In this paper we report the synthesis

In this paper, we report the synthesis and microbiological evaluation of a series of novel chromogenic sugar-based enzyme substrates based upon catechol, 2,3-dihydroxynaphthalene and 6,7-dibromo-2,3-dihydroxynaphthalene 9 cores (Fig. 1).14, 15 2,3-Dihydroxynaphthalene is inexpensive and available in large quantities (>100 g) from several commercial suppliers thus making this an ideal starting material for the synthesis of enzyme substrates. Additionally, halogen atoms can be introduced into this ring-system remote from the hydroxyl-groups, i.e. at the 6,7-positions, whereas the introduction of halogen atoms into known substrates such as compounds 6 and 7 would only be possible adjacent to the hydroxy-groups which may have a detrimental effect on glycosidase activity. We anticipated that the introduction of halogen atoms would be beneficial for reducing 23401 zip code kinase of chelates in solid (agar) media. We envisaged that catechol-derived substrates would have potential applications in liquid media (where the resulting metal chelates would require appreciable aqueous solubility) and that the increased size of the naphthalene-derived substrates 9 would potentially generate a more insoluble end-point better suited for use in solid (agar) media, where diffusion of the chelate must be localised within colonies of microorganisms. The sugar components of structures 9 have been chosen to target a broad range of enzymatic activities across a range of clinically important pathogenic microorganisms. The sugar moieties together with illustrative applications in diagnostic microbiology include: (i) β-d-glucopyranosides (for the detection of enterococci and Listeria monocytogenes), (ii) β-d-galactopyranosides (for the detection of coliforms), β-d-glucuronides (for the detection of Escherichia coli), N-acetylhexosaminides (for the detection of the pathogenic yeast, Candida albicans) and β-d-ribofuranosides (for the detection of Staphylococcus aureus, including MRSA). Catechol β-d-ribofuranoside has previously shown efficacy for S. aureus detection in liquid media.
Synthesis of substrates Catechol 2,3,4,6-tetra-O-acetyl-β-d-glucopyranoside 1018, 19, 20, 21 was prepared from catechol in low yield using a Michael-type glycosidation procedure (Scheme 2). A Zemplén deprotection of compound 10 gave the required β-glucosidase substrate 11. The proton-NMR spectral data of compounds 10 and 11 were consistent with those reported in the literature with large anomeric coupling constants confirming the β-configurations at the anomeric centres.20, 22 The direct reaction of glucose and catechol has been reported to give a 95:5 ratio of α:β anomers in low (11%) overall yield. 2,3,4,6-Tetra-O-acetyl-β-d-galactopyranoside 12 was prepared using a Michael-type glycosidation reaction and after deprotection, the β-galactosidase substrate 13 was obtained. The coupling constant for the anomeric proton (7.7 Hz, d6-DMSO) and the chemical shift of C-1 (104.0 ppm, d6-DMSO) in the proton and carbon NMR spectra of compound 13 respectively, confirmed the presence of a β-glycoside. The tetraacetyl derivative 12 has been described previously in the literature and was reported as comprising a mixture of both α- and β-anomers. Somewhat surprisingly, the substrate 13 appears to be novel although the synthesis of its isomer with the α-configuration has been claimed but no NMR-spectral data was disclosed to support this assignment. Additionally, the large negative optical rotation (–33° in DMSO) reported for this proposed α-anomer structure is more aligned to the value expected from a β-d-galactopyranoside. The protected glucuronide derivative 1425, 26 was prepared from catechol following a similar procedure to that reported in the literature. Deprotection of compound 14 under basic conditions, followed by acidification using an ion-exchange resin, afforded the required β-glucuronic acid derivative which was conveniently isolated as the cyclohexylamine salt 15. The impure glucuronic acid has been proposed as a catechol metabolite produced from rabbits but this compound was not characterised directly. Methylation, per-acylation and finally hydrolysis of the metabolite gave 2-methoxyphenol (guaiacol) which suggested the rabbit metabolite was a mono-glucuronide.