X-Glc (5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside)

X-Glc (5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside, CAS 15548-60-4) is a synthetic chromogenic substrate widely used in molecular biology for specific detection of β-glucosidase (EC 3.2.1.21) activity, producing an insoluble intense blue precipitate (5,5′-dibromo-4,4′-dichloro-indigo) upon enzymatic hydrolysis that allows unambiguous colony visualization on agar plates. With formula C14H15BrClNO6 (MW 408.63 g/mol), this white crystalline compound features a β-D-glucopyranosyl moiety O-linked to the 3-position of 5-bromo-4-chloro-1H-indol-3-ol, stable under neutral conditions until cleaved by microbial β-glucosidases from Enterococci, Streptococci, Klebsiella, Enterobacter, and Listeria species, yielding the aglycone that spontaneously dimerizes/oxidizes to the intensely colored indigo dye detectable by eye without instrumentation. Unlike fluorogenic alternatives, X-Glc enables permanent archival staining, quantitative plaque assays, and differential chromogenic media (e.g., for coliforms/cellulase producers), with >1000x signal amplification localized to enzyme source for histochemical mapping in tissue sections and bacterial lawns. Incorporated into commercial media like ChromoCult and Rapid’E. coli 2 Agar, it supports food safety testing, environmental monitoring, and functional screening of glycoside hydrolase libraries, offering superior contrast to X-Gal (β-galactosidase) while resisting endogenous plant/microbial interferents through haloaromatic steric protection.

Appearance

• White to off-white crystalline powder.
• Odorless solid; blue product insoluble precipitate.

Source

• Chemical synthesis via Koenigs-Knorr coupling of protected bromochloroindolyl bromide + glucose.
• Commercial suppliers: Sigma-Aldrich (≥97%), TCI (>98% HPLC).

Molecular Weight and Structure

• Formula: C14H15BrClNO6; MW 408.63 g/mol.
• (2S,3R,4S,5S,6R)-2-(5-bromo-4-chloro-1H-indol-3-yloxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol.

Sugar Specificity

• Highly selective β-D-glucopyranoside for β-glucosidases (GH1/GH3 families).
• Minimal cross-reactivity with β-galactosidases/α-glucosidases.

Biological Activity

• Chromogenic reporter; no intrinsic toxicity.
• Detects enzyme activity >10^3-fold via insoluble blue product.

Purity and Microbial Contamination

• ≥97-99% (HPLC/TLC); microbiology grade.
• Synthetic; non-sterile reagent.

Identity and Quality Control

• ¹H-NMR (anomeric H-1 ~5.5 ppm d, J=7.8 Hz); mp 249-251°C.
• Specific rotation [α]D -55° (c=1, DMF).

Shelf Life and Storage

• 3 years at -20°C desiccated; stable in DMF/DMSO.
• Light/moisture protected powder.

Applications

• β-Glucosidase chromogenic media (Enterococci, Listeria detection).
• GH enzyme screening, histochemical staining.

Key Characteristics

• Insoluble blue precipitate; eye-readable without equipment.
• Archival stable; superior to fluorogenic substrates.

Citation Links

• https://cymitquimica.com/cas/15548-60-4/
• https://pubchem.ncbi.nlm.nih.gov/compound/84982
• https://www.tcichemicals.com/IN/en/p/B5393
• https://www.chemicalbook.com/ChemicalProductProperty_EN_CB0683827.htm
• https://www.glentham.com/en/products/product/GC5699/
• https://www.chembk.com/en/chem/5-BROMO-4-CHLORO-3-INDOLYL-%CE%B2-D-GLUCOPYRANOSIDE%20(X-GLC)[9]
• https://www.sigmaaldrich.com/US/en/product/sigma/b4527
• https://pubchem.ncbi.nlm.nih.gov/compound/5-Bromo-4-chloroindol-3-yl-beta-D-glucopyranoside
• https://www.merckmillipore.com/deepweb/assets/sigmaaldrich/product/documents/417/570/b8049dat.pdf
• https://pubchem.ncbi.nlm.nih.gov/compound/5-Bromo-4-chloro-3-indolyl-beta-d-glucuronide