Staudinger Ligation

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Staudinger ligation is to capture the intermediate of azaylide with electrophilic groupsgenerally located on triaryl phosphines, and act in aqueous medium to obtain the intramolecular amido phosphine oxide after rearrangement.

Introduction

Staudinger ligation is a metal-free catalytic click reaction between organic azide and phosphine and it belongs to a bio-orthogonal reaction. It has the following three elements: the reaction must occur in aqueous solution, the catalyst or reactant involved in the reaction is non-toxic, the two functional groups do not exist in the biological system and do not cross react with any functional group in the biological system.

  • Ligation mechanism

In staudinger reduction reaction, the reaction of azide and triphenylphosphine (TPP) firstly releases nitrogen to generate intermediate phosphine imine, and then spontaneously hydrolyzes in aqueous solution to generate primary amine and stable triphenylphosphine oxide (TPPO). The reaction mechanism of staudinger ligation is different from the classical reduction reaction. After improvement, TPP is constructed by using the ester located in the ortho position of the phosphine atom in the reactant through intramolecular cyclization reaction as an electrophilic trapping agent, which can capture the nucleophilic azalactium salt intermediate as an amide under the condition of aqueous solution, thus realizing the covalent "binding" of the two molecules. Traceless staudinger liagtion is a further improvement on the basis of staudinger liagtion. Its "traceless" is reflected in the formation of amide bonds in the hydrolysis step and the removal of TPPO from the final product[1]. (Figure 1)

A) Staudinger reduction reaction; B) Staudinger ligation; C) Traceless staudinger ligation Figure 1 A) Staudinger reduction reaction; B) Staudinger ligation; C) Traceless staudinger ligation

  • Influence factor

The influence of reaction influencing factors on the yield mainly lies in whether the reaction is promoted to proceed in the direction of staudinger ligation.

  1. Polarity of solvent in reaction system
  2. Position and structure of functional groups in reactants
  3. PH of functional groups in reactants
  4. Water solubility of functional groups in reactants
  5. Stability of functional groups in reactants

Application

As a representative of metal-free catalytic click reactions, staudinger ligation plays an important role in various complex biological systems. At present, Staudinger ligation has been widely used in biomarkers, target material delivery and so on.

  • Application in biomarkers

Phosphine and azide in staudinger ligation have bio-orthogonal characteristics. The chemical inertia of azide determines its molecular size and high stability under physiological conditions, making it very suitable for biological coupling and widely used for biomolecule labeling.

Staudinger ligation can be used in marking nucleic acid molecule such as RNA and DNA. For example, RNA is labeled with a fluorescent substance modified with an azide group, and the imaging of RNA in mammalian and bacterial cells is realized by templated stoodinger ligation. Staudinger ligation mediated glycan labeling provides a new possibility for realizing the visualization of glycans participating in various biological processes in the native environment and detecting intracellular interactions. Staudinger ligation doesn’t affect phage viability, and enrichment could be easily achieved after ligation. Therefore, staudinger ligation can selectively modify protein without changing its function, and can be applied to produce highly uniform PEGylated proteins, and surface immobilized proteins.

  • Application in biosensors

Because the ester leaving groups generated in the staudinger ligation can be designed for specific biological applications, staudinger ligation is often used in fluorescent biosensors, providing a fast and convenient method to detect nucleic acids and small molecules.

For example, a reduction triggered fluorescent probe is used for the detection of oligonucleotides. The new fluorescent compound in the probe is a kind of rhodamine derivative, which is labeled on the DNA strand complementary to the target strand. The azide group in the probe can be connected with the TPP labeled on the other DNA strand by staudinger ligation. After the azide group is reduced, a fluorescent signal appears. The probe has been successfully applied to detect oligonucleotides at single nucleotide level in solution and endogenous RNA of bacterial cells.

Reference

  1. Yangzi Zhang; et al. Research progress on biosensing and drug delivery mediated by the staudinger ligation [J]. Biotechology Bulletin. 2019, 35(1):187-198.

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