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Fragment linking is a topic we’ve discussed several times. One of the more interesting approaches is template-directed synthesis, in which a protein causes two fragments in close proximity to react with one another (see here for example). In a recent issue of Angew. Chem. Int. Ed., Beat Ernst and colleagues at the University of Basel provide a new variant of this theme, without requiring structural information about the protein.

The researchers were interested in a protein called myelin-associated glycoprotein, or MAG, which blocks axonal regrowth. They started with an NMR screen to determine which members of a fragment library bind to MAG as assessed by a phenomenon known as transverse magnetization decay; essentially, small molecules that bind to a protein behave like large molecules in showing a rapid decay in magnetization, so an increased magnetization decay of fragments in the presence of protein suggests binding. A number of fragments were identified as binders, but the site of binding was not determined.

To find molecules that could be linked, the researchers took a known ligand, the sialic acid derivative 1 (see figure – albeit larger than a fragment), and modified this to contain a spin-label. Spin-labels are small moieties that contain an unpaired electron and, just like large molecules, cause an increase in magnetization decay, but only to molecules within close proximity. The two effects are additive, and thus the researchers could determine which fragments bind to the protein in close proximity to the spin-label-containing derivative of compound 1. In fact, the distance dependence is so pronounced that different protons on the fragment can show different effects, thus indicating which portion of the fragment is close to the spin label (see here for a similar approach using ILOE). In this case, the researchers found that a nitroindole fragment (see figure) had its 5-membered ring positioned closer to the spin label than its 6-membered ring.



Knowing the relative positions of the two ligands, the researchers modified them so they could be linked together. They added functional groups with different linker lengths to create several analogs, replacing the spin label with an alkyne and adding an azide to the nitroindole fragment. They then incubated all the analogs together in the presence of the protein. Analysis of the reaction by HPLC-MS after three days at 37 degrees revealed one prominent product, with a mass consistent with compound 7. Two isomers of this product can be formed, with syn and anti configurations around the triazole, and the researchers synthesized both of them. Interestingly, the anti isomer (shown) had a Kd for MAG of 190 nM, while the syn isomer bound roughly 10-fold more weakly.

Although the ligand efficiency of the final compound is low, sugar-based molecules typically have low ligand efficiencies, and maintaining the same efficiency as starting compound 1 is impressive. However, the ligand efficiency of the final molecule is probably lower than the second-site ligand: the researchers don’t report its affinity, but since it would likely need to be 10 mM or better to be detected its ligand efficiency is probably at least 0.23 kcal/mol/atom.

Still, the final product is sufficiently potent that it could make a useful biological probe. Moreover, the approach is notable in not requiring structure of the protein – a rare and attractive feature for fragment linking.