I usually use reagent K for everything (recently I discovered using too
little gives poor crudes; more is better assuming you can precipitate the
product). Your choices of reagents are probably sound.
Are you sure the Pbf group is on Arg and hasn't migrated to a sensitive
residue (Trp, Tyr, others)? Ms/Ms sequencing (difficult of a 21 mer; you
probably don't want to bother with trypsin affording smaller peptides) or
Edman (close enough to the N-terminus to get you info?) should tell you
exactly what's modified.
If you re-cleave and see no changes, I'd bet on migration (Friedel-crafts
acylation) rather than incomplete deprotection.
If you need to remove the Pbf peptide, (assuming it's still on Arg), ion
exchange should work well. You now have 2 different zeta potentials at
acidic pH.
David H. Singleton
Scientist
Pfizer Central Research
PO Box 8118-101
Eastern Point Road
Groton, CT 06340
-----Original Message-----
From: Jane Nagel [mailto:jnagel@genetics.com]
Sent: Friday, July 30, 1999 9:41 AM
To: Recipients of ABRF List
Subject: Arg-Pbf rescue
Hello all,
I have been cursed with a peptide which has some residual Pbf protecting
group left on an Arg residue (it is a 21-mer). The peptide synthesizes
beautifully, one peak on HPLC and usually one peak on MALDI-TOF but the
removal of the Pbf is somewhat hit and miss. I have never encountered a
problem with its removal before so I have been surprised to see it on this
peptide. I make a bunch of this stuff and the cleavage either goes to
completion or it gives me this portion of still Pbf protected peptide even
when batches are cleaved side by side.
As it happens, the Pbf protected bits elute at roughly the same % as the
clean stuff so I had hoped to try a rescue measure and re-cleave the already
cleaved peptide. I looked through the ABRF archive and could not find any
instance of this being attempted before so here I am, asking for your
assistance. I have had excellent results removing residual Trt from cleaved
peptides by stirring them in 99% TFA and 1% TIS, anyone have any experience
with Pbf removal in the same general fashion? I was thinking of using 95%
TFA + 2.5% EDT + 2.5% TIS.
Your input would be much appreciated!
Thanks in advance,
Jane
Jane A. Nagel MSc
Genetics Institute
Cambridge, MA
1 888 577 1500 x 8597
jnagel@genetics.com
!
!