FW: PEPSYN; solvation c/DMSO & pNA peptides

Everson, Anita [PRI] (AEverson@prius.jnj.com)
Wed, 1 Apr 1998 14:26:30 -0500

We routinely use DMSO/NMP with our resin; addition of the DMSO actually
helps our couplings, which is why we use it. It does swell the resin;
how well, I'm not sure. I haven't had a problem using neat DMSO for a
coupling. As to PNA peptides, that is something I'm banging my head on
right now. The only way we have found out how to do it is to synthesize
the peptide minus the C-terminal amino acid on chlorotrityl resin (from
Anaspec); leave the FMOC on, then cleave with low % of TFA. This leaves
the side chain protecting groups on the des-C-term peptide since odd
things can couple onto various places of the peptide in subsequent
steps. Then we couple the (C-term amino acid)-PNA 2:1
((AA)-PNA:peptide) in solution at a concentration of 10mg/ml. Remove
the side chain protecting groups and purify. Problems: the (AA)-PNA (we
get ours from Anaspec) is extremely expensive; removal of piperdine
after deprotecting the N-term (we've tried precipitating the peptide
with cold ether, but all our PNA peptides have been 3 or 4 mers, so
precipation has not been effective even with t-butyl ether; we usually
go with HPLC purification); getting a good HPLC separation from the
final mix can be a challenge, as the peaks all seem to want to coelute
with each other. If anyone else has any light to shed on this, I would
appreciate their input!

> ----------
> From:
> mahrenholz@biochem.purdue.edu[SMTP:mahrenholz@biochem.purdue.edu]
> Sent: Tuesday, March 31, 1998 9:29 AM
> To: Recipients of ABRF List
> Subject: PEPSYN; solvation c/DMSO & pNA peptides
>
> Dear folks,
>
> This forum has been so helpful in the past, I wish to spring two more
> questions regarding peptide synthesis:
>
> 1. A client wishes to couple his active azide-compound to a completed
> peptide which is still on-resin. The Fmoc is the only group removed,
> the
> peptidyl- HMP resin (ABI) is dried and in a flask, ready to go.
> Apparently, the azide can only be solubilized effectively in neat
> DMSO. I
> wonder about the ability of DMSO to effectively swell the resin - if
> anyone
> can enlighten me I would be grateful.
>
> 2. Another problem: I have done a little poking around, but have
> come up
> empty-handed with regard to protocol for producing p-nitroaniline
> amides at
> the COOH-terminus of peptides (pNA peptides). This seems to be used
> in
> proteolytic assays, where the release of the p-nitroaniline can be
> followed
> conveniently using visible wavelengths. Anyone able to point the way?
>
> Thanks for any help,
>
> Alan
>
> =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=
> Alan Mahrenholz, Ph.D.
> Dept. Biochemistry
> 1153 S. University St.
> Purdue Unversity
> West Lafayette, IN 47907-1153
>
> voice: (765) 494-6383
> lab: 494-6540
> fax (765) 494-7897
>
>