As far as purification, I have not needed to do so on
my fragments, also 10 to 12 residues in length. As
far as analysis of them, I use a simple gradient of
0.1% TFA in water (A) and Acetonitrile (B). The
column used is a Waters Symmetry (150mm), or even a
Phenomenex Nucleosil column (250mm) and my fragments
come off at about 15 minutes (your actual results may
vary....) on a 15-55%B gradient. They are very
clean...like 90+% by area and can be used 'as is'. Be
sure to break your peptide at non-racimizable residues
like glycines...and if they are not available or
inconvienent, Leucine is the next best place.
I also do a full test cleave on my fragments (small
portion of the resin) so I can get a mass spec and
another HPLC of it to make sure it is what it's
supposed to be as well as being able to tell where the
fragments run as potential impurities from truncations
due to incomplete couplings etc.
As far as the couplings...let them go a LONG time to
ensure maximum coupling. Realize that the longer the
chain gets, the slower the coupling due to solubility
problems of the lengthening chain.
Two good papers on the subject are:
Benz, H. "The Role of Solid Phase Fragment
Condensation (SPFC) in Peptide Synthesis" Synthesis,
April 1994 pp 337-358.
Riniker, B., Florsheimer, H.F., Sieber, P., Kamber, B.
"A General Strategy for the Synthesis of Large
Peptides: The Combined Solid-Phase and Solution
Approach." Tetrahedron vol. 49, No. 41, pp.
9307-9320, 1993.
One other paper that may help in racimization
suppression is:
Rydanov, M.G., Klimenko, L.V., Mitin, Y.V. Suppression
of epimerization by cupric (II) salts in peptide
synthesis using free amino acids as amino components.
J.Peptide Res., 1999, 53, 322-328.
Hope these help.
Regards,
Steven R. Johnson, B.S. Chem.
--- Jan Lukszo <lukszo@ncifcrf.gov> wrote:
> Dear ABRFers,
>
> Facing the synthesis of the relatively long (>70 AA)
> peptide I have decided
> to do it by a mixed approach, comprising of the
> stepwise addition of AA and
> a few fully protected peptide fragments of 10-11AA
> each. Would anybody
> experienced in peptide synthesis by fully protected
> peptide fragment
> condensation methodology share with me/us some
> insights about analysis and
> purification of these fully protected (Fmoc-ON)
> fragments by HPLC. Details
> about types of columns (packings)used, solvents,
> gradients, detection, etc.
> would be greatly appreciated.
>
> JL
> Dr. Jan Lukszo
> Peptide Analysis and Synthesis Unit
> NIH/NIAID/OSD/SBS
> Twinbrook II
> 12441 Parklawn Drive
> Rockville, MD 20852
> phone:(301)496-3786
> fax:(301)480-2618
> E-Mail: lukszo@ncisun1.ncifcrf.gov
>
>
=====
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