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

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