Practical Spectrophotometric Assay for the dapE-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase, a Potential Antibiotic Target

A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) is described. This assay employs N ⁶ -methyl- N ² -succinyl-L,L-diaminopimelic acid ( N ⁶ -methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N ⁶ -methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono- N ⁶ -methylated-L,L-SDAP as an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N ⁶ -methyl-L,L-SDAP, was synthesized from the tert -butyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I)(COD)( S , S )-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza ( Hi DapE) including captopril (IC ₅₀ = 3.4 [± 0.2] μM, 3-mercaptobenzoic acid (IC ₅₀ = 21.8 [±2.2] μM, phenylboronic acid (IC ₅₀ = 316 [± 23.6] μM, and 2-thiopheneboronic acid (IC ₅₀ = 111 [± 16] μM. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics.