Hi, everyone!
There is a problem about CuAAC efficiency that troubles me a lot. I modified a water soluble comb-like copolymer polyallylamine-graft-PEG (PAA-g-PEG) with azidoacetic acid (about 15 mol% of mainchain monomer). I want to use CuAAC to conjugate some alkyne modified biomolecules (dyes, peptides, anything.) in the next step, but I found the efficiency of my click reaction is really low. Like I added alkyne-Alexa dye to my copolymer, but less than 20% of the dye was found to be clicked on the copolymer. I tried to modify the conditions, but improvement is very limited. Here are my conditions,
1) 150 uM Azide (which means 1 mM main chain monomer)
2) 60 uM Alkyne-biomolecule.
3) 200 uM CuCl2
4) 1 mM THPTA.
5) 5 mM Amoniguanidine (tried to protect the amino groups of my copolymer from being crosslinked by the by-product of ascorbic acid)
6) 2.5 mM ascorbic acid.
7) R.T.
8) 24 h incubation.
9) HEPES buffer, pH 7
In papers, lots of authors said they could get perfect yield by CuAAC. I wonder why the efficiency of my CuAAC is so low. Could it be due to too many amino groups on the PAA main chain that formed complex with copper? Or because of the steric hindrance effect of the PEG side chain? Does anyone have any idea about how to improve the efficiency?
Another detail is that in the beginning of my reaction, the mixture is clear, but about 1-2 h of incubation, there is some insoluble precipitate. What could it be?
Thank you very much for any comment!
Dear Wancheng, many thanks for sharing this interesting chemical problem with other RG members. In this case it is very difficult to judge from the outside what perhaps went wrong in your reaction. This is a very special question which should normally be discussed on-site in detail with your supervisor. All I can do right now is suggest to you two potentially useful review articles which might help you in your analysis:
CuAAC: an efficient Click Chemistry reaction on Solid Phase
Article CuAAC: an efficient Click Chemistry reaction on Solid Phase
and
A Hitchhiker’s Guide to Click-Chemistry with Nucleic Acids
Article A Hitchhiker’s Guide to Click-Chemistry with Nucleic Acids
The second article is even freely accessible as public full ext on RG. You can request the full text of the first article form one of the authors directly via RG. All three of them have RG profiles.
I hope this helps. Good luck with your research and best wishes, Frank Edelmann
Thank you very much for your invaluable suggestions, Prof. Frank T. Edelmann ! I will read those papers!
Wencheng,, did you do calculation to see the density of the dye for 20% of the dye clicking on the polymer? Which Alexa dye did you use?
Hi, Guobing Xiang . Yes, I used the UV module of GPC to calculate the amount of dye. I used Alexa Fluor 647 Alkyne.
Wancheng
Alexa Fluor 647 is heavily negatively charged with 4 SO3H groups, if two dye molecules are very close in space, charge repulsion may affect the loading?
Just FYI, I maybe incorrect!
I see. Never thought about that! I will consider it. Thank you very much for your invaluable comments, Dr. Guobing Xiang !
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