I have a 27-residue peptide, and its specific function is mostly attributed to the N-terminus. So I wanna leave N-terminus untouched and link a fluorescent dye preferentially to the terminal carboxyl group or one of two Glu residues located in C-terminus. Do you know any dye and protocol suitable for this purpose?
The best way is to have the peptide synthesized with the fluorophore where you want it. The next best way is to have the peptide synthesized with a cysteine residue (only one) at the C-terminus . This residue can be labeled with a fluorophore using maleimide or alkyl halide chemistry.
Thank you Dr. Shapiro. But the peptide is ready there and I have to label it just as it exists, no alternative choices.
Thanks Afred. Which coumarin is appropriate? How about coumarin-6? Suggest any protocols??
Reagents, such as hydrazides, are available for coupling to carboxylic acids, but they are not specific to the C-terminus and will also react with glu and asp side chains. Fluorescent derivatives can be found here:
https://www.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook/reagents-for-modifying-groups-other-than-thiols-or-amines/derivatization-reagents-for-carboxylic-acids-and-glutamine.html
7-Amino-4-methylcoumarin (AMC) can be used as a fluorescence at C-terminal,,,generally the peptide sequence divided into two fragment ,the C-terminal AA couplling with AMC as one part [Peptide Research [1996, 9(2):92-96], the remains as the other, fully protected;,, then the two fragment condensated in solution phase,,,,
just as Dr Shapiro said above you also can try Hydrazines method,,,
if you have more question pls contact me [email protected] ,
Dear fellows, Thanks for information and support.
I think first of all I should look for the right dye in the lab shelf ;-).
I'll be in touch if need arises
Also the commercially available Fluoresceinamine could be useful for that purpose. This compound is similar to FITC but it contains a reactive amine group, so you can introduce in your peptides as a C-terminal Dye
http://www.sigmaaldrich.com/catalog/product/aldrich/201626?lang=es®ion=ES
Hi, Farzad:
Clearly, your main problem is to get you peptide temorary protected to be able to manipulate C-terminus selectively. And it is not possible to to give you advice regarding this mater without knowing amino acid composition of your peptide. In case you have Asp and/or Glu in the sequence, there is no good solution to your problem. Otherwise, you may try exhaustive total acylation using Boc2O in excess in aprotic media (say, Pyridine-DMF) which will produce mixed anhydride of protected peptide directly in one pot, suitable for acylation of amino-derivative of the dye. But there may be serious solubility problems.
Hi dear Igor,
Thanks for the hint
Fortunately, internal Asp or Glu residues are not of any trouble in the case of my peptide.
Regards.
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