Two nonhydrolyzable prolyl adenylate analogs, 5′-O-[N-(L-prolyl)-sulfamoyl]adenosine (L-PSA) and 5′-O-[N-(D-prolyl)-sulfamoyl]adenosine (D-PSA), were prepared in three steps from 2′,3′-di-O-isopropylideneadenosine. Both of these compounds inhibited thein vitroactivity ofEscherichia coliand human prolyl-tRNA synthetase (ProRS). The human enzyme used in this study was derived from the carboxy-terminal domain of the multifunctional humanEPRSgene. TheKATPi values forL-PSA, determined using the ATP–PP i exchange assay, are very similar for both synthetases (≈ 1–2 nM). TheKProi values, on the other hand, vary approximately seven-fold between the two synthetases (0.6 nMfor human and 4.3 nMforE. coli). TheK i values measured for theD-PSA analog are much higher (51–470 nM) for all cases examined; however, the same species-specific differences are observed with respect toKProi. These results indicate possible structural differences in or near the active sites of the two enzymes that may be exploited in the future design of compounds that function as species-specific synthetase inhibitorsin vivo.