Evaluation of cyclohexenoesculetin-beta-D-galactoside and 8-hydroxyquinoline-beta-D-galactoside as substrates for the detection of beta-galactosidase

Abstract

We describe the synthesis of two new substrates for the detection of ␤-galactosidase and evaluate their performance in comparison with that of 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal). Of 171 Enterobacteriaceae strains that were able to hydrolyze X-Gal, 166 (97.1%) also hydrolyzed cyclohexenoesculetin-␤-D-galactoside whereas only 96 (56.1%) showed evidence of hydrolysis of 8-hydroxyquinoline-␤-D-galactoside. No false-positive results were observed with either substrate.

19 F‐NMR detection of lacZ gene expression via the enzymic hydrolysis of 2‐fluoro‐4‐nitrophenyl β‐D‐galactopyranoside in vivo in PC3 prostate tumor xenografts in the mouse 1

“…At 2 mM there was an ϳ80% survival after 96 h for WT cells, whereas considerable cell loss was observed after 96 h exposure to concentrations of Ͼ1 mM for the lacZ cells with only 10% survival at 2 mM. Conversion of OFPNPG to OFPNP by stably transfected human prostate cancer PC3-lacZ cells could also be detected by 19 F CSI (Fig. 3).…”

“…Addition of OFPNPG to a suspension of PC3-lacZ cells at 37°C led to rapid cleavage of the galactoside and release of the yellow aglycone OFPNP. Both substrate and product were detectable by 19 F-MRS, and a time course is shown in Fig. 2.…”

“…Primary monoclonal anti-␤-gal antibody (Promega) and anti-actin antibody (Sigma) were used as probes at a dilution of 1:5000, and reacting protein was detected using a horseradish peroxidase-conjugated secondary antibody and ECL detection (Amersham, Piscataway, NJ, USA). 19 1 H coupling has been observed at either field, no decoupling was applied. T1 was measured for both substrate and product as a mixture in saline at 37°C at 9.4 T using a standard saturation recovery sequence.…”

“…PCR and Western blot are the most commonly used techniques for evaluation of gene expression, and can be used for quantitation, but are highly invasive (requiring a biopsy). Multiple colorimetric reporter substrates for ␤-gal have been demonstrated, and some are commer-cially available for histology and in vitro detection (15)(16)(17)(18)(19).…”

“…We previously presented the successful synthesis and evaluation of fluoro-nitro-phenyl-galactopyranosides to detect ␤-gal activity in vitro by 19 F NMR spectroscopy and imaging (23)(24)(25)(26)(27)(28). In particular, 2-fluoro-4-nitrophenyl ␤-d-galactopyranoside (OFPNPG) is highly responsive to the action of ␤-gal, and cleavage to form the aglycone 2-fluoro-4-nitrophenol (OFPNP) results in a pH-dependent chemical shift of 4 -6 ppm for the fluorine resonance.…”