Hi Michael: Coupling a fluor to the N-terminus of a peptide, follows the same
rules for other aspects of peptide synthesis, namely the chemistry for coupling
and the orthagonal protection scheme. This means that for a blocked peptide on
SPPS, a free amino group is amenable for different types of on-column,
on-instrument or off-instrument coupling startegies. Next the fluor should be
stable to the cleavage cocktail and lastly do you want a masked or unmasked
fluor. Next the nature of the fluor will determine choices.
For example if you can get or make a preactivated fluor (OSu or other) it
simplifies coupling. Next is the coupling through carboxyl or other group on
the fluor. Simplest fluors such as fluorescien come in many flavors such as
FITC etc. There are number of amino-targeted fluors from companies such as
Molecular Probes (Oregon). There are others targeting -SH, OH groups. A
chemcial such as NBD-Cl will react to just about any reactive side chain of a
free peptide.
There are several working protocols available for labeling peptides. I am
certain there must be a few in the ABRF archives. This question has been
addressed by a number of excellent suggestions on the bulletin board before. I
am enclosing one protocol that I have used successfully on many peptides bound
to resin. This is from Sue Hantmann at Perseptive.
Fluorescein-succinimidyl-ester coupling to peptides (Fluor from Molec. Probes)
1. Deprotect final amino acid
2. Wash 5 X 1 ml DMF
3. Dissolve 11 mg Carboxy fluorescein-succinimidyl ester in 11
µl of
diisopropylethylamine (DIPEA, Flammable storage) and 300 µl of DMF. (Should be
bright red).
4. Add above solution to peptide-resin, cover tube with aluminum foil. Let
stand with occasional shaking for 2 hours, or overnight.
5. Wash column with 10 X 1 ml DMF, followed by several washes with 1:1
DMF/DCM. Wash thoroughly with DCM (should be essentially colorless).
6. Dry peptide and deblock with Reagent K. Ether precipitate deblocked and
cleaved peptide. Lyophilize twice from water.
7. Check by HPLC and Mass Spec. Purify if needed.
8. Keep labeled peptides desiccated and away from light.
Gautam Sarath
N-226, The Beadle Center, 19th and Vine Street
University of Nebraska, Lincoln
Lincoln, NE 68588-0664
Tel: 1-402-472-2928; Fax: 1-402-472-7842
http://www.biotech.unl.edu/Proteins/index.html
http://www.pigment.unl.edu/dept/sarath/sarath.html
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