Casting light on target-guided synthesis

Target-guided synthesis, in which a protein templates the formation of its own inhibitor, is a concept first proposed decades ago. There are roughly two flavors. Dynamic combinatorial chemistry (DCC) involves reversible formation of the product, and we wrote in 2017 about some of the challenges. Kinetic target-guided synthesis (KTGS) involves irreversible chemistry, for which the options are limited. The classical click chemistry azide-alkyne cycloaddition is so slow that reactions usually take days, which can be a problem for delicate proteins. A recent (open-access) paper in Angew. Chem. Int. Ed. by Cyrille Sabot et al. describes a bright way to accelerate things.

 

The researchers turned to photochemistry, specifically diazirine chemistry. Illuminating 3-trifluoromethyl-3-phenyldiazirines leads to loss of nitrogen and formation of highly reactive carbenes. The carbenes are so hot that they can react indiscriminately with proteins, as we described here. However, the reaction with thiols is faster than the reaction with other functional groups on proteins, so the researchers reasoned that a library of thiols could out-compete the protein.

 

The carbonic anhydrase bCA-II was chosen as a model protein. Sulfonamide-containing molecules such as compound 5 are known to be good inhibitors. This “anchor” molecule was incubated at 60 µM with seven different diazirines, each at 400 µM, in the presence or absence of 30 µM bCA-II and then irradiated with 365 nM light for a few minutes. Most of the reactions produced similar amounts of product in the presence or absence of bCA-II, but compound 1b yielded about threefold more of compound 2d in the presence of bCA-II, suggesting the reaction was being templated by the protein. 

 

Control experiments lend credence to this hypothesis. First, adding a known competitive bCA-II inhibitor reduced the formation of compound 2d to background levels. Second, other proteins did not cause a similar enhancement in the formation of compound 2d. Finally, conducting the experiment with phenylmethanethiol (ie, a variant of compound 5 lacking the sulfonamide moiety essential for interaction with bCA-II) did not cause an enrichment of the photochemical product in the presence of the enzyme.

 

Chiral HPLC was used to show that compound 2d was slightly enriched for the (R)-enantiomer, with an enantiomeric excess of around 10%, when the reaction was conducted in the presence of bCA-II but not in the absence. The two enantiomers were synthesized and tested, and the (R) form did indeed have slightly better activity (300 nM vs 330 nM).

 

This is a thoughtful, well-conducted investigation. But it makes me even less sanguine about the practicality of KTGS for finding new chemical matter, for several reasons. First, the efficiency of the reaction is poor: the researchers calculate the yield of compound 2d at around 1% of the enzyme concentration, so low that they used single-ion monitoring (SIM) mass spectrometry to detect it. Because of this low efficiency, the concentration of enzyme used needs to be quite high.

 

The most serious strike against KTGS is the fact that all of the diazirines generated potent (sub-micromolar) inhibitors. One of them was even slightly better than compound 2d but did not show enrichment in the presence of bCA-II. False negatives seem to be a major problem, as we’ve written previously.

 

One caveat to my caveats is that compound 2d is only marginally more potent than the starting compound 5. NMR experiments conducted with diazirine 1b suggest binding to the protein, though the affinity was not quantified. Perhaps a different fragment linking system, in which both fragments have measurable affinity for the target, would be better suited to demonstrate the utility of KTGS. For now, this paper does a nice job highlighting its drawbacks.