I have an 18aa azido-peptide conjugated at the N-terminus (cysteine). Synthesis and folding were validated through HPLC and mass spec. Multiple attempts were made with Dylight-phosphine conjugates and the azido-peptide to enact the Staudinger ligation (1:1 mol ratio). After incubation, the product is isolated by analytical HPLC and retains color of the dye. Upon mass spec (ESI), the product generates m/z of the unconjugated starting material.
Trials were run using different phosphine dyes at 24 hrs and 76 hrs. at room temp. Solvated in DMF as recommended by Thermo scientific.
Does anyone have experience with successful fluorescent bioconjugation of peptides? Or can offer explanations as to what might be going wrong with my experiments? Any help is appreciated.
If your cysteine has a free thiol, the sulhydryl may be preferentially reacting with the phosphine, and then later hydrolyzing to give starting material. In that case, you may be able to overcome the competing reaction by using excess phosphine dye reagent.
My experience suggests that a spacer moiety, incorporated between the peptide functional region and the N-terminal dye conjugation site, helps prevent steric inhibition.
Did you consider ordering your peptide directly with the required fluorescent dye and the N-terminal cysteine for conjugation?
John Carter thank you for the recommendation! I forgot to mention there is a 6-azidohexanoic acid spacer linked to the N-terminal cysteine. I haven't tried a molar excess of the phosphine dye reagent because of the cost but I suppose I have no other options at the moment!
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