Kevin Howland
Protein Science Facility Manager
Research School of Biosciences
University of Kent
Canterbury CT2 7NJ
Tel: +44-1227-764000 ext 7987
Fax: +44-1227-763912
email: k.howland@ukc.ac.uk
At 07:57 27/01/99 -0500, Singleton, David H wrote:
>A noticeable improvement in quality came from using PEG based resins and
>washing at the completion of every coupling with 1:1 TFE/DCM. This
>supposedly allows the peptide to adopt an alpha helical structure, thereby
>negating the propensity for resin collapse. (B-sheet)
>
>The least expensive of these would be PEG resin. I have examples,
>especially phosphopeptides, where this made a 100% difference.
>
>Why the need for such a high concentration of capping? Couldn't you drop to
>5% and see the same effect? Also, add some HOBt to your activator. Excess
>will make forming the active ester easier and supposedly HOBt suppresses
>racemization.
>
>Note: These observations are from experience on an ABI433, but these truths
>should be universal.
>
>My 2 cents worth,
>David H. Singleton
>Scientist
>Pfizer Central Research
>Eastern Point Road
>Groton, CT 06340
>
>
>
>-----Original Message-----
>From: mcada002@mc.duke.edu [mailto:mcada002@mc.duke.edu]
>Sent: Tuesday, January 26, 1999 6:33 PM
>To: Recipients of ABRF List
>Subject: Optimal chemistry for Rainin Symphony
>
>
>
> To: All using the Rainin Symphony Peptide Synthesizer
>
> I have been using this instrument for about seven months now and am
> over all very pleased with the ease at synthesizing multiple peptides
> simultaneously.
>
> However, I do have some peptides that do not synthsize well at all.
> I am not in a position to spend time and dollars for R&D. Although, I
> have done that to some extent. I would like to improve my
> "success/failure" ratio.
>
> Some difficulties I have noticed:
>
> a. coupling phosphorylated amino acids - even with extended
> coupling times.
> b. peptides with multiple Prolines.
> c. hydrophobic peptides.
> d. ??? and some that I cannot see a known reason for the poor
> quality.
>
> I have found that using 0.1M HOBT/20% piperidine/NMP as deprotect
> reagent does help with the Asp-X difficulties.
>
> I have several years experience with the ABI peptide synthesizers
> which use a bit different activator solution (HBTU/HOBT/NMP, catalyzed
> with DIEA). I wonder if the activator used with the Symphony is as
> robust as the one used on the ABI instruments. Is there an alternative
> that has been proven more effective than the one I am currently using?
>
> Currently, I am using:
>
> Activator: 400mM NMM/150mM HBTU in NMP
> Deprotectant: 0.1M HOBT/ 20% piperidine/ NMP
> Amino acid concentration: 150mM (used 200mM routinely for a while,
> and switched with no noticeable drop in quality.
> Capping: 50% Acetic Anhydride/NMP
>
>
> Coupling time: 40 minutes (2 x 30 min. for double coupling)
> Deprotect time: 2 x 15 minutes
> Capping time: 10 minutes
>
> Resins: Usually loaded Chlorotrityl resins ( PAL for C-term amides)
>
> I would appreciate some discussion on the different protocols you are
> using with good success.
>
> Thanks in advance!
>
>
> Millie McAdams/ HHMI Biopolymer Facility @ DUMC
> 919-684-2652
> FAX 919-684-5458
> email: mcada002@mc.duke.edu
>
>