A cell-permeable probe for the labelling of a bacterial glycosyltransferase and virulence factor

Chemical probes for bacterial glycosyltransferases are of interest for applications such as tracking of expression levels, and strain profiling and identification. Existing probes for glycosyltransferases are typically based on sugar-nucleotides, whose charged nature limits their applicability in intact cells. We report the development of an uncharged covalent probe for the bacterial galactosyltransferase LgtC, and its application for the fluorescent labelling of this enzyme in recombinant form, cell lysates, and intact cells. The probe was designed by equipping a previously reported covalent LgtC inhibitor based on a pyrazol-3-one scaffold with a 7-hydroxycoumarin fluorophore. We show that this pyrazol-3-ones scaffold is surprisingly stable in aqueous media, which may have wider implications for the use of pyrazol-3-ones as chemical probes. We also show that the 7-hydroxycoumarin fluorophore leads to an unexpected improvement in activity, which could be exploited for the development of second generation analogues. These results will provide a basis for the development of LgtC-specific probes for the detection of LgtC-expressing bacterial strains.


Control experiment with CIP.
Compound 7a was tested for potential inhibition of the calf intestinal alkaline phosphatase (CIP) used in the inhibition assays.CIP was obtained commercially and used as received.

Synthetic protocols.
General.All chemical reagents were obtained commercially and used as received.Target compounds and synthetic intermediates were purified by flash chromatography and characterized by TLC, 1 H-NMR, 13 C-NMR, and ESI-MS.Flash chromatography columns were packed wet.Thin layer chromatography (TLC) was performed on precoated aluminium plates (Silica Gel 60 F254, Merck).Compounds were visualized by exposure to UV light (254/365 nm).NMR spectra were recorded on a Bruker BioSpin at 400 MHz ( 1 H) or 100 MHz ( 13 C).
Mass spectra were recorded at the EPSRC National Mass Spectrometry Service Centre, Swansea.Compounds 1, 2, 4 and 5 were synthesised as previously reported. 1,2

Figure S1
Figure S1Fluorescence emission spectra of 7a a

Figure S2
Figure S2 Dose-dependent inhibition of LgtC by probe 7a and inhibitor 1 a

1 H and 13 C
NMR spectra for 7a-d and 8 (final compounds), and 6a-d, 9a, ) and heated to reflux for 1h.The reaction was cooled to room temperature, diluted with ice water, and placed in an ice bath.Sodium nitrite (414 mg, 6.0 mmol) was added, and the reaction was stirred for 10 mins.Sodium azide (594 mg, 9.0 mmol) was added in portions, and the solution was stirred for another 15 mins.The yellow precipitate was filtered off, washed with cold water, and dried under reduced pressure to afford the final compound as a yellow solid in 62% yield (350 mg, 1.86 mmol).