We synthesized the peptide Thr-Ser-Lys-Tyr on Wang resin by Fmoc chemistry. After the cleavage/deprotection with a mixture of TFA/TIS/anisole/water and precipitation of the peptide by MTBE/petroleum ether we obtained the desired product with a certain amount of +96 by-product, as ESI-MS with a direct injection showed. These products eluted in two different peaks from the preparative HPLC column (eluent 0-20% acetonitrile in 0.1% acetic acid), but the first peak of the desired product after a short storage totally converted into +96 by-product. We tried to reveal what was happened by MS/MS of the +96 by-product, but the only thing we found out was that Tys residue is not modified.
Just an idea...
Have you considered a modification of the aliphatic hydroxyl groups with TFA?
A TFA-modification would show a mass shift of 95.98230 Da.
If this would be the case, you should be able to cleave the TFA quite easyliy by hydrolysis (as proof of principle)
Greets
Hi, Ekaterina,
sometimes the TFA binds to free amine moieties (at Lys or N-terminus) or hydroxyl groups (Thr or Ser). Does the +96 Da side product elute later from the prep column? This might indicate masking of free amine moieties when eluting at low pH.
You can test for bound TFA by raising the pH. The amides and esters of TFA are not stable at high pH.
Best, Jan.
Thank you all,
The exact mass addition is equal to 95.982, the modified product elutes from the C18 column AFTER the desired one, MS/MS signals for all aminoacids except Tyr were ambiguous, so I can suppose it means NH2-group trifluoroacetylation. I wondered whether it was possible in the environment we used for peptide cleavage and purification. Some time ago we prepared the peptide Thr-Ser-Lys-Tyr-Arg (neokyotorphin, and the present peptide represents its fragment), and we had no such a problem with it, though we purified it in 0.1% TFA environment. It seems to be sequence-dependent ...?
A question for Jan Ungewiss: What pH do we need to use to destroy TFA amide? Is 0.1% or 1% NH3 solution enough?
Best regards for all,
Ekaterina.
For a test I would add enough NaOH to a sample to achieve a pH of 12. Sudden analysis will show if the M+96 Da peak is still observeable or not.
For preparative purposes pH should be 10 or higher (but check for peptide stability!).
Best,
Jan.
Just to let to know our results. Maybe, it will be useful for someone.
Unfortunately, we did not succeed in de-trifluoroacetylation with NaOH or NH3. High pH led to the peptide destruction. Synthesis in solution from alpha-Boc- and -Z-amino acids was also unsuccessful, since the final side-chain deblocking by H2/Pd reduction resulted in the modifications of Tyr with Bzl and OH in Thr and Ser with CH3 groups (when methanol was used as a solvent, even at 10%), and they could not be blocked by any scavenger. At last, we used SPPS on 2-Cl-Trityl resin and two-step resin cleavage and deblocking: resin cleavage by acetic acid in DCM followed by side-chain deblocking by HCl/dioxane.
So many problems with a such short peptide... But we got it in 99% purity as a pharmacologically suitable salt and in a good yield just after ether precipitation, without any HPLC!
It is not uncommon to see TFA conjugated to the peptide during peptide synthesis, which is chemistry/sequence dependent.
See:
A facile strategy to prevent trifluoroacetylation of N-terminal proline peptides
Article A facile strategy to prevent trifluoroacetylation of N-termi...
Generation and screening of combinatorial peptide libraries designed for rapid sequencing by mass spectrometry
Article Generation and screening of combinatorial peptide libraries ...
Hi Tim,
Thank you for references, they are very useful for me.
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