For the first time, yttrium triflate was used as an efficient green catalyst for the synthesis of α-aminophosphonates through a one-pot three-component Birum–Oleksyszyn reaction.
The α-aminophosphonate UAMC-00050, a newly developed
trypsin-like
serine protease inhibitor, is a lead compound for the treatment of
dry eye syndrome and ocular inflammation. The medicinal chemistry
route developed at the University of Antwerp possessed several problems
hampering the scale-up such as poor yields for some of the steps,
hazardous reagents, and environmental footprint. Herein, we report
an optimized route for the UAMC-00050, in which environmental unfriendly
solvents were excluded, hazardous reagents were replaced with safer
alternatives, and are more efficient in terms of atom economy. Every
reaction step was optimized to reach a higher yield, and design of
experiment was used to find the optimum conditions in the last step.
Furthermore, all the flash chromatography purifications of intermediates
were replaced with plug filtration, slurry purifications, or crystallization.
The overall yield was increased from 3% in the medicinal chemistry
route to 22% in the process development route.
Activity-based probes (ABP) are molecules that bind covalently to the active form of an enzyme family, making them an attractive tool for target and biomarker identification and drug discovery. The present study describes the synthesis and biochemical characterization of novel activity-based probes targeting trypsin-like serine proteases. We developed an extensive library of activity-based probes with “clickable” affinity tags and a diaryl phosphonate warhead. A wide diversity was achieved by including natural amino acid analogs as well as basic polar residues as side chains. A detailed enzymatic characterization was performed in a panel of trypsin-like serine proteases. Their inhibitory potencies and kinetic profile were examined, and their IC50 values, mechanism of inhibition, and kinetic constants were determined. The activity-based probes with a benzyl guanidine side chain showed the highest inhibitory effects in the panel. Surprisingly, some of the high-affinity probes presented a reversible inhibitory mechanism. On the other hand, probes with different side chains exhibited the expected irreversible mechanism. For the first time, we demonstrate that not only irreversible probes but also reversible probes can tightly label recombinant proteases and proteases released from human mast cells. Even under denaturing SDS-PAGE conditions, reversible slow-tight-binding probes can label proteases due to the formation of high-affinity complexes and slow dissociation rates. This unexpected finding will transform the view on the required irreversible nature of activity-based probes. The diversity of this library of activity-based probes combined with a detailed enzyme kinetic characterization will advance their applications in proteomic studies and drug discovery.
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