Pepsyn, +14 adduct of N-terminal Cys

FranÁoise Baleux (baleux@pasteur.fr)
Wed, 27 Oct 1999 14:00:54 +0100

>From: "Singleton, David H" <david_h_singleton@groton.pfizer.com>
>Old-To: "'abrf@aecom.yu.edu'" <abrf@aecom.yu.edu>
>Subject: Pepsyn, +14 adduct of N-terminal Cys
>Date: Tue, 26 Oct 1999 16:01:45 -0400
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>Precedence: bulk
>
>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
>

FranÁoise Baleux
Institut Pasteur
UnitÈ de Chimie Organique
28 rue du Dr Roux
75724 Paris cedex 15

Tel: (33) 145 68 83 96
Fax: (33) 145 68 84 04
e.mail: baleux@pasteur.fr