Maleimide Modifier (5') can be used to directly incorporate an active maleimide moiety onto the 5'-end of an oligonucleotide. The maleimide is separated from the 5'-end nucleotide base by a 2-carbon spacer arm to reduce steric interaction between it and the oligo. Maleimide-labeled oligos are typically used to form conjugates with thiol-labeled moieties, or Diels-Alder cycloaddition products.
As the maleimide moiety itself is not stable over the long-term, Gene Link provides lyophilized maleimido-oligos with all base protecting groups removed and as a maleimide-2,5-dimethylfuran cycloadduct.The cycloadduct serves to protect the maleimide from degradation. The customer then converts the cycloadduct to the active maleimide via a retro-Diels-Alder reaction using toluene and heat. The conversion protocol is provided with the modified oligo. If required the protected oligo should be aliquoted in smaller portions and stored dry.The retro-Diels-Alder reaction should be performed immediately prior to conjugation.
Deprotection by heating in Toluene
Gene Link provides the oligo in a dried lyophilized form. The maleimide is provided in a protected state and deprotection by Retro Diels-Alder reactions is required before use by the end user for conjugation.
The Retro Diels-Alder reactions involves dehydration by co-evaporation with anhydrous acetonitrile and anhydrous toluene. The Retro Diels-Alder reaction requires anhydrous conditions and any significant level of moisture can cause incomplete deprotection, hydrolysis, and/or addition of water to the maleimide. The evaporation of the toluene leaves a white residue ready for conjugation. A detailed procedure is shipped with the product. An abbreviated procedure is described below.
1. Suspend lyophilized oligo in 1 mL anhydrous Acetonitrile. It will be a suspension as oligo will NOT dissolve.
2. Evaporate using a speedvac.
3. Resuspend oligo in 1.5 mL Toulene.
4. Incubate for 4 hrs at 90o C.
5. Cool to room temperature.
6. Evaporate toluene using a speedvac.
7. The oligonucleotide is now ready for conjugation.
Detailed deprotection Retro Diels-Alder reactions protocol
Retro Diels-Alder Reaction protocol.