If you are biotinylating with Biotin-NHS, and using HBTU to couple it, that
is your problem. We had the same problem. We then synthesized (FMOC SPPS
with Merrifield resin) the peptide up to the addition of the biotin &
checked the integrity of the synthesis. It was OK. Couple the biotin-NHS
with HBTU and voila, deletion peptide with biotin. Something to do with the
NHS and HBTU. Now, this didn't happen every time we biotinylated using this
method, only every once in a while. Going back and looking at all the
sequences this happened to, we could find no common amino acid or stretch of
amino acids present to explain what was going on. It appears to be
"sequence dependent"; those magic words used to explain why peptides screw
up when you don't know why. We too are a production lab and don't have any
time to look into the why of things. We went back to manually coupling
Biotin-NHS with HOBt only and have had no problems since.
Anita Everson
Peptide Lab
R W Johnson PRI
La Jolla, CA
(858)784-3061
aeverson@prius.jnj.com
> -----Original Message-----
> From: Carter, Mark [SMTP:MCarter@axcellbio.com]
> Sent: Tuesday, August 08, 2000 12:52 PM
> To: Recipients of ABRF List
> Cc: Chemistry
> Subject: PepSyn
>
> 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.com
>
> AxCell 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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