Covalently bind OH- group of a toxin to a stationary phase

[Craig Venter]

Hey guys I work with cylindrospemopsin which is a cyanobacterial toxin (see first figure). I want to know how it interacts with mammalian proteins so I wanted to do a “pull down” assay, using the toxin as bait for target proteins.

 

The uracyl group has been proven to be the binding portion to the toxin’s targets. The sulfate group is not involved with the toxic mechanism so I could hydrolyze it with HCl or other acid. This would leave an OH- group to work with. The molecule will have two OH- groups after hydrolysis.

 

I would like to covalently bind this terminal OH- group to a stationary phase (like magnetic beads or spe column) so I can then use the other portion of the toxin as bait for target proteins. Since OH- is not that reactive, maybe I could substitute it for another functional group?

 

What do you recommend?

[hypervalent_iodine]

Sensitivity of other groups aside, the biggest issue I see with your proposed scheme is that you have another secondary OH in there. How do you propose to react one and not the other? I would protect that first and then free your other OH.

 

If you wish to then react that centre with something else, then you could substitute it for a halogen and react that way rather than react the OH directly. Depending on what you wanted to do, you would need a strong base for your reaction (with the OH), which would be complicated by the alpha hydrogen on the other side of the molecule (Edit: I was looking at your drawing, where you do not have the eneone present in the final product, which is there in the digital file; be careful of how you draw things!).

 

One other alternative is to convert it to an ester with an amine base and something like a cyclic anhydride, or maybe an acid chloride. You can functionalize and attach to solid support pretty easily from there. I believe there is extensive literature on this, and it is probably your simplest option. You'll still need to protect the other OH, however.

[hypervalent_iodine]

One other question that has just occurred to me. Do you have any feeling for what type of protein it might bond to and where within the protein it would bind? One issue that might affect your results is if the binding site for the toxin is buried deep inside. This would be problematic for what you are proposing, since the resin bead would limit how far in the toxin can penetrate. I'm not too familiar with pulldown assays and my biochemistry knowledge is sketchy, so perhaps this is a non-issue?

[Craig Venter]

Hi thanks for your quick reply and concern. That centered OH- is an issue. I am afraid that if I halogenate the molecule, the uracyl group could end up modified. There is an easier way to get around it. The strain of cyanobacteria also produces a chemical variant of this toxin (see figure 4 ). I am also isolating this deoxy variant, which has very similar toxic effects on mammalian cells. I can use the deoxy variant instead of modifying the cylindrospermopsin.

This way I will only have one OH- group to worry about.

This toxin inhibits protein synthesis, so we believe it may bind with ribosomes or other proteins related to the translation process. I am not familiarized with pull down assays myself, but I am willing to try different polimers and linkers which might improve my chances.

PS: Sorry for the double bond missing on the drawing, you were right.

[hypervalent_iodine]

Great.

 

Your carbamide should be fine if you are halogenating, but it probably isn't necessary to go that route. My next question is how familiar are you with chemical synthesis and do you have the right set up? I myself deal with a lot of nitrogen heavy compounds and can tell you from experience that they are not especially easy to work with. Purification is probably the most prohibitive aspect, as it is entirely hit and miss. Sometimes it will be incredibly easy, other times have caused me to rethink my synthesis completely. As you will need your compound to be pure for your biological work, make sure you have a handle on this aspect.

 

As well, since I strongly suspect your compounds will be new compounds, you will need to make sure that each step you do has a full suite of characterisation data to go with it. Journals normally require this for publication. Apologies if you already knew all that.

[Craig Venter]

Hi M Viper, I'm back. So our lab does not focuses on organic synthesis, we basically isolate compunds from microalgae, purify them and define chemical structure (LCMSMS mostly) and biological properties. Lots of analytical chemists and pharmacysts in the lab, but we don't have organic synthesis gurus. So characterization at least won´t be a problem.

 

Do you think that the OH- group resulting from hydrolyzing SO4 on Fig.4 could react to a solid phase with divinyl sulfone or epoxy attached?

 

If not what kind of substitution would you go for if you where in my shoes? Thanks!

Edited February 8, 2017 by Craig Venter

[CharonY]

Just as a completely different side note, but make sure that you have a control assay in place. Pull-downs can be notoriously unspecific. Especially high-abundance proteins, such as ribosomes tend to get co-purified very frequently.