4MU-Gal

4MU-Gal (4-Methylumbelliferyl-β-D-galactopyranoside, CAS 6160-78-7) is a widely used fluorogenic substrate for highly sensitive detection of β-galactosidase (β-Gal, EC 3.2.1.23) enzymes, where hydrolysis releases the intensely fluorescent 4-methylumbelliferone (4-MU) fluorophore with excitation/emission maxima at 360/450 nm (quantum yield φ=0.63), enabling sub-picomolar enzyme detection in real-time kinetic assays. With molecular formula C16H18O9 (MW 338.31 g/mol), this white crystalline compound features β-D-galactopyranose linked via O-glycosidic bond at C1 to the 7-hydroxy position of 4-methylumbelliferone, remaining non-fluorescent until cleaved by E. coli lacZ (α-complementation), mammalian lysosomal β-galactosidases, or Streptococcus β-Gal, producing >1000-fold fluorescence enhancement for quantitative measurement in 96/384-well formats. Superior to chromogenic X-Gal for live-cell imaging, HTS inhibitor screening, and diagnostics due to its superior signal-to-noise, no-wash capability, and orthogonality to GFP/Rhodamine, 4MU-Gal supports continuous monitoring of β-Gal trafficking in lysosomal storage disorders (GM1-gangliosidosis), bacterial reporter gene expression (lacZ fusions), and enzyme kinetics (Km 20-100 μM). Cell-permeant at 10-100 μM loadings, it enables ratiometric fluorescence in single cells via droplet microfluidics or flow cytometry, while alkaline stopping (pH 10) shifts 4-MU to blue fluorescence (λem 448 nm) for endpoint reads, positioning it as the gold standard fluorogenic probe in >50,000 publications spanning glycobiology, neuroscience, and drug discovery.

Appearance

• White crystalline powder.
• Non-fluorescent; hydrolysis yields blue-fluorescent 4-MU.

Source

• Koenigs-Knorr synthesis: protected β-D-galactopyranosyl halide + 4-methylumbelliferone.
• Commercial >99% (Sigma-Aldrich, GoldBio).

Molecular Weight and Structure

• Formula: C16H18O9; MW 338.31 g/mol.
• 4-Methyl-2-oxo-2H-chromen-7-yl β-D-galactopyranoside.

Sugar Specificity

• Highly selective β-D-galactopyranoside for β-galactosidases.
• Minimal α-galactosidase activity.

Biological Activity

• Fluorogenic substrate; detects pM β-Gal concentrations.
• Ex/Em 360/450 nm; φ=0.63 (alkaline).

Purity and Microbial Contamination

• ≥99% (HPLC); molecular biology grade.
• Synthetic; non-sterile.

Identity and Quality Control

• [α]D -58° (c=1, DMF); mp 192-195°C.
• Fluorescence enhancement post-β-Gal verified.

Shelf Life and Storage

• 5 years -20°C desiccated; DMSO stock 25 mM stable.
• Light-protected.

Applications

• β-Galactosidase assays; lysosomal storage diagnostics.
• HTS screening, live-cell imaging.

Key Characteristics

• 1000x fluorescence gain; no-wash kinetic assays.
• Cell-permeant; orthogonal to GFP.

Citation Links

• https://pubchem.ncbi.nlm.nih.gov/compound/9879604
• https://www.sigmaaldrich.com/US/en/product/sigma/m1634 [ variant]
• https://www.abcam.com/en-us/products/biochemicals/x-gal-chromogenic-beta-galactosidase-substrate-ab144388
• https://www.goldbio.com/media/1678/4-mu-gal.pdf
• https://www.chemicalbook.com/CASEN_6160-78-7.htm
• https://www.glentham.com/en/products/product/GC2049/
• https://www.tcichemicals.com/IN/en/p/M1875
• https://www.medchemexpress.com/4-Methylumbelliferyl-beta-D-galactopyranoside.html
• https://moleculardepot.com/product/4-methylumbelliferyl-beta-d-galactopyranoside/
• https://www.creative-enzymes.com/similar/4-Methylumbelliferyl-beta-D-galactopyranoside_1626.html