Ok, my turn to pose a question...(Using one of my life
lines...I'll ask the audience Regis...)
I need to synthesize a 12 residue peptide (net charge
-2) with Met at the 10 position, and a cyclic Cys-Cys
at positions 2 and 8 with an aromatic AA between the
Cys and the Met.
When I cleaved the linear (bis-Acm), I used
TFA/H20/TIS and observed only a small amount of Met(O)
(+16) by Mass Spec. I also noted a couple of small
deletion masses but not their +16 peaks. HPLC then
Mass Spec of collected impurity peaks did not show
+16s indicative of Met(O), nor did the major.
(Artifact of the direct infusion Mass Spec when done
as the crude?)
Problem: I seem to be getting oxidation of the Met
both on standing (as solid TFA salt, purified once),
and during cyclization (attempted both crude and
purified) in Aqueous AcOH (4%) at 2mg/mL, with I2/MeOH
as the reagent. (HPLC, and Mass Spec agree on that
issue.)
Obviously I cannot use the traditional methods i.e.
DTT, of Met reduction as it'll pop the ring open.
Here's my question...
I would like to try making the 9 residue fragment,
cleave it protected, do the cyclization, purify, then
couple that onto the Met-AA-AA-resin, then cleave the
whole peptide with TFA/H2O/TIS cocktail (or just
TFA/TIS), then purify/desalt.
Now the killer question...will the Cys-Cys bond
survive both the coupling and the cleavage steps, or
will it pop open during either of the two steps?
Any other suggestions would be MOST appreciated.
Regards,
Steven Johnson
Research Associate, B.S.Chem
Biomeasure, Inc. Milford, Massachusetts, USA
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