Six transition-state or bisubstrate analogue inhibitors (6-11) have been designed, synthesized, and tested against aspartate transcarbamoylase (ATCase). Several of these inhibitors, 7-9, were designed as analogues of N-(phosphonoacetyl)-L-aspartate (PALA, 5a) and incorporated a tetrahedral sulfur group (-S-, -SO-, -SO2-) alpha to a phosphonic acid moiety. Synthesis of 7-9 was accomplished with a new reagent, diethyl (mercaptomethyl)phosphonate (19). Thiol addition of 19 to diethyl itaconate or other olefins proves a new general synthetic route to (thiomethyl)-phosphonate analogues of acyl phosphates or diphosphate anhydrides. Analysis of the observed inhibition kinetics with ATCase and structural modeling studies indicate that increased steric size of the sulfur moieties in the sulfide 7, sulfoxide 8, sulfone 9, and sulfonamide 10 may cause these compounds to be less potent inhibitors of Escherichia coli ATCase than N-(phosphonoacetyl)-L-aspartate (PALA, 5a). The pKa of the carbonyl groups (or S-analogue thereof) may be a key factor in determining the affinity of ATCase for inhibitor. The distance from the alpha-carbon to the phosphorus atom was judged to be a less important factor in determining the tightness of inhibitor binding since no significant change in the inhibition constant (Ki) occurred upon elimination of the alpha-methylene group in sulfide 7 to give sulfide 11. The ester analogue of PALA (5a), O-(phosphonoacetyl)-L-malic acid (6), exhibited a Ki of 2 X 10(-6) M.