We have synthesized amino labeled oligos at our lab, and performed post syntetic conjugation to ATTO 647 (NHS), following the original protocol we got from the supplier.
We purified the oligo by HPLC, but we obtained two peaks with readings at both 260nm and 650nm, but with slightly different colors (one dark blue, one slightly greenish blue).
Does anyone have experience with conjugating oligos to ATTO (NHS) dyes and could provide some insights? What is the expected chromatogram?
Hello Taissa Engel, no idea what you have done. But this is what I could tell you: If you have two peaks in HPLC you have definitely two different compounds. Either in the process of oligo synthesis or during the conjugation two different possibilities gave two compounds. You should exactly analyze, which of the two compounds is that you need for further work.
Hi, Joachim R. Grün ... Thanks for yor answer!
Yes, I definitely believe that I have two compounds there, but I'm trying to brainstorm here to investigate this issue. After synthesis, the amino oligo was purified by HPLC before being conjugated with the fluorophore. The oligo was extremely pure, free of synthetic flaws, traces of amino labeling and salts. Therefore, I believe that it was something that occurred during conjugation, possibly some degradation of the fluorophore.
We followed the protocol to the letter, but the protocol suggested a pH between 8 and 9, and our reaction was done at pH 9. However, after we noticed the separation of these two peaks (with two different colors), we went back to the literature to investigate, and we found that the fluorophore is unstable at pH greater than 8.5. So why does the protocol suggest a pH between 8 and 9? I'm a little irritated by this. Anyway... I would like some insights from someone who has experience with these ATTO dyes, to see if there are other tips to optimize my post-synthetic conjugation (besides pH below 8.5), and also to know what to expect from the chromatographic profiles, yield, stability, etc.
We have a lot of experience with fluorescent probe synthesis, but we have always used phosphoramidite dyes, and we are now starting the synthesis of fluorescent probes labeled in post-synthetic conjugation, this was the first attempt. Tips and references are welcome!
They might be steroisomers, try to get mass to verify. The color difference might be concentration difference.
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