Chiral fragments – and poll!

Chirality underpins all life. Nineteen of the twenty amino acids contain at least one stereocenter, as do all nucelosides, sugars, and most metabolites. The very first fragment I ever found was chiral, but that is not typical, at least judged by those that show up in publications. Only 5 of the 27 fragment to lead success stories published in 2015 started with a fragment containing a chiral center. This probably reflects what people choose to screen and pursue. Chiral centers can lead to challenging chemistry, and chiral centers also add to molecular complexity.

All of which brings us to the topic of our new poll: do you include chiral fragments in your primary screening collection? If so, do you include both enantiomers? Please vote in the poll to the right.

If you do include chiral fragments, do you screen racemic mixtures? Crystallography can sometimes reveal which enantiomer is active if the quality of the structure is good enough, but woe betide anyone screening racemic mixtures by ITC! In a new paper in Magn. Res. Chem., Claudio Dalvit (University of Neuchatel) and Stefan Knapp (Goethe University Frankfurt) show that fluorine NMR can also be used to screen racemic mixtures.

As Teddy wrote more than five years ago, ¹⁹F NMR is “just like ¹H NMR”. Most applications of ¹⁹F rely on detecting the line broadening that occurs when a fluorine-containing fragment binds to a protein. However, the chemical shift of the fluorine atom(s) can also change, particularly if the ligand forms hydrogen bonds to the protein. This “chemical shift perturbation” can be large enough to be detectable.

In the absence of protein, ¹⁹F NMR shows the same signal for different enantiomers, so a racemic ligand containing a single trifluoromethyl group gives a single sharp peak. However, upon addition of a protein that binds one enantiomer, the signal splits into two; one remains sharp and retains essentially the same chemical shift, while the other becomes broader and moves. The researchers show this both theoretically and experimentally with a racemic fragment that binds to the bromodomain BRD4. Adding a high-affinity ligand that binds to the same site displaces the fragment, causing the two signals to again converge.

Unfortunately there is no X-ray structure of the ligand bound to the protein, and the two pure enantiomers were not tested individually. And of course, unlike crystallography, ¹⁹F NMR does not reveal which enantiomer in a racemic mixture binds. Still, enantioselective binding can itself be indicative of specific binding, as opposed to various artifacts, and the researchers recommend that “racemates should always be included in the generation of the fluorinated fragment libraries.” What do you think?