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Glycosides of fluorinated p-nitrophenol offer improved sensitivity for detection of β-galactosidase and β-glucuronidase in Escherichia coli and other Enterobacterales

Lookup NU author(s): Dr John Perry

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).

Abstract

© 2026 The AuthorsWe describe the synthesis and evaluation of six halogenated nitrophenyl glycosides for detection of β-galactosidase and β-glucuronidase enzyme activity among Enterobacterales (“coliforms”) and Escherichia coli, respectively. These were evaluated alongside the established substrates; o-nitrophenyl-β-D-galactopyranoside (ONPG), p-nitrophenyl-β-D-galactopyranoside (PNPG) and p-nitrophenyl-β-D-glucuronide (PNP-GUR). The evaluation was performed using 30 isolates of Enterobacterales including 19 isolates of E. coli. Hydrolysis of 2-fluoro-p-nitrophenyl-β-D-galactopyranoside (2-fluoro-PNPG) yielded a significantly stronger yellow coloration after a six-hour incubation period compared to hydrolysis of ONPG and PNPG, potentially allowing for a more sensitive detection of Enterobacterales. Similarly, hydrolysis of the novel substrate 2-fluoro-p-nitrophenyl-β-D-glucuronide sodium salt (2-fluoro-PNP-GUR Na) by producers of β-glucuronidase also yielded a significantly stronger yellow colouration, potentially allowing for a more sensitive detection of E. coli. The yellow chromophore 2-fluoro-PNP retained high colour intensity at reduced pH when compared to o-nitrophenol and p-nitrophenol. Both substrates potentially offer enhanced sensitivity for the detection of Enterobacterales and E. coli in environmental samples as markers of faecal pollution.

Publication metadata

Author(s): Burton M, Cains C, Fenwick DJC, Foster A, Preece CL, Saleem S, Stanforth SP, Turner HJ, Turnbull G, Perry JD

Publication type: Article

Publication status: Published

Journal: Bioorganic and Medicinal Chemistry Letters

Year: 2026

Volume: 135

Print publication date: 01/06/2026

Online publication date: 05/02/2026

Acceptance date: 02/02/2026

Date deposited: 16/02/2026

ISSN (print): 0960-894X

ISSN (electronic): 1464-3405

Publisher: Elsevier Ltd

URL: https://doi.org/10.1016/j.bmcl.2026.130573

DOI: 10.1016/j.bmcl.2026.130573

Data Access Statement: All experimental data is provided in the manuscript and associated supplementary information

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