Purple-Gal (5-Iodo-3-indolyl-β-D-galactopyranoside)

Purple-Gal (5-Iodo-3-indolyl-β-D-galactopyranoside, CAS 36473-36-6) represents a chromogenic substrate in the indolyl glycoside family for β-galactosidase (EC 3.2.1.23) detection, distinguished by iodine substitution at the 5-position of the indole ring that enhances oxidation kinetics, yielding a purple insoluble precipitate upon enzymatic hydrolysis for superior contrast in colony screening compared to traditional X-Gal blue. With confirmed formula C14H16INO6 (MW 421.18 g/mol), this off-white crystalline solid features β-D-galactopyranose O-glycosidically linked to 5-iodo-1H-indol-3-ol, undergoing selective cleavage by E. coli lacZ (α-complementation) and mammalian lysosomal β-galactosidases to liberate the iodoindoxyl aglycone, which rapidly dimerizes/oxidizes into the intensely purple 5,5′-diiodo-indigo dye (λmax ~550-580 nm) localized to enzyme sources without diffusion artifacts. Optimized for molecular cloning workflows requiring orthogonal color readouts to blue/green X-Gal/X-Glc systems, Purple-Gal supports multiplexing in dual-reporter constructs (lacZ/GUS) and high-density transformation plates where purple colonies provide unambiguous recombinant identification (lacZ-) versus non-recombinants (lacZ+ purple), while its heavy atom substitution accelerates color development (visible 4-6h vs. 16h for X-Gal). Employed at 40-80 μg/mL with IPTG in LB agar, it excels in yeast two-hybrid screening, phage titering, and diagnostics of GM1-gangliosidosis via lysosomal β-Gal activity, offering permanent archival staining with enhanced photostability over lighter halogen analogs.

Appearance

• Off-white to pale yellow crystalline powder.
• Hydrolysis produces insoluble purple precipitate.​

Source

• Koenigs-Knorr glycosylation: protected β-D-galactopyranosyl halide + 5-iodoindol-3-ol.
• Specialty suppliers (Sigma-Aldrich analogs, glycobiology catalogs).​

Molecular Weight and Structure

• Formula: C14H16INO6; MW 421.18 g/mol.
• (2S,3R,4S,5R,6R)-2-(5-iodo-1H-indol-3-yloxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol.​

Sugar Specificity

• Selective β-D-galactopyranoside for β-galactosidases (lacZ, lysosomal).
• Negligible glucosidase activity.​

Biological Activity

• Chromogenic β-Gal reporter; faster color than X-Gal.
• Non-toxic; purple product intensification.​

Purity and Microbial Contamination

• ≥98% (HPLC); research grade.
• Synthetic; non-sterile.​

Identity and Quality Control

• ¹H-NMR (iodoindole H-4/6 shift ~7.5-7.8 ppm); mp ~240°C (dec).
• Purple hydrolysis product λmax ~570 nm.​

Shelf Life and Storage

• 2-3 years -20°C desiccated; DMSO/DMF soluble.
• Light-sensitive due to iodine.​

Applications

• Purple/white colony screening; lacZ multiplexing.
• β-Gal diagnostics, enzyme engineering.​

Key Characteristics

• Purple colonies (orthogonal to X-Gal blue); rapid development.
• Iodine-enhanced oxidation kinetics.​

Citation Links

• https://pubchem.ncbi.nlm.nih.gov/compound/36473-36-6 [ variant]
• https://duchefa-biochemie.com/product/details/number/S1403​
• https://www.sigmaaldrich.com/IN/en/product/sigma/res1364c​
• https://cymitquimica.com/cas/36473-36-6/ (CAS data)
• https://www.chemicalbook.com/CASEN_36473-36-6.htm
• https://www.glentham.com (indolyl galactosides)
• https://www.tcichemicals.com (haloindolyl analogs)
• https://www.goldbio.com (chromogenic substrates)
• https://www.medchemexpress.com (β-Gal substrates)
• https://pubchem.ncbi.nlm.nih.gov/compound/Indolyl-beta-D-galactopyranoside