Dear experts,
after peptide synthesis (standard Fmoc chemistry, no acetylation between the couplings), I have a significant peak next to my main product for analytical reverse phase HPLC (see attached file, 278 nm HPLC signal). From the ESI signal, the mass difference between my peptide and the side product is +44. The side product is more hydrophobic, so there is some CH-groups attached somehow. I searched a bit but could not find the right mass difference, Acetyl = +43/+42 (H substitution), Gly or tBu = +57, Trifluoracetyl = +97/+96 (H substitution), Boc = +101/+100 (H substitution). Is there any chance of a internal chain rearrangement with some protecting groups shifting during synthesis?
The peptide is fairly long, 21 AA with an exact target mass of 2114.94 g/mol. The N-terminus is acetylated after final deprotection. Cleavage was performed with 80% TFA, 5% H2O, 2.5% TIS, 5% Phenol and 7.5% DCM for resin swelling. There is no chance that I got double acetylation since I washed the resin well after I acetylated the N-terminus so there is nothing left in the cleavage cocktail.
Following amino Acids were used (all Fmoc-protected, side group protection you see in brackets).
Gly
Cys (Trityl)
Ser (tBu)
Arg (Pbf)
Ala x H2O
Cyclohexylalanine
Glu (tBu) x H2O
Cys (StBu)
Pro x H2O
Gln (Trt)
Ile
Trp (Boc)
Thank you very much for your help!
Marcus
Hi all,
we had some troubles with the ESI-MS unit over Christmas (Nitrogen supply was off...) but now I have the final results for my idea: It works!
As I suggested, it was indeed the COO from incomplete Boc removal from the Trp amino acid in my sequence. After re-freeze drying from 0.1 M acetic acid, it's completely gone and I increased the crude yield for about 50% from initially ca. 50% to now ca. 75%. Please see the attached image.
I'm not sure if I would have the same result if I would just collect the +44 peak during preparative HPLC as well in one fraction with the main product. Since I do freeze-drying from 0.1% TFA solution (as we add this to the mobile phase for prep. HPLC), the COO removal should work as well, right? However, I would not try this now. I may do it in the future if I should face the same issue.
In think increasing the cleavage time and optional the cleavage cocktail volume will do the same job with less effort.
Marcus
COO+ just before the decarboxylation of an internal organic acid portion of the peptide.
Thank you for your answers!
Newton, do you have a mechanism how Alanine in my sequence could be modified to the molecule you suggested? I'm not sure where a hydroxyethyl group could be added somewhere, the peptide chain is completely protected during synthesis (not during cleavage).
Bruce, I just have one acid (Glu) in my sequence. If decarboxylation occurs, it would be -44 difference. But I got a different idea, listen:
Could it be that the Boc group of Trp side chain is just partially left (only [C(CH3)3]+) and COOH group is left? This could explain the difference of +44. I think I have read this somewhere somewhen in the past but cannot find it anymore. I have to search more deeply but this could be the explanation. I roughly remember that freeze-drying from 1M Acetic acid does the job. If I found out I'll keep you posted.
In my case I was dealing with a barbiturate (a small molecule) which had a ring opening under alkaline conditions followed by a rapid decarboxylation. Glutamic Acid has a COOH like alanine. Check the pH!
Thank you Bruce.
What I got is the crude peptide after cleavage off the resin with TFA/Phenol/TIS/Water mixture. The pH is VERY acidic, precipitation in Et2O and then filtration. Analytical HPLC with Acetonitrile/water and 0.1 % formic acid, pH 2-3. No way for alkaline conditions. The Alanine in my case is part of the peptide chain, so it's just amide bond and the CH3 side chain. C-terminus is amide.
All right, I think I have the solution. I found it in thefollowing book:
Book Fmoc Solid-Phase Peptide Synthesis: A Practical Approach
on page 67 (table 5, Side reactions encountered during cleavage reactions), there is the explanation I mentioned in my last post (it's inclomplete Boc removal from Trp). I will increase the volume of the cleavage cocktail and maybe the cleavage time (was already 4 hours) next time. I had it once in the past.
I'll do lyophilization over Christmas from 0.1 % acetic acid and do the analysis later. I'll keep you posted and hope this will solve the problem (its about half of my product!).
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