From: Jonas Nilsson (jonni$##$ifm.liu.se)
Date: Thu Oct 09 2003 - 04:22:42 EDT
Dear All,
I've come across an intresting problem.
I'm trying to make an makromolecular acyl donor by using a 42 aa
polypeptide as a scaffold. The scaffold contains only one primary amine
(Lysine) and one thiol (Cysteine). I modify the polypeptide by treating it
with acylating reagents (N-hydroxy succineimide esters (NHS) and
p-Nitrophenyl esters (PNP)). Treatment with one equivalent of NHS ester
will acylate the Lysine highly specifically. Treatment with more acylating
reagent (NHS or PNP) will then acylate the cysteine. This gives me the
opportunty to place two different acylgroups on my scaffold.
The aim is then to put a ligand on thy lysine, that have affinity to a
target, and a transferable group on the cysteine that due to thioester
lability can migrate to the target by the means of proximity effects.
However I have experienced a remarkable stability of the thioester that
inhibits the migration. Treating the thioesters with base (pH>12 for 3
days) or even hydroxyl amine will not cleave the thioester! Actually the
amidebonds of the peptide starts breaking up, but the acyl on cysteine sits
there rock solid.
I had the impression that thioesters are quite labile and I can't really
explain this remarkable stability. I've done this on two different
polypeptides with different sequences. I've also used two different
acylgroups with the same results.
Do you have any experience with the stability to thioesters? Can you
present a rationale for my inability to hydrolyse the acyl-cysteine bond?
The neigbourhood of the cysteine have the sequences ..DAAAJAECLARHFEAFAR..
and ..DAAQLAEQLACRFEAFAR.. and my acylgroups are butyrate (PNP-ester) and
N-(p-sulfonamoylbenzoyl)-6-amino-hexanoate or with a different name
6-(4-Sulfamoyl-benzoylamino)-hexanoate (Smiles:
O=S(C1=CC=C(C(NCCCCCC([O-])=O)=O)C=C1)(N)=O) (NHS-ester).
Please share your knowledge with me.
/jN
-- _______________________________Jonas Nilsson, Ph D student
IFM Organisk kemi Linköpings universitet SE-58183 Linköping
ph: +46-(0)13-282583 fax: +46-(0)13-122587 email: jonni$##$ifm.liu.se _______________________________
_______________________________________________ ORGLIST - Organic Chemistry Mailing List Website / Archive / FAQ: http://www.orglist.net To post a message (TO EVERYBODY) send to everybody$##$orglist.net To unsubscribe, send to everybody-request$##$orglist.net the message: unsubscribe your_orglist_password your_address List coordinator: Joao Aires de Sousa (coordinator$##$orglist.net)
This archive was generated by hypermail 2.1.4 : Thu Oct 09 2003 - 05:47:01 EDT