I have coupled Gemcitabine to a cyclic peptide. I got the corrrect mass to the last fmoc protected amino acid. But after fmoc deprotection and cleavage from resin I am not getting the coupled peptide. Can anyone help me? Whether gemcitabine shows any unstability?
It depends how you did the deprotection, what was the concentration/excess of the amine you used, at what temp. and for how long, etc. There may be a number of possible reactions that your original conjugation product may undergo when treated with an organic base.
Thanx Jacek for the reply. I used 20 percent piperidine in DMF for 10 min at RT for the fmoc deprotection, washed with ether for 3-4 times. Then I used the cocktail TFA/Phenol/Water/Tips (88/5/5/2) for 3 hrs for the peptide cleavage. Precipitated the peptide in ether and then dissolved in small amount of water and again evaporated in rotavapor at 50 deg C.The I checked the mass of the evaporated sample. I got the mass of remaining peptide (gemcitabine broke down) again cyclised. But i didnt get the mass of gemcitabine in the spectra.
Could you elaborate on your coupling reaction conditions? Are you monitoring the coupling reaction to determine its completeness before attempting peptide cleavage? It is difficult to provide an answer without knowing these things.
Thanks Rinku for the additional information. Without knowing the particular peptide structure under consideration, I could only recommend changing the FMOC deprotection, as well as the cleavage steps to much milder ones. By that I mean (1) a smaller excess of base (we do not know the volume, but it seems many fewer equivalents may be possible); (2) try to find a way to monitor step 1; (3) a smaller excess of acid in step 2 (we do not know the volume, but it seems many fewer equivalents and/or shorter times are possible; perhaps a milder acidic cleavage conditions are amenable); (4) monitor step 2 ! (LCMS?); and (5) do not ever heat your product above 25 degC, store it at around 0 degC. The observed stability may also depend on how all the operations are done (time, temperatures, anaerobic atmosphere, etc.).
Thank you very much Jacek. I will follow your suggestions.
With Regards
Dear Adam A Profit...I did peptide coupling reactions with TBTU (5 eqvnt) and DIPEA (10 eqvnt) for 1 hr and Gemcitabine wit DIC and HOBt (5 eqvnt each) for 3-4 hrs.I got mass also till the fmoc protected gem coupled peptide. I did fmoc deprotection with 20% piperidine and TFA C
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