Date: Tue, 7 Jan 1997 11:21:47 -0800 (PST)
From: "PPMAL (Hathaway/Krapf)" <ppmal@cco.caltech.edu>
To: "ABRF Hypermail (Dirk Krapf)" <abrfhyp@cco.caltech.edu>
Subject: Putting a spacer in a disulfide bond?? -Reply (fwd)
Date: Mon, 06 Jan 1997 13:27:53 -0500
From: Margaret Schott <SCHOTTM@gunet.georgetown.edu>
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Subject: Putting a spacer in a disulfide bond?? -Reply
Perhaps something in what follows will be of use for the problem of how
to insert a spacer between cysteines, if you haven't already got going on
other suggestions! I've spent a fair amount of time in the past on the
chemistry of antibody interchain disulfide bond reductions, alkylations,
and cross-linking. A good overview article for protein cross-linking
reagents is in Bioconjugate Chemistry (1990) 1, 2-12 (Means and
Feeney). It seems that the same could apply to peptides.
Generally cysteines are most reactive under neutral (pH 6-8) aqueous
conditions with maleimide and iodoacetamide compounds, as both have
good specificity for -SH over -NH2 in proteins. Bromo- and
chloroacetamides would be decreasingly reactive but nonetheless have
been used extensively by Robey et al. for the synthesis of "peptomers".
Unfortunately there does not appear to be a readily available source for
bis-iodoacetamides, although some of these compounds were
synthesized and reported several years ago by a Japanese group
(having approx. 6-8 carbons in the chain as I recall).
Bismaleimide compounds are commercially available and may be the best
bet for rapid and specific reactions, and have been used extensively for
cysteine cross-linking in proteins. The downside for your application is
that the tethers may result in more "chemical baggage" than you are
seeking for your molecular locking extension. Pierce sells
bismaleimidohexane (Cat #22319), and Aldrich sells 1,2- and
1,4-phenylenedimaleimide. An excellent source would also be Molecular
Biosciences (Huntsville, AL 205-533-7992). Their catalog lists several
bismaleimides with different tether lengths as short as two carbons (and
even a tris-maleimide!).
A bis-vinyl sulfone (BioLink 6) with a 6-carbon tether is also available
from Molecular Biosciences but my past reading of the literature is that
vinyl sulfones are less reactive.
Another possibility, although more exotic, is an ETAC equilibrium transfer
alkylation cross-link reagent developed by Richard Lawton (U. Michigan)
several years ago and still carried by Aldrich in their Rare Chemicals
supply (Cat. #S-13499, 25 mg/$40). The name is
[2-(4-nitrophenyl)allyl]trimethylammonium iodide] and it was reported in
JACS (1979) 101, 3097 and Aldrichimica Acta (1986) 19,22. (It has been
discontinued but appeared in the 1994-95 catalog). This compound
would provide a propyl linkage (but with an aminophenyl side chain on the
central carbon after reduction step). It has been used to cross-link
partially reduced ribonuclease; some reaction with lysines also occurred.
Margaret E. (Peggy) Schott
Georgetown University-Lombardi Cancer Center
Macromolecular Synthesis and Sequencing Lab
(202) 687-1684