[DJNorman]

(I was advised to move this post to this board from the main OC one. If someone could remove my previous post I would appreciate it. Thanks.)

Hi there, I've synthesised Fmoc-Phe-Lys and it's still a bit acidic after work-up.

I was thinking there is maybe some residual TFA from the deprotection step, however, rotary evaporation and ether washes haven't affected the pH. So I was thinking maybe the TFA has formed a salt with the amine of the lysine group? ie R-NH₃^+-OOCCF₃.

I was considering doing a wash with NaHCO₃ followed by a brine wash. Would this work? Are there any other ways that the pH would be lower than expected?

Thanks for any help, it's really appreciated!

[BobfromNC]

As long as you have the Fmoc protection still on the peptide, I would think that it would still partition into DCM or chloroform, so washing a solution of it in a chlorinated solvent gently with aqueous base may remove traces of TFA.   Another solution can be to dissolve in ether and add HCl in ether or dioxanes and evaporate, if the HCl salt is better. 

[ziqquratu]

Will the presence of TFA be a problem? In many cases, it's common to simply use peptides as their TFA salt.

If TFA would be a problem, could you use the HCl salt? If so, simply dissolving the peptide in an excess of 1M HCl then removing the solvent should get rid of the TFA, leaving you with the HCl salt.

To get rid of all acids, I'd lean towards ion exchange - pass it through a short column of Amberlyst or Dowex resin, where the peptide should stick, wash the TFA out with a little water, then elute the peptide with aqueous ammonia. Removing the solvent (presumably by lyophilisation) should also get rid of the ammonia!

Finally, if you think a simple base wash is the way to go, just try it on a small amount to ensure that your peptide is actually soluble in DCM or similar - the fact that it's an amino acid (terminal COOH and lysine side chain) could make it insoluble.

Edit:
My bad on that last part - you've got a free acid group in your molecule, so a base wash will simply deprotonate that, leading to a different salt, but one that's still probably water soluble (and certainly DCM insoluble)! Try the ion exchange, if the presence of a lysine salt is not tolerable.

[DJNorman]

Thanks for the input!

I went for the sodium bicarbonate wash in DCM. It worked but reduced my yield too far for it to be practical.

Cells don't seem to appreciate the TFA unfortunately I'll have a look into ordering an ion exchange, thanks again.