I am trying to couple my mPEG-COOH polymer (Mw 2000) with TAT peptide (Mw 1339) using selective carbodiimide reaction. I ran different trials using EDC + NHS with different solvents such as DMSO alone, a combination of DMSO+ DMF and water alone. Here is a brief procedure for reaction.
1. mPEG-COOH, EDC and NHS is dissolved in a solvent (water or all above mentioned solvents) for 24h for -COOH gp activation.
2. TAT is dissolved either in DMSO or water and further added into the reaction.
3. pH of the reaction mixture was adjusted to 9.0 using triethylamine and keep stirred for 24h
4. After this free TAT and polymer were removed using dialysis medium by running for 24-48h.
5. dialysate was freeze dried to get final product
As IR and NMR techniques are not suitable for the determination of the extent of conjugation between mPEG-COOH and NH2-TAT peptide because TAT is itself contains lots of amide bonds which overlaps CONH amide bond between polymer and peptide. To overcome this issue, I used MLDI/TOF analysis for my product which is showing more of free TAT peptide and free polymer.
Can someone advice me how can I increase the extent of conjugation? I used fluorescamine method to determine the extent of conjugation also which is also predicting less conjugation of the peptide with the polymer.
Visit: https://tools.thermofisher.com/content/sfs/manuals/MAN0011309_NHS_SulfoNHS_UG.pdf It looks like you ran the reaction too long since "NHS esters have a half-life of 4-5 hours at pH 7, 1 hour at pH 8 and only 10 minutes at pH 8.6." I suggest dissolving the mPEG-COOH in 0.1M MES (2-[morpholino]ethanesulfonic acid), 0.5M NaCl, pH 6.0 then follow the instructions for NHS activation, note the reaction only takes 15 minutes! Then bring the pH up and add the TAT-NH2 peptide. I wouldn't use triethylamine to bring the pH up since it could be contaminated with a small amount of diethyl amine that will compete with the TAT peptide to add to the mPEG-COOH. Use the sodium bicarbonate buffer mentioned in the protocol, especially if you are doing MALDI/TOF on the product.
Dear Bruce.
Thank you so much for your kind attention and giving me a fact about this. I will try this reaction.
I am curious to know that As we are running reaction a room temperature and it is with TAT Peptide. would it be enough to further run reaction only for 2 h while reacting with primary amine.
Many Thanks
Bhav
You could probably run the reaction with TAT peptide longer than 2 hours; I would suggest you quench any remaining EDC with 2-mercaptoethanol (or use size exclusion) from the activation step to prevent the TAT peptide from reacting with itself. Perhaps you could follow the progress of the amine coupling by MALDI/TOF and that way you would know when the coupling was complete. Note there are 3 primary amines (n-terminal, and 2 epsilon lysine) in the TAT peptide and any one (or more) of them could couple to the activated mPEG-COOH.
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