Resorufin beta-D-galactopyranoside
[CAS# 95079-19-9]

Identification

• Classification: Pharmaceutical intermediate >> Heterocyclic compound intermediate >> Pyran compound
• Name: Resorufin beta-D-galactopyranoside
• Synonyms: 7-(beta-D-Galactopyranosyloxy)-3H-phenoxazin-3-one
• Molecular Formula: C₁₈H₁₇NO₈
• Molecular Weight: 375.33
• CAS Registry Number: 95079-19-9
• SMILES: C1=CC2=C(C=C1O[C@H]3[C@@H]([C@H]([C@H]([C@H](O3)CO)O)O)O)OC4=CC(=O)C=CC4=N2

Properties

• Solubility: Very slightly soluble (0.23 g/L) (25 °C), Calc.^*
• Density: 1.71±0.1 g/cm³ (20 °C 760 Torr), Calc.^*
• Boiling point: 651.6±55.0 °C 760 mmHg (Calc.)^*
• Flash point: 347.9±31.5 °C (Calc.)^*
• Index of refraction: 1.724 (Calc.)^*

^* Calculated using Advanced Chemistry Development (ACD/Labs) Software.

Safety Data

• Hazard Symbols: GHS07 Warning
• Risk Statements: H302-H315-H319-H335
• Safety Statements: P261-P264-P270-P271-P280-P301+P312-P302+P352-P304+P340-P305+P351+P338-P330-P332+P313-P337+P313-P362-P403+P233-P405-P501

Discovery and Applications

Resorufin beta-D-galactopyranoside is a synthetic chemical compound widely used as a fluorogenic substrate in biochemical assays, particularly for detecting the activity of the enzyme beta-galactosidase. The molecule consists of a resorufin moiety linked to a beta-D-galactopyranoside sugar unit. Upon enzymatic hydrolysis by beta-galactosidase, the glycosidic bond is cleaved, releasing free resorufin, a fluorescent compound that can be quantitatively measured.

The discovery of resorufin beta-D-galactopyranoside as a substrate is part of the broader development of fluorogenic enzyme substrates designed to provide sensitive and specific detection of enzyme activities in biological samples. Resorufin itself is a well-known fluorophore with excitation and emission wavelengths suitable for many fluorescence detection systems. Its conjugation to a sugar moiety creates a non-fluorescent compound that becomes strongly fluorescent only after enzymatic cleavage, enabling a clear signal-to-noise ratio in assays.

This substrate has found extensive application in molecular biology, microbiology, and clinical diagnostics. It is commonly used to monitor beta-galactosidase activity in gene expression studies where the lacZ gene is employed as a reporter. In such contexts, cells expressing beta-galactosidase convert resorufin beta-D-galactopyranoside into resorufin, allowing fluorescence-based detection and quantification of gene expression levels. This method offers advantages over chromogenic substrates by providing higher sensitivity and enabling real-time monitoring.

Resorufin beta-D-galactopyranoside is also utilized in bacterial detection and identification assays. Many bacteria express beta-galactosidase, and the substrate facilitates rapid and sensitive detection through fluorescence measurement, improving the speed and accuracy of microbial diagnostics. Additionally, the substrate is employed in high-throughput screening assays to evaluate inhibitors of beta-galactosidase or related glycosidases, contributing to drug discovery efforts.

In enzymology, this compound helps characterize beta-galactosidase kinetics and activity under various conditions, including pH, temperature, and the presence of inhibitors or activators. The fluorescence signal generated by resorufin allows precise kinetic measurements with minimal interference from other sample components.

Manufacturing of resorufin beta-D-galactopyranoside involves chemical synthesis techniques that attach the galactopyranoside unit to the resorufin fluorophore via a glycosidic bond. The product is supplied in a purified form suitable for laboratory use, often as a powder or concentrated solution.

Safety considerations include handling the compound under appropriate laboratory conditions to avoid exposure and contamination. The compound’s stability and solubility characteristics are well-documented to ensure reliable assay performance.

In summary, resorufin beta-D-galactopyranoside is a valuable tool in biochemical and molecular biology research. Its discovery and application highlight the importance of fluorogenic substrates in enabling sensitive, specific, and versatile assays for beta-galactosidase activity across diverse scientific fields.

References

1988. A single‐cell assay of β‐galactosidase activity in Saccharomyces cerevisiae. Cytometry, 9(4).
DOI: 10.1002/cyto.990090418

1999. beta-galactosidase assay using capillary electrophoresis laser-induced fluorescence detection and resorufin-beta-D-galactopyranoside as substrate. Biomedical Chromatography, 13(8).
DOI: 10.1002/(sici)1099-0801(199912)13:8<516::aid-bmc918>3.0.co;2-u

2017. Planar yeast estrogen screen with resorufin-β-D-galactopyranoside as substrate. Journal of Chromatography A, 1497.
DOI: 10.1016/j.chroma.2017.03.047