[Craig Venter Clone]

Hey guys I work with cylindrospemopsin which is a cyanobacterial toxin (see first figure). I want to see 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 a 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 would you do gentlemen?

[Arkcon]

I'm not an expert, but what you want to do sounds similar to fuctionalizing CPG or polystyrene resin with a nucleoside base.  That's detailed here:  http://www.atdbio.com/content/17/Solid-phase-oligonucleotide-synthesis#Resin-functionalization  It all seems to be there:  an -OH group connected by a succinate group to an amino fuctionalized porous solid bead in two steps.

[Arkcon]

Or you can go hog wild: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1431690/

[kriggy]

You could convert it to sulphonyl chloride and then to some PEG linker bound to resin. There is tons of possible resins. I suggest you to read something on solid phase synthesis and give it a try. The biggest problem might be that if you try to convert the SO₃^- to chloride, your secondar alcohol might get converted too.

[wildfyr]

The typical method to convert to sulfonyl chlorides is with thionyl chloride or PCl5, both of which will convert the alcohol. If you do your acid hydrolysis, then you won't get any specificity for tethering for one secondary alcohol vs the other. I don't know if that's OK with you. I actually did my PhD in connecting complex functionalities to polymer surfaces, and you've got an interesting one here.

An activated ester like NHS or PFPA in the presence of DMAP might be able to do transesterification and tether your compound. I'm a little worried about this approach because there are a couple amines in there that could be reactive and outcompete the alcohol.

If you want something exotic, you could TBDMS protect the alcohol and convert some polystyrene sulfonate resin to polystyrene sulfonyl fluoride (boil resin in thionyl chloride, then stir it in cold KFHF in water and MeCN), and do SuFEx to tether your molecule. Such an attempt would be pretty novel and make people sit up since the reaction is quite new. It would also preserve your functionality to a large degree, since you would end up with sulfonate ester connections, similar to the sulfate group currently on the molecule.

[Craig Venter Clone]

The typical method to convert to sulfonyl chlorides is with thionyl chloride or PCl5, both of which will convert the alcohol. If you do your acid hydrolysis, then you won't get any specificity for tethering for one secondary alcohol vs the other. I don't know if that's OK with you. I actually did my PhD in connecting complex functionalities to polymer surfaces, and you've got an interesting one here.

An activated ester like NHS or PFPA in the presence of DMAP might be able to do transesterification and tether your compound. I'm a little worried about this approach because there are a couple amines in there that could be reactive and outcompete the alcohol.

If you want something exotic, you could TBDMS protect the alcohol and convert some polystyrene sulfonate resin to polystyrene sulfonyl fluoride (boil resin in thionyl chloride, then stir it in cold KFHF in water and MeCN), and do SuFEx to tether your molecule. Such an attempt would be pretty novel and make people sit up since the reaction is quite new. It would also preserve your functionality to a large degree, since you would end up with sulfonate ester connections, similar to the sulfate group currently on the molecule.

Thanks for all the help guys. So wildfyr I also purify a similar molecule from the same bacterial culture. Is a structural analog of the previus molecule, but without the OH- group in the middle (see figure 4). It has a similar toxicity, and I can use it to test the PCl5 or the SuFEx approach.
Organic synthesis is not my thing, our lab is focused on isolation and purification of metabolites. Have you done this before or do you have a protocol to recommend? It would be of great help if you can point me to some relevant references.

Also I got another question for you wildyr, and all of you guys out there. Do you think that a solid phase or matrix activated with Divinyl Sulfone could crosslink the OH- group created after hydrolyzing SO4 on figure 4?

[wildfyr]

The divinyl sulfone question is a good one. Now that I look at your molecule with fresh eyes, I think you actually don't have a nucleophilic amine, which means no competitive reactions. So divinyl sulfone with a base like triethylamine or DBU might be perfect for attaching the alcohol version of either of these molecules to a solid phase.