Dear Dave,

I believe that instead of a linear peptide in the final product, I may have
branched peptides in addition to the linear peptide.   That was confirmed by
sequence analysis of the same peptide made with side-chain protected Glu.
PTH-glutamic acids in the "regular" peptide synthesized with side-chain
protected Glu, eluted at the expected time, and there was no drop in
sequencing yields after Glu positions.  With regards to your comment about
"an increase in mass", I did see an increase in mass and am in the process
of determining (if possible) what residues could be in this "branch"

Thank you so much for your response to my questions.  The ABRF Discussion
Group is indeed a great invention.

Best regards,

Nga

-----Original Message-----
From: dave chin [mailto:dave200108@yahoo.com]
Sent: Wednesday, March 20, 2002 8:50 PM
To: ABRF Discussion List
Subject: Re: Peptide Synthesis + Amino Acid Sequencing Questions

Try changing over to acetic acid by using a RP with
acetic acid or repeated lypholyzation redissolving in
dilute HOAc or gel filtration using P gel and HOAc
buffer to get acetate salt form instead of TFA salt
form.
If you have made a branched peptide, it should be
reflected in an increased mass.  As you suggested a
decrease in yield is also an indication of a branch
structure.  Like the branched ABRF test peptide for
sequencing c.a. 1989, except it was branching on a
lysine residue.
In using HBTU/HOBt the excess also reacts and blocks
the amino terminal residues. The carboxy could be
activated, but would have nothing to imediately react
with until the next deprotection and/or making a suc.
intermediate (pyro-) and then isoasp or isoglu (also
causing a drop in Edman sequencing yields).

David Chin
--- "Nguyen, Nga Yen" <nguyen@cber.FDA.gov> wrote:
> I would like to seek advice and assistance from the
> ABRF Discussion group
> regarding a problem that I encountered with peptide
> synthesis and subsequent
> sequencing of a 36-amino acid peptide.
>
> Peptide Questions: The peptide was made with
> 15N-glutamic acid (6 glutamic
> acids are present in the sequence) and 15N-aspartic
> acid (there is only one
> aspartic acid in the sequence).  The 15N acidic
> amino acids from the company
> which provided us with the products, do not have
> protecting groups for their
> side chains.  We only found out after the peptide
> was made.  The peptide was
> cleaved with 95% TFA + scavengers and purified by
> reverse phase HPLC.  A
> major component (with ~90% of total areas of all
> observed HPLC peak) was
> collected by HPLC when a shallow gradient of ~0.5%
> acetonitrile/minute was
> used.  
>
> 1.  This purified peptide exhibited a mass (by
> Maldi) which is ~297 - 300
> amu higher than expected.  Could this be a product
> of ion-pairing with TFA
> since the peptide has 2 lysines and one His? Would I
> be able to exchange TFA
> with acetic acid if I incubate the peptide in acetic
> acid at a concentration
> 50% of higher, at RT for 2 hours?
>
> 2.  What is the potential for the side chain
> unprotected -COOH to
> participate in chain elongation/termination?  and to
> what extent?  Synthesis
> yielded the full length peptide as indicated by
> N-terminal Edman degradation
> and AA composition (please see question below).
>
> 3. What is known about the relative coupling
> affinity of HBTU (or other SPPS
> coupling agents) for the side chain carboxyl vs. the
> alpha carboxyl?  From
> my conversation with some peptide chemists, the side
> chain carboxyl group is
> also activated, and possibly to the same degree as
> the alpha -COOH.
>
> 4. If you have used acidic amino acids without side
> chain protecting groups
> in SPPS (intentionally or not), could you share your
> experience with me,
> please?  
>
> Amino Acid Sequencing Question:  Although N-
> terminal amino acid sequencing
> yielded the expected N-terminal sequence, sequencing
> yields dramatically
> dropped after the first two glutamic acids were
> encountered in the sequence
> (positions 10 and 11 from N-terminus), indicating
> that the % of free alpha
> amino group needed for sequencing also dropped,
> possibly as a consequence of
> glutamic acid cyclization (possibly through bond
> formation between the
> activated side chain -COOH and the -NH2 of the
> resin-bound peptide).  
>
> Thank you very much.
>
> Best regards,
>
> Nga
>
>
>
>
>
>
>
>  
>
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