These linkers are based on a tetra-aza scaffold having four acetic acid arms (ubiquitous chelator DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), each of which is linked to a para-nitro-phenylphosphonate (referred to as the DOTA-(amide)4-(EG)n-tetraphosphonate or DOTP). The phosphonate group is an irreversible inhibitor of the fungal esterase cutinase and reacts with an active-site serine residue (Ser 120) to give a phosphonate linkage. Linkers can bind 4 molecules of cutinase with different length of tether (L) m=3, n=5 (M) m=1,n=5, (S) m=1, n=1 giving rise to a megamolecule
This paper describes the synthesis, characterization, and modeling of a series of molecules having four protein domains attached to a central core. The molecules were assembled with the "megamolecule" strategy, wherein enzymes react with their covalent inhibitors that are substituted on a linker. Three linkers were synthesized, where each had four oligo(ethylene glycol)-based arms terminated in a para-nitrophenyl phosphonate group that is a covalent inhibitor for cutinase. This enzyme is a serine hydrolase and reacts efficiently with the phosphonate to give a new ester linkage at the Ser-120 residue in the active site of the enzyme. Negative-stain transmission electron microscopy (TEM) images confirmed the architecture of the four-armed megamolecules. These cutinase tetramers were also characterized by X-ray crystallography, which confirmed the active-site serine-phosphonate linkage by electron-density maps. Molecular dynamics simulations of the tetracutinase megamolecules using three different force field setups were performed and compared with the TEM observations. Using the Amberff99SB-disp + pH7 force field, the two-dimensional projection distances of the megamolecules were found to agree with the measured dimensions from TEM. The study described here, which combines high-resolution characterization with molecular dynamics simulations, will lead to a comprehensive understanding of the molecular structures and dynamics for this new class of molecules.
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