This paper describes the discovery of a new linker that facilitates target identification experiments. They synthesized a diazo reagent that is substituted in the alpha position with different electron-withdrawing groups and also with an alkyne. The diazo reagent can be attached to a natural product containing hydroxyl groups in an efficient and chemoselective way using Rhodium catalyst, this natural product can be subsequently tagged with biotin via click chemistry to facilitate affinity chromatography.
Most of the time, these experiments require a lot of structure activity relationship to find a position on the molecule where the linker can be attached without modifying the bioactivity. The really interesting thing about this paper is that the diazo reagent was selectively attached to the most accessible hydroxyl group of diverse natural products and the activity was maintained, yielding probes ready to be used in affinity chromatography without the need of SAR.
While I think this is a great method to easily purify and isolate proteins, the major limitation would be in where the free alcohol was during inhibition. For example, if the only alcohols are deep within an active site of the protein of interest, it may be difficult to attach the chemical tag.
ReplyDeleteYes. They functionalized the alcohol (with the alkyne-diazo ligand) before incubation of the small molecule with the cell extract, so if the alcohol was deep in the binding site, it is more likely to be important for the affinity. So they would probably have observed a significant decrease in activity of the functionalized small molecules, and these probes probably couldn't be used for affinity chromatography.
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