ACS Medicinal Chemistry Letters
Letter
We used a chromogenic substrate for NagZ, p-nitrophenyl β-
D-N-acetylglucosamine, for evaluation of our iminosaccharides
as potential NagZ inhibitors. Kinetic evaluation of compounds
3−6 with NagZ identified 3 as a potent competitive inhibitor
(Ki = 300 15 nM). The magnitude of this Ki is comparable to
the Ki values reported previously for 3 against several β-N-
acetylglucosaminidases.6,20 Compounds 4, 5, and 6 were much
less effective competitive inhibitors (respective Ki values of 51
4 μM, 35 3 μM, and 33 2 μM). The poorer inhibition by
4, 5, and 6 is understandable in terms of their C3 substitution,
compared to the unsubstituted NAG moiety of the endogenous
substrate for NagZ.
The significance of NagZ-dependent exoglycosidase catalysis
has increased following the discovery of its central role28,29 in
peptidoglycan recycling by E. coli.1,30 Peptidoglycan recycling is
monitored by many of the Gram-negative bacteria for the
purpose of controlling induction of the expression of β-
lactamase enzyme, in response to cell wall damage by β-lactam
antibiotics.31 Small molecule inhibition of NagZ attenuates β-
lactamase expression with concomitant improvement in in vitro
β-lactam efficacy, in both E. coli and P. aeruginosa.32−34 Given
the successful application of structure-based design toward
potent and selective GlcNAc pyranosylidene aminocarbamate
inhibitors of NagZ35,36 and the proven synthetic strategies
toward improved iminosaccharide inhibitor efficacy,6−16,20,37,38
our observation of potent NagZ inhibition by a piperidine-
based GlcNAc iminosaccharide augurs well for future
application of the iminosaccharide for small molecule inter-
rogation of these pathways.36,39 Moreover, as it has now been
proven that peptidoglycan recycling occurs in at least one
Gram-positive bacterium (Bacillus subtilis)40 and engages a
mechanistically distinct NagZ ortholog,41 this inhibitor class
may have broad-spectrum implications toward efforts to
preserve clinical relevance for the β-lactam antibiotics.42
ACKNOWLEDGMENTS
■
We thank T. Wencewicz and M. Miller (University of Notre
Dame) for the P. aeruginosa PAO1 strain and B. Galan
(National Research Council-CIB) for a gift of the pET28a
plasmid.
́
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ASSOCIATED CONTENT
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S
* Supporting Information
Experimental procedures, characterization data for all new
compounds, including the 1D (1H, 13C NMR, DEPT) and 2D
NMR spectra (H−H COSY and C−H HETCOR), and the
crystallographic information file (CIF) of compound 15. This
material is available free of charge via the Internet at http://
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Acetamido-1,2-dideoxynojirimycin: an improved synthesis. Tetrahe-
dron 1993, 49, 9605−9612.
AUTHOR INFORMATION
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(12) Gradnig, G.; Legler, G.; Stutz, A. E. A novel approach to the 1-
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Corresponding Author
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*Telephone: (+1)5746312933. Fax: (+1)5746316652. E-mail:
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Funding
This work was supported by a grant from the National
Institutes of Health and by postdoctoral fellowships by the
Spanish Ministry of Science and Innovation (to B.B.) and a
Pew Latin American Fellowship in the Biomedical Sciences,
supported by The Pew Charitable Trusts (to L.I.L.). The Mass
Spectrometry & Proteomics Facility of the University of Notre
Dame is supported by Grant CHE-0741793 from the National
Science Foundation.
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iminoglycitols from amino acids. J. Org. Chem. 2002, 67, 3184−3193.
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strategy for the synthesis of iminoglycitols from amino acids. Angew.
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(16) Steiner, A. J.; Schitter, G.; Stutz, A. E.; Wrodnigg, T. M.;
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Notes
The authors declare no competing financial interest.
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dx.doi.org/10.1021/ml2002746 | ACS Med. Chem. Lett. 2012, 3, 238−242