intermediates that quickly rearrange in aqueous conditions to form stable amide bonds between reactant molecules.

Applications

Biomolecular Crosslinking

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.

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NHS-Azide Reagents

NHS-Phosphine Reagents

Metabolic Labeling

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.

Example of bioorthogonal metabolic labeling for biotinylation.
Example strategy for in vivo metabolic labeling with biotin using azide-phosphine reagents. When azido-sugar derivatives are supplied to living cells, they are incorporated into glycoproteins by endogenous post-translational modification mechanisms. The azide-tagged molecules can then be selectively labeled or conjugated to phosphine-activated molecules, in this case, a derivative of biotin. If the azido sugars were supplied to cells being studied for response to a particular treatment, the biotin affinity-tag could be used to purify and analyze differences in glycosylation resulting from the treatment regime.

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Metabolic Labeling

 

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Metabolic Labeling Reagents

Azido-Sugar Metabolic Labeling Reagents

Phosphine-PEG3-Biotin Labeling Reagent

DyLight Fluorescent Phosphine Labeling Reagents

 

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Photoreactive Amino Acids

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.

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Comparison of azide-phosphine (Staudinger) ligation to click chemistry

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.
 
Written and/or reviewed by Douglas Hayworth, Ph.D.

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