Staudinger ligation

This reaction has since been used successfully to synthesize amines in countless numbers of organic compounds and still remains one of the most common organic reactions performed today. Often azides are thought of as hidden amines, because the azide is relatively inert to other reactants until it is revealed through the Staudinger reaction. 

There now are available a number of alkyl azide compounds that may be used in click chemistry reactions and the Staudinger ligation processes. It is not recommended, however, to use aryl azide compounds, as these are light sensitive and photoreactive as well as highly susceptible to reduction in the presence of thiols. Unfortunately, at the time of this writing there are fewer choices in aryl phosphine compounds to participate in this reaction, as commercial sources of labeling reagents are limited. 

Grow cells ( 1 X 105 cells/ml) for 3 days in appropriate media containing a 20 pM concentration of acetylated azidoacetylmannosamine. 

Wash the cells at least twice with 0.1 percent fetal bovine serum in 10mM sodium phosphate, 0.15M NaCl, pH 7.4 (PBS) to remove excess azido-sugar. 

Add to the washed cells 60 pi of a 5 mM concentration of the phosphine derivative to couple to the azido-sugar groups on the cell surface (e.g., biotin-PEG-phosphine). 

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Kosiova I, Janicova A, Kois P (2006) Synthesis of coumarin or ferrocene labeled nucleosides via staudinger ligation. Beilstein J Org Chem 2 2-23... 

Figure 11.22 Azido-sialic acid-containing glycans can be labeled in vivo with biotin-PEG-phosphine using the Staudinger ligation reaction, which forms an amide bond.

Figure 13.4 APTS-modified surfaces may be further derivatized with amine-reactive crosslinkers to create additional surface characteristics and reactivity. Modification with NHS-PEG4-azide forms a hydrophilic PEG spacer terminating in an azido group that can be used in a click chemistry or Staudinger ligation reaction to couple other molecules.

Figure 17.12 Azido derivatives of sugars can be used as monomers for glycan and carbohydrate synthesis by cells. Such modifications can be probed using click chemistry or Staudinger ligation reactions.

Figure 17.18 The Staudinger ligation reaction uses a modified phosphine derivative containing an electrophilic group that acts as a trap for the nucleophilic nitrogen in the intermediate aza-ylide. The resultant shift yields an amide bond derivative between the phosphine-containing molecule and the azide-containing molecule.

Staudinger ligation techniques also can be used to detect post-translational modification of proteins in vivo. Hang et al. (2007) developed a method to monitor fatty acid acylation of proteins using azido-fatty acids fed to cells. The two major types of fatty acid acylation,... 

Figure 17.19 An azido-sialic acid derivative that gets incorporated into glycans in cells can be labeled specifically with a biotin-phosphine tag using the Staudinger ligation process. The result is an amide bond linkage with the glycan.

Figure 17.20 An azido-palmitic acid derivative can be added to cells to obtain palmitoylated proteins that contain an azide group able to participate in the Staudinger ligation reaction. Biotinylation of these post-translationally modified sites then can be done in vivo using a biotin-phosphine reagent.

Another important variation of the Staudinger ligation reaction described above involves the use of cleavable aryl groups on the triphenylphosphine component, which allows for... 

Figure 17.22 Certain unique phosphine derivatives can be used in the design of modification or conjugation reagents to create a traceless Staudinger ligation process, wherein the phosphine group is lost and an amide bond between an azide-containing molecule and the phosphine-containing molecule results.

The phosphanes useful in this process are built from acyl derivatives of compounds such as those shown in Figure 17.22. During the Staudinger ligation process, once the azide reactant forms the aza-ylide with the phosphine, electrophilic attraction induces the nitrogen to attack the electron deficient carbonyl, which in turn causes release of the phosphonium group and forms the amide bond (Figure 17.23). 

Figure 17.23 A traceless Staudinger ligation process involves the formation of an intermediate aza-ylide with subsequent attack of the nucleophilic nitrogen atom on the neighboring electrophilic group. The formation of an amide bond then occurs concomitant with the loss of the phosphine component, thus forming a zero-length crosslink between the two molecules.

Figure 18.10 NHS-PEG-azide compounds can be used to modify amine-containing proteins or other molecules for subsequent conjugation using either the click chemistry reaction or Staudinger ligation.

Click chemistry or Staudinger ligation equipped heterobifunctional crosslinkers containing a hydrophilic PEG spacer provide a stable, yet highly efficient means to conjugate or immobilize biomolecules. The PEG spacer provides water solubility, low nonspecific binding character, and low immunogenicity. 

Figure 18.11 NHS-PEG4-azide can be used to modify an amine-containing molecule to create an amide derivative terminating in azido groups. The azide modifications then can be used in a click chemistry reaction that forms a triazole linkage with an alkyne-containing molecule. Alternatively, the azide derivative can be used in a Staudinger ligation reaction with a phosphine derivative, which results in an amide bond linkage.

Kiick, K.L., Saxon, E., Tirrell, D.A., and Bertozzi, C.R. (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc. Natl Acad. Sci. USA 99, 19-24. 

Kohn, M., and Breinbauer, R. (2004) The Staudinger ligation-A gift to chemical biology. Angew. Chem. Int. 43, 3106-3116. 

Nilsson, B.L., Kiessling, L.L., and Raines, R.T. (2001) High-yielding Staudinger ligation of a phosphi-nothioester and azide to form a peptide. Org. Lett. 3, 9-12. 

Xu, J., DeGraw, A.J., Duckworth, B.P., Lenevich, S., Tann, C.-M., Henson, E.C., Gruber, S.J., Barany, G., and Distefano, M.D. (2006) Synthesis and reactivity of 6,7-dihydrogeranylazides reagents for primary azide incorporation into peptides and subsequent Staudinger ligation. Chem. Biol. Drug Des. 68, 85-96. 

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