Fragment-linking on proteins: amide formation

Among fragment-linking approaches, protein-templated reactions have a special appeal. When two fragments that bind next to one another on a protein react, the protein essentially creates its own ligand. The reaction can be reversible, as in dynamic combinatorial chemistry, though the choice of chemistries tends not to be very drug-like, necessitating replacement of the linker for lead development to proceed. Irreversible chemistries, such as cycloadditions (“click chemistry”), give more stable linkers, but these still tend to be unusual. In a recent open-access paper in Angew Chem., Jörg Rademann and collaborators at the Freie Universität Berlin and Philipps-Univesität Marburg have extended the approach to amides – one of the most vanilla chemistries out there.

The researchers used factor Xa as a model protein. Using information in the literature they designed compound 15, found that it was a low nanomolar inhibitor, and obtained a crystal structure of the molecule in complex with the enzyme. Cleaving one of the amide bonds provides two fragments, compounds 16 and 17, each with very weak activity. Put another way, linking these two fragments leads to superadditivity of binding.

Chemists often make amides by “activating” a carboxylic acid and reacting it with an amine. We usually want this to happen quickly, but here the researchers wanted the reaction to only occur when the two fragments bind next to one another on the surface of the protein.

The team made a dozen activated esters of compound 1 and incubated 5 mM of each of these with 0.285 mM of amine 14, in the presence or absence of 14.5 nM factor Xa. (Each combination of reactants would produce compound 15.) After two hours protein was added to the no-protein controls and fluorogenic substrate was added to all the samples, which were tested for enzyme activity. Not surprisingly, inhibition was complete for particularly active esters (such as 4-nitrophenyl ester), whether or not protein had been present during the incubation. Similarly, the presence of protein also didn’t matter for particularly inactive esters (such as methyl ester): inhibition was minimal because very little product 15 formed. But for two Goldilocks esters (phenyl ester and 2,2,2-trifluoroethyl ester), inhibition was greater when protein was present during incubation.

The researchers used mass spectrometry to quantify the amount of compound 15 formed. For the 2,2,2-trifluoromethyl ester, after 1.7 hours the concentration of compound 15 was 10 nM in the presence of factor Xa, but only 0.15 nM in its absence. (The phenyl ester gave less selective results.)

Of course, as these numbers suggest, product formation could be limited by the concentration of the enzyme, possibly complicating detection. More significantly, even relatively unreactive esters could react with the dozens of nucleophiles on proteins, potentially inactivating or denaturing them. Still, this is an intriguing approach, and it will be fun to see whether it works in more challenging systems.