S-Gal

S-Gal (3,4-Cyclohexenoesculetin-β-D-galactopyranoside, CAS 182805-65-8), commercially known as S-Gal®, represents a superior chromogenic substrate to X-Gal for β-galactosidase (β-Gal, EC 3.2.1.23) detection, hydrolyzing the β-glycosidic bond to release 3,4-cyclohexenoesculetin that chelates ferric ions (Fe³⁺) forming an intense black insoluble precipitate with strong T₂* MRI contrast enhancement, enabling both histological visualization and noninvasive molecular imaging. With molecular formula C19H22O9 (MW 394.38 g/mol), this white crystalline solid features β-D-galactopyranose linked at C1-O to the enol form of cyclohexenoesculetin—a coumarin derivative with vicinal hydroxy groups at positions 3,4—exhibiting rapid cleavage by E. coli lacZ (α-complementation), mammalian lysosomal β-galactosidases, and viral β-Gals (kcat/Km >10⁴ M⁻¹s⁻¹), producing the catechol-like aglycone that quantitatively binds Fe³⁺ (1:2 stoichiometry) to generate superparamagnetic nanoscale precipitates detectable by eye, absorbance (λmax 590 nm), and MRI (ΔR₂* >100 s⁻¹mM⁻¹). Unlike X-Gel’s diffusion-prone indigo, S-Gal’s nondiffusible black product localizes precisely to enzyme sources, supporting high-density colony screening (>10⁴ CFU/plate) with 2-4x faster color development (visible at 4h vs. 16h), while Fe³⁺ complexation enables multimodal readout (histology/MRI/optical) for in vivo reporter gene imaging in lacZ-transfected tumor xenografts. Used at 100-200 μg/mL with 0.5-2 mM ferric ammonium citrate (FAC), it revolutionizes molecular imaging by bridging traditional histology with clinical MRI, with applications in gene therapy monitoring, oncolytic virotherapy tracking, and real-time β-Gal pharmacodynamics without ionizing radiation.

Appearance

• White crystalline powder.
• Hydrolysis + Fe³⁺ yields black insoluble precipitate.​

Source

• Glycosylation of 3,4-cyclohexenoesculetin with protected β-D-galactopyranosyl donor.
• Commercial (Sigma-Aldrich S9811 >98%).​

Molecular Weight and Structure

• Formula: C19H22O9; MW 394.38 g/mol.
• (2S,3R,4S,5R,6R)-2-(3,4-dihydroxy-3,4-dihydro-2H-benzo[h]chromen-6-yl)oxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol.​

Sugar Specificity

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

Biological Activity

• Chromogenic/MRI reporter; Km 50-150 μM (E. coli β-Gal).
• Fe³⁺-dependent T₂* contrast (ΔR₂* >100 s⁻¹mM⁻¹).​

Purity and Microbial Contamination

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

Identity and Quality Control

• ¹H-NMR (β-anomeric H ~5.0 ppm d, J=7.8 Hz); mp 210-215°C.
• Black Fe³⁺ complex formation verified.​

Shelf Life and Storage

• 2 years -20°C desiccated; water-soluble sodium salt available.
• Light/metal-protected.​

Applications

• β-Gal colony screening; lacZ MRI reporter imaging.
• Gene therapy monitoring, tumor tracking.​

Key Characteristics

• Black Fe³⁺ precipitate; faster than X-Gal (4h visible).
• Multimodal (histology/MRI); nondiffusible.​

Citation Links

• https://onlinelibrary.wiley.com/doi/10.1002/mrm.22400​
• https://pmc.ncbi.nlm.nih.gov/articles/PMC2924164/​
• https://pubmed.ncbi.nlm.nih.gov/20572145/​
• https://www.chemicalbook.com/ChemicalProductProperty_EN_CB12562019.htm​
• https://www.vulcanchem.com/product/vc241049​
• https://www.medchemexpress.com/3-4-cyclohexenoesculetin-β-d-galactopyranoside.html​
• https://www.chemsrc.com/en/cas/182805-65-8_1270621.html​
• https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/377/874/s9811dat.pdf​
• https://www.medchemexpress.cn/cas/182805-65-8.html​
• http://do.labnovo.com/mdlnumber/MFCD03458481