Dear Rinku,

this should not happen, removal of Pbf requires high TFA concentration and rather long time (say 2-4h) in contrast to Mtt which requires (ca 1% TFA in DCM) for 3-5 min. your next question is a bit unclear in the sense that you dont mention details as to which AA was coupled next and what masses did you obtain.Nonetheless, if Pbf is removed you should see a reduction of 252 mass units. be carefull if you have more protection groups sensitive to low acids (boc, Trt etc) you will also loose them but this loss would also be more than just two water molecules. hope this helps.

best regards,

Parvesh

Thank you Dr Parvesh Wadhwani for you reply. In my sequence Asp(Oall), Arginine(Pbf) and Lysine(Mtt) is there.I deprotect OAll group from Asp and then cyclise with NH2 of Lys (after fmoc deprotection). Then I deprotect Mtt in 1% TFA in DCM and then couple nex Fmoc-Asp(Oall)-OH. I did mass and  got the required product (50% compared to base peak), but also I got another mass with 2 molecules of water less (this is showing 100% base peak). Thats where I am confused. Should I continue with next amino acid coupling?

Dear Rinku Baishya.., No., With 1%TFA  the Pbf protection never possible to get remove.,  if you have doubt in mass spectrum around 250 mass reduction is there, and here you said about 100% base peak for 2mole of water molecule, you no need to worry about this peak., you continue next., be careful about sequenced amino acids side chain protecting group also.,

Good Luck...

Regards R.Selva....

You haven't mentioned whether you have observed an extra water molecule loss in the totally deprotected peptide or in -Asp(OAll)-containing peptide. If you have the peptide with Asp with  free beta carboxyl group, it can form aspartimide that forms an extra peak with an extra 18 Da loss (compared to the desired product) in MS. If you have Asp-Asp fragment... it is highly prone to the aspartimide formation, and you need to take special precautions to avoid it.  

Thanx Selva Chemist (Bio) for the reply. I will try with your advice.

Thanx Ekaterina Kolesanova also. I have two Asp in the sequence.One Asp I cyclise with Lys alpha amino (fmoc amiino removed), another Asp I use for coupling another compound. But both the amino acids are flanked by other amino acids. I have observed an extra water molecule loss in the cleaved peptide (that have the Asp(Oall) group).

For this you should protect the Second Asp group with specific protecting group and after did your reaction you remove this, so you may avoid this problem, rest regarding to your question Selva Chemist Answer will be best for you.