The protected maleimide modification is deprotected using a retro Diels-Alder reaction. This can be done with microwave irradiation or by heating in toluene. The second approach is described below. Retro Diels-Alder reactions should be conducted in anhydrous conditions as exposure to significant levels of moisture can cause incomplete deprotection, hydrolysis and/or addition of water to the maleimide.
1. Suspend lyophilized oligo in anhydrous toluene.
2. Let sit for 4 hrs at 90°C.
3. Cool to room temperature.
4. Evaporate toluene.
The oligonucleotide is now ready for conjugation to a thiol compound.
Hi,
We ordered a couple of mal modified oligos from you and activated them according to this protocol.
They work well indeed!
Few questions however
1. We find notoriously difficult to resuspend these oligos in water after activation by toluene. Is there any way to circumvent that?
2. Is there any need to replace the toluene during activation – to remove the furan?
Thanks in advance for your help
Dear Iov,
Thank you for contacting us. I’ve asked our Manufacturing team to review your questions. I will let you know their response once they’ve had a chance to research.
Best regards,
Elizabeth
Dear Iov,
Maleimide oligos should readily go into solution by raising the pH to 8 and sonicating if necessary. It should be noted that depending upon the quality of the toluene, there may be some nonvolatile impurities which won’t go into water and may appear to be oligo. This can be confirmed if toluene from the same bottle is rotovaped from a clean round bottom or vial to determine if there’s any residue left afterward.
With regards to adding additional toluene, that shouldn’t be necessary, the furan will be sufficiently dilute to prevent the reverse reaction from occurring (the forward Diels-Alder reaction).
Please let us know if you have any further questions.
Best regards,
Tiffany
Product Specialist I
They certainly DON’T go readily into solution. Besides if everything is done in eppendorf tubes, plastic gets a bit melted by toluene and 90C and in the end lumps of plastic remain in aqueous phase after solubilization.
I think you have a FANTASTIC product. But a bit more is needed to make it user friendly.
The best conditions we found – is instead of evaporating toluene wash the pellet 10 times with ethanol. This makes it a bit more ready for solubilization.
But still there is need for more precise directions with respect to these oligos
Dear Iov,
Thank you for sharing the conditions you found successful. I apologize that the recommended conditions did not work.
Best regards,
Elizabeth