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Superiority of Staudinger Ligation Chemistry



Staudinger ligation for biomolecular conjugation.

Experiments demonstrate the advantages of azide-phosphine chemoselective ligation over azide-alkyne click chemistry.

Chemoselective ligation involves using unique pairs of mutually specific reactive chemical groups to accomplish molecular conjugation. Click conjugation (azide-alkyne) and Staudinger ligation (azide-phosphine) are competing technologies for chemoselective ligation. Staudinger ligation is the most effective and the most compatible method for live-cell labeling and proteomics applications.

 

Similarities:

  • Chemoselective – both chemistries provide specific reaction to azide-labeled molecules
  • Bioorthogonal – both chemistries provide the same options for in vivo incorporation of azido-tagged derivatives of metabolic building blocks
  • Activated reagents – both chemistries are supported by the availability of ready-to-use fluorescent dyes, affinity tags and other reagents for detection or crosslinking

Differences:

  • Number of reaction components – Staudinger ligation requires only the azide-phosphine reagents in aqueous solution at physiological pH; by contrast, the commercially available copper-catalyzed click chemistry requires five different reagents
  • Toxic and oxidizing effects of copper – the copper component required for the alkyne-based click chemistry can be toxic to cells, may adversely affect the biology of interest, and damages glycosylation; by contrast, Staudinger ligation does not require copper and does not damage biomolecules.

 

Thermo Scientific DyLight Phosphine-activated Dyes detect metabolically incorporated azido-sugars more effective than Click-iT* Alexa* Fluor Alkynes.
Intracellular detection of metabolically incorporated azido-mannosamine in fixed cells. A549 cells were incubated with azido-acetylmannosamine and then fixed with 4% paraformaldehyde. The cells were then probed with DyLight 650-Phosphine (A) or with Click-iT* Alexa* Fluor 647 DIBO Alkyne (B). The experiment data demonstrate that DyLight-Phosphine clearly reveal azido-mannosamine (green) incorporation in the golgi compartment within fixed cells. (Blue: Hoechst 33342)

 

Experimental Details:

Both phosphines and alkynes chemoselectively react with azides. Thus, the same azido components can be used in experiments to compare the ligation chemistries. In one experiment, Thermo Scientific Azido Sugars were supplied to the cells to profile their metabolic incorporation into glycoprotein subclasses. The Thermo Scientific DyLight 550- and 650-phosphine reactive dyes label azido-sugars with equal or better signal than a similar fluorescent dyes activated with alkyne Click-iT Chemistry (Life Technologies, Inc). Background staining of control lysates was also lower when using the phosphine dyes, even when used at higher concentrations than the alkyne dyes (data not shown).

Thermo Scientific DyLight Phosphine-activated Dyes detect metabolically incorporated azido-sugars more effective than Click-iT* Alexa* Fluor Alkynes.
Thermo Scientific DyLight Phosphine-activated Dyes detect metabolically incorporated azido-sugars more effectively than Click-iT Alexa Fluor Alkynes. A549 cells were incubated with 40µM azido-acetylmannosamine in cell culture media for 72 hours and the cells were used for either live- or fixed-cell labeling. Panel A. Cells were washed, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton* X-100. Metabolically incorporated azido-mannosamine was labeled with either DyLight 550-phosphine-activated or Alexa 555 alkyne-activated dyes according to the manufacturer's protocol and fluorescence intensity of each dye determined. Panel B. Live cells were incubated with DyLight 650-Phosphine or Alexa Fluor 647 DIBO Alkyne according to the manufacturer?s protocol and fluorescence intensity of each dye determined.

 

Thermo Scientific DyLight Phosphine-activated Dyes detect metabolically incorporated azido-sugars more effective than Click-iT* Alexa* Fluor Alkynes.
Comparison of reactive dyes for in vivo detection of metabolically labeled sugars. HK-2 cell were metabolically labeled by incorporation of azido-acetylgalactosamine, azido-acetylglucosamine or azido-acetylmannosamine and the live cells were probed with DyLight 650-Phosphine or Alexa Fluor 647 DIBO Alkyne. The degree of labeling for incorporated azido-sugar was measured using a Thermo Scientific ArrayScan Vti demonstrating that DyLight-phosphine reactive dyes perform better in live cell labeling than alkyne-reactive dyes.

Some click chemistry (alkyne) reactions require copper (I) as a catalyst, which is typically generated in situ from copper (II) sulfate with the aid of a reducing agent and stabilizing ligand. Reduced metals are oxidizers and can generate free-radicals in aqueous buffers, resulting in protein inactivation and enhanced susceptibility to proteases. One common oxidation adduct of protein side chains is the formation of carbonyls (e.g., ketones and aldehydes), which can be detected by biotin-hydrazide staining. To compare the effect of click chemistry reaction conditions on proteins, alcohol dehydrogenase, HSA and ribonuclease A were incubated with Click-iT Buffers. After only 1 hour at room temperature, there was significant biotin-hydrazide labeling, indicating oxidation of amino acid side chains compared to the control or phosphine samples.

Azide-Alkyne click chemistry causes glycoprotein oxidation.
Azide-alkyne click chemistry with copper causes glycoprotein oxidation. Purified proteins (0.5mg/mL) were incubated for 1 hour with Click-iT Reaction Buffers (Life Technologies Inc.) B-D or A-D (lanes 3 and 4 in each panel). Identical protein samples were also incubated with Staudinger phosphine acid (P3) or 5% hydrogen peroxide mixed with 100µM iron(II) perchlorate [Fe(II):H2O2], a required component of the click chemistry reaction. Following incubation, the untreated control reagents (C, lane 1) and test samples were reacted with biotin-hydrazide, which conjugates to oxidized polysaccharides. The extend of biotinylation (and therefore oxidation) was measured by Western blot using a streptavidin-HRP conjugate and chemiluminescent substrate (top panels). Gels were also stained with Thermo Scientific GelCode Blue Stain Reagent to confirm equal protein load (bottom panels).

 

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