Pepsyn, +14 adduct of N-terminal Cys

Singleton, David H (david_h_singleton@groton.pfizer.com)
Tue, 26 Oct 1999 16:01:45 -0400

We are synthesizing a peptide (67AA) with an amino-terminal free cys
(Non-acetyl). Upon cleavage and LC-ESMS analysis, we observe 2 peaks in
60/40 ratios. The major component is the desired MW; the minor component is
+14Da.

We resynthesized the peptide leaving the N-terminal FMOC group in place as a
possible purification handle. (This trick works well with a diphenyl column
purification followed by FMOC removal) This peptide shows ONLY desired
component and no +14Da.

We synthesize these using FMOC-Ser(tBu)-PEG-PS resin with HBTU/HOBt
activations.

I'm familiar with thiazolidine formation from extraneous formaldheyde
(His(BOM)) in tBOC cleavages, but don't understand this. Possibilities:

1.) Could methanol used during resin drying be effecting my tBu protected
cysteine? If so, is this S-Me reduceable? (I doubt that)
2.) Could residual MeOH from the resin dry be S-alkylating during Reagent K
cleavage? Why wouldn't a more protic TFA stop this?
3.) Extraneous sources of formaldehyde? I usually use "good" reagents.
4.) Others, Ideas?

Thanks in advance for any suggestions. I can't avoid the free cys as this
peptide is being used for chemical ligations.

David H. Singleton
Scientist
Pfizer Central Research
PO Box 8118-101
Eastern Point Road
Groton, CT 06340