Document Type
Article
Publication Date
4-26-2018
Publication Title
PLoS One
Volume
13
Issue
4
Pages
1-24
Publisher Name
PLOS
Abstract
A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) is described. This assay employs N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-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, N6-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 (HiDapE) including captopril (IC50 = 3.4 [± 0.2] μM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] μM, phenylboronic acid (IC50 = 316 [± 23.6] μM, and 2-thiopheneboronic acid (IC50 = 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.
Recommended Citation
Heath, Tahira K.; Lutz, Marlon R. Jr.; Reidl, Cory T.; Guzman, Estefany R.; Herbert, Claire A.; Nocek, Boguslaw P.; Hotz, Richard C.; Olsen, Kenneth W.; Ballicora, Miguel A.; and Becker, Daniel P.. Practical Spectrophotometric Assay for the dapE-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase, a Potential Antibiotic Target. PLoS One, 13, 4: 1-24, 2018. Retrieved from Loyola eCommons, Chemistry: Faculty Publications and Other Works, http://dx.doi.org/10.1371/journal.pone.0196010
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Copyright Statement
© The Authors 2018
Comments
Author Posting. © The Authors 2018. This article is posted here for personal use, not for redistribution. The article was published in PLOS One, vol. 13, no 4, 2018, https://doi.org/10.1371/journal.pone.0196010.