I am synthesizing 7 amino acid long peptide sequence with CTC resin, 50oC coupling for 10 min. On cleavage (TFA/TIPS/Water 95:2.5:2.5) I got peptide with good yield. However, It has 68 Da more mass than the calculated mass of the peptide. I had made 3 peptide sequences with CTC resin with slight variations ( one amino acid change), all of these have 68 Da more mass. I think it is coming something from resin. What could be the reason for this observation. Please suggest the method to avoid this.
Dear Deepika Gupta ,
I found the following site: https://peptidechemistryportal.com/peptide-mass-shift/ and perhaps the following link explains your observation:
https://peptidechemistryportal.com/aspartimide-formation/ The following question here on RG might be of interest as well. Hope this helps you a bit further.
Best regards.
Rob Keller is correct - it is likely occurring due to aspartimide formation. This suggests you have Asp/Asn next to a Gly or similarly vulnerable residue. If you are seeing complete conversion to +68 Da species in a 7 amino acid peptide, that suggests aspartimide formation is exceptionally favored. Some things you can do to reduce this side-product: 1) Add Oxyma to your deprotection (see: http://www.ub.edu/chembiolab/pdf/Peptide%20Science%202012,%2098,%2089.pdf ). 2) Reduce your deprotection times and temperature - especially after the vulnerable residues. 3) Use an alternative side-chain protecting group on Asp/Asn - typically bulkier side chains protecting groups are thought to provide better protection. Also see this recent publication from Bode et al. (https://www.nature.com/articles/s41467-020-14755-6 )
Thanks a lot Rob Keller and James M Fulcher for the answer.
My peptide has amino acids-Valine, Phenylalanine, Lysine, Alanine only. The coupling agents are DIC/Oxyma and for Fmoc deprotection I have used Piperazine. The same peptide sequence I had synthesised with Rink amide resin multiply times with desired calculated mass in good yield. I need peptide with Carboxylic acid terminal, therefore, I switched to CTC resin. Even with wang resin also I got right mass but in this case yield was very less.
I think there is no possibility of aspartimide formation. There should be only one carboxylic acid that too at the terminal.
Any suggestions?
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