Synthesis and evaluation of new substrate analogues of Streptomyces R61 DD-peptidase: Dissection of a specific ligand

Good substrates of the Streptomyces R61 DD-peptidase, such as glycyl-L-α-amino-ε-pimelyl-D-alanyl-D-alanine, 1 (Anderson, J. W.; Pratt, R. F. Biochemistry 2000, 39, 12200-12209), contain the glycyl-L-α-amino-ε-pimelyl side chain. A number of thia variants of this structure have been synthesized by means of a disconnection strategy whereby the appropriate thiols were reacted with either acryloyl-D-alanyl-D- alanine or haloalkanoyl-D-alanyl-D-alanines. Kinetics studies of the hydrolysis of these compounds by the R61 DD-peptidase showed that the presence of the N-terminal glycylammonium ion and the pimelyl-α-carboxylate are very important for efficient catalysis. The results of deletion of the C-terminal D-alanine indicate a promising direction toward new inhibitors. Shorter (one methylene less) and longer (one methylene more) analogues of 1 are also poor substrates. Molecular modeling and dynamics studies suggest that the higher mobility of the active site residues and the modified substrates in enzyme-substrate complexes may be the dominant factor in this loss of reactivity. The general conclusion is that essentially all of the structural elements of the side chain of 1 are required to produce a good substrate. This result has important implications for the design of inhibitors of DD-peptidases.