Maleimide Deprotection for Oligonucleotide Conjugation

Bio-Synthesis offer two types of maleimide functionalize oligo 

• Protected Maleimide by direct chemical synthesis
• Ready-to-Use maleimide modified oligo

The maleimide-functionalized oligonucleotide is supplied in a protected form and must undergo Retro Diels–Alder deprotection prior to conjugation. This reaction requires anhydrous conditions, as moisture can cause incomplete deprotection, hydrolysis, or undesired side reactions with the maleimide group.

The process involves co-evaporation with anhydrous acetonitrile and toluene, followed by thermal activation. After completion, the product forms a white residue ready for conjugation.

Below is an abbreviated protocol for deprotection procedure

1. Suspend the lyophilized oligonucleotide in 1.0 mL anhydrous acetonitrile. (Note: The oligo will remain as a suspension and will not fully dissolve.)
2. Evaporate the suspension to dryness using a speedvac.
3. Resuspend the dried oligo in 1.5 mL anhydrous toluene.
4. Incubate the solution at 90 °C for 4 hours under anhydrous conditions.
5. Cool the reaction mixture to room temperature.
6. Evaporate the toluene completely using a speedvac. 
7. The oligonucleotide is now ready for conjugation to thiol-containing molecules via the exposed maleimide group.