Anomalous cleavage/elimination during Fmoc assembly?
I know how C-terminal proline can cause diketopiperazine cleavage (with
elimination of a cyclic dipeptide after deprotection of the
penultimate/second amino acid). But recently I've seen what looks like a
slightly similar reaction at the N-terminus of a peptide chain, and with no
proline.
For our HTS ligand peptides, we commonly incorporate a tag and spacer at the
N-terminus. It is: biotin-Lys(dansyl)-Lys-Lys-Gly. This structure is
simply assembled stepwise onto various peptides of about 20 amino acids via
standard SPPS. (I say it's standard SPPS. We use HBTU chemistry on
polyethylene Mimotopes Inc. crowns. The model we employ bears a
hydroxyethyl-methylacrylic graft and a typical RAM linker. We make
thousands of individual non-combinatorial peptides per month.)
Now the weird observation is that, for almost every peptide we make with
this N-terminal structure, around 10% of the crude material lacks the entire
tag/spacer structure except for biotin (as determined via LC-MS analysis).
I.e., instead of having biotin-Lys(dansyl)-Lys-Lys-Gly on the N-terminus,
these peptides have only biotin. If there are any single amino acid
deletions mixed in there, they must be well below 1%, since we can't find
any even when we look specifically for them. I.e., e.g., we don't see ANY
peptide with biotin-Lys-Lys-Gly or biotin-Lys(dansyl)-Lys-Gly. So I've come
to doubt that it's simply poor coupling.
Before I decide to give up and believe that this phenomenon is due to some
kind of voodoo aggregation or backbone structure (which would seem to have
to exist in all the peptides individually, PRIOR to addition of the
spacer/tag), I'm entertaining the possibility that the whole
Lys(dansyl)-Lys-Lys-Gly structure is somehow cleaving itself off. So, I'm
considering doing experiments with timed incubation with individual
components of the biotinylation cocktail. Then we can look in the
supernatant for the fragment, possibly cyclized. My lab people are
suggesting that we look into different spacer sequences and
fluoro/chromophores.
But we're an industrial lab, not an academic one. And the fact is that I'd
really rather just get the answer than try to get a little paper out of
this. So if anybody has seen anything like this before I'd love to hear
their stories. Particularly if there's a logical explanation. And
especially if there's a simple solution.
Mark
-- . . . . . Dr. John Mark Carter MCarter@AxCellBio.comAxCell Biosciences Corporation 826 Newtown-Yardley Road, Suite 100 Newtown, PA 18940-1721 office 267.757.1223 lab 267.757.1230 FAX 267.757.1301 . . Axcell Biosciences is a wholly owned subsidiary of the Cytogen Corporation. . . . . . "Ohne Analyse, keine Synthese." [Without analysis, no synthesis.] -Friedrich Engels, Anti-D¸hring, 1878
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