During a chemical synthesis of peptides by the F-moc method, the following happened: all the initial steps of the synthesis occurred as planned, but in the cleavage, TFA is used. In the lab, there was TFA at 0.3% and 99%. I did not find the right concentration to use. So I opted for TFA at 0.3%. Then I centrifuged with ice ether, but no pellet appeared, only two phases, like water and oil. The protocol says to discard the supernatant, so I discarded only the first phase that probably corresponds to the ether and froze the second phase. So I would like to ask if anyone among you has experience with the chemical synthesis of peptides and knows if the concentration of TFA I used is really wrong. And also, if the non-formation of the pellet means that the synthesis was not successful or if the peptide may be in any of the phases that formed during centrifugation.
Hi the reason for not getting as expected product was :
Peptide - Polymer Resin Covalent bond much stronger , so need to use TFA in higher concentration.
For better results
Use >97% TFA + 1% Scavenger (TIS like this based on amino acids nature) + 1.5%Water.
If any questions let me know
Some of my research paper Full Text available here in RG - look you may get clear details and protocols
All The Best For Your Research & Success
- Dr.R.Selvam
Ana, did you check to see the completion of the de-Fmoc? I thought you shuld use piperidine to remove Fmoc, TFA is for removal of Boc.
Do you have any further informations about the synthesis?
Which resin did you use? Is the used 0.3% TFA on aqueous basis?
Ana, conditions for cleavage of a synthetic peptide (removal from the solid phase) are determined by the linker, which typically is pre-loaded on the resin when you buy it. Rink amide is the most common linker, producing a peptide with a C-terminal amide group. Rink requires at least 70% TFA. Your cleavage cocktail also should include scavenger reagents based on the amino acid side chains present, as side chain protecting groups are typically removed concomitant with cleavage. For peptides without Cys, Met, or Trp, 5% water is an excellent scavenger, with 95% TFA. For challenging side chains Reagent K usually works well, although it includes 1,2-ethanedithiol which is a stench (TFA/thioanisole/water/phenol/1,2-ethanedithiol at 82.5 : 5 : 5 : 5 : 2.5). Rinse the crude peptide precipitate thoroughly with ice cold methyl t-butyl ether to remove the cleavage cocktail and scavengers.
After your attempt with 0.3% TFA, your peptide probably is still safely on the resin. If you kept it, you can go back and try cleavage again with a proper reagent.
Totally agree with John Carter 's comment.
You cannot see the pellet because your peptide is likely still bound to the resin, since the concentration you used is simply too small to detach it. Use the 99% TFA. Would be useful to know which resin you used, though. Hope it helps!
Hi the reason for not getting as expected product was :
Peptide - Polymer Resin Covalent bond much stronger , so need to use TFA in higher concentration.
For better results
Use >97% TFA + 1% Scavenger (TIS like this based on amino acids nature) + 1.5%Water.
If any questions let me know
Some of my research paper Full Text available here in RG - look you may get clear details and protocols
All The Best For Your Research & Success
- Dr.R.Selvam
Use > 95% TFA : The reaction was little exothermic , after that extract with Cold Diethylether - you will get fine product. ii agree with R. Selvam and John Carter
Regards
Dr. Athira R S
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