Biomolecule labeling is a widespread approach in biological research. Labeling with click chemistry reactions is the most promising technique in the field of bioscience, where rather complex systems such as cells, tissues, and organisms are under investigation. Both Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted alkyne-azide cycloaddition (SPAAC) are widely utilized for biomolecule labeling.
Plenty of experiments with cells, tissues, or protein solutions involve buffers containing sodium azide (NaN3) as a preservative.
Here we report how sodium azide can interfere with click chemistry reactions. The influence of sodium azide on both CuAAC and SPAAC has been investigated by the Lumiprobe team. We found out that the degree of labeling (DOL) of the protein used in the experiment decreased significantly when azide was present in the reaction medium, and the outcome was dependent on NaN3 content.
1) Alkyne-modified ovalbumin was conjugated with TAMRA azide (3x excess) via CuAAC Click reaction. NaN3 content varied from 0.03 wt% to 3 wt%.
| Sample | #1 | #2 | #3 | Positive control (with alkyne-modified ovalbumin) | Negative control (with unmodified ovalbumin) |
| NaN3 content | 3 wt% (460 mM) | 0.3 wt% (46 mM) | 0.03 wt% (4.6 mM) | no NaN3 | no NaN3 |
| DOL | 0.497 | 0.741 | 0.766 | 0.821 | 0.002 |
The following reagents by Lumiprobe were used: Protein labeling buffer (with THPTA), 1.5x, Alkyne NHS ester, TAMRA azide.
2) Azide-modified ovalbumin was conjugated with sulfo-Cyanine3 DBCO (3x excess) via SPAAC Click reaction. NaN3 content varied from 0.03 wt% to 3 wt%.
| Sample | #1 | #2 | #3 | Positive control (with azide-modified ovalbumin) | Negative control (with unmodified ovalbumin) |
| NaN3 content | 3 wt% (460 mM) | 0.3 wt% (46 mM) | 0.03 wt% (4.6 mM) | no NaN3 | no NaN3 |
| DOL | 0.125 | 0.111 | 0.460 | 0.841 | 0.103 |
The following reagents by Lumiprobe were used: Protein labeling buffer (with THPTA), 1.5x, Azidobutyric acid NHS ester, sulfo-Cyanine3 DBCO.
As can be seen from the tables, DOL in the case of CuAAC decreased significantly with increasing NaN3 concentration. SPAAC is much more sensitive to NaN3 presence in the reaction buffer, so the reaction efficiency dropped dramatically even at azide concentration as low as 0.03 wt% (4.6 mM).
In the case of SPAAC, the degree of the labeling observed in the presence of 3 wt% of azide and 0.3 wt% of azide is similar to the DOL of the control reaction with unmodified albumin. The research we performed before had shown that some non-specific labeling via click chemistry occurred with DBCO reagents because of the reaction between cycloalkynes and cysteine residues of the protein.
Conclusion: NaN3 affects the efficiency of both CuAAC and SPAAC click chemistry reactions; SPAAC is sensitive even to low concentrations of inorganic azide. Being an inorganic azide NaN3, along with other present organic azides, reacts with alkynes in the reaction buffer. Therefore, one should avoid using NaN3 as a preservative while performing click reactions, especially for strain-promoted (SPAAC) reactions.