ONP‑α‑Gal

ONP‑α‑Gal (CAS 19887‑85‑5), systematically 2‑nitrophenyl‑α‑D‑galactopyranoside, is a synthetic α‑galactosidase‑specific chromogenic substrate in which a 2‑nitrophenyl aglycone is linked via an α‑glycosidic bond to the anomeric carbon of α‑D‑galactose in the pyranose form. When hydrolyzed by α‑D‑galactosidase (EC 3.2.1.22) or other α‑galactoside‑cleaving enzymes, the glycosidic bond is cleaved to release 2‑nitrophenol and α‑D‑galactose. 2‑Nitrophenol has a yellow‑orange color and a convenient absorbance maximum in the 340–360 nm region in mildly acidic to neutral pH, enabling continuous spectrophotometric monitoring of enzyme activity. ONP‑α‑Gal is used in enzyme‑kinetic studies (Km, Vmax), inhibitor‑screening assays, and α‑galactosidase‑activity profiling in microbial, plant, and mammalian systems. The vacuum‑foil packaging helps protect the compound from moisture, light, and air, preserving its stability and performance in biochemical assays. The reagent is compatible with standard glycosidase‑buffer systems and is particularly useful for kinetic assays because 2‑nitrophenyl‑glycosides generally exhibit lower spontaneous hydrolysis than 4‑nitrophenyl analogs, reducing background signal.

Appearance

• Off‑white to pale yellow crystalline solid.
• Forms a clear, colorless solution in aqueous buffer; develops a yellow‑orange color only after enzymatic hydrolysis.

Source / synthesis

• Synthetically produced by glycosylation of 2‑nitrophenol with α‑D‑galactopyranose or a protected α‑D‑galactoside, followed by deprotection and crystallization from water or aqueous alcohols.
• Commercially manufactured by carbohydrate‑chemistry and biochemical‑reagent suppliers (e.g., ChemSynlab, various ChemicalBook‑linked vendors) as a high‑purity α‑galactosidase substrate.

Molecular weight and structure

• Molecular formula: C12H15NO8C12H15NO8.
• Molecular weight: 301.25 g/mol.
• Structure: 2‑nitrophenyl α‑D‑galactopyranoside, with an α‑glycosidic linkage between the aromatic 2‑nitrophenyl group and the anomeric oxygen of the D‑galactose ring in the (2R,3R,4S,5R,6R) configuration.

Sugar specificity 

• Specifically hydrolyzed by α‑D‑galactosidases and other α‑galactoside‑cleaving enzymes.
• Commonly used to distinguish α‑galactosidase activity from other glycosidase activities in enzyme‑profiling panels and kinetic‑assay systems.

Biological activity

• Serves as a chromogenic reporter: the rate of 2‑nitrophenol release under defined pH and temperature conditions reflects α‑galactosidase activity.
• Enables determination of kinetic parameters (Km, Vmax), inhibitor‑IC₅₀, and comparative activity profiling of different α‑galactosidases, including enzymes from microbial, plant, and mammalian sources.

Purity and microbial contamination

• Typically supplied at ≥98% purity as indicated by supplier data sheets and chemical‑property listings (e.g., “2‑nitrophenyl‑α‑D‑galactopyranoside, min. 98%”).
• Described as a non‑sterile, research‑grade solid, suitable for use in standard enzymatic assays under clean‑laboratory conditions.

Identity and quality control

• Identity confirmed by molecular formula, molecular weight, UV‑Vis spectral profile (2‑nitrophenol‑type absorbance), and IUPAC‑style structural data consistent with 2‑nitrophenyl‑α‑D‑galactopyranoside.
• Quality‑control generally includes checks for appearance, solubility, and performance in an α‑galactosidase activity assay (e.g., colorimetric or spectrophotometric assay under defined buffer and temperature conditions).

Shelf life and storage

• Shelf life is generally 12–24 months when stored as a dry powder at −20 °C in a tightly sealed, light‑protected container, with particular benefit from vacuum‑foil packaging to minimize moisture and oxidation.
• Aqueous working solutions are best prepared fresh or stored at 4 °C for short periods, as elevated temperature and pH extremes can increase nonspecific hydrolysis and background absorbance.

Application

• Enzyme‑kinetic assays and specificity studies of α‑D‑galactosidases from bacterial, yeast, and plant sources.
• Use in α‑galactosidase‑profiling panels and glycan‑metabolism investigations (e.g., raffinose‑family oligosaccharide breakdown).
• Inhibitor‑screening and enzyme‑mechanism‑elucidation experiments involving α‑galactosidases relevant to food, feed, and industrial‑enzyme applications.

Key characteristics 

• Colorimetric/UV‑absorbing readout based on 2‑nitrophenol release, enabling spectrophotometric quantification of α‑galactosidase activity with lower background hydrolysis than 4‑nitrophenyl counterparts.
• Defined α‑D‑galactopyranoside linkage providing selectivity for α‑galactosidases over many β‑glycosidases.
• Vacuum‑foil‑packed, ≥98%‑pure crystalline powder with a known molecular formula and weight, suitable for quantitative enzyme assays and standardized protocols.

Citations 

• ChemicalBook – 2‑Nitrophenyl‑α‑D‑galactopyranoside (ONP‑α‑Gal, CAS 19887‑85‑5, MF C₁₂H₁₅NO₈, MW 301.25, purity ≥98%): https://amp.chemicalbook.com/ProductChemicalPropertiesCB1102338_EN.htm
• ChemicalBook compound‑entry page (CAS 19887‑85‑5, 2‑nitrophenyl α‑D‑galactopyranoside, formula and weight): https://www.chemicalbook.com/ChemicalProductProperty_US_CB1102338.aspx
• ChemSynlab – 2‑Nitrophenyl‑α‑D‑galactopyranoside (ONP‑α‑D‑galactoside, CAS 19887‑85‑5, sugar‑chemistry product): http://www.chemsynlab.com/cn/product/19887-85-5.html
• Alpha‑galactosidase‑activity‑definition page (context for α‑galactosidase‑substrate‑use, though with 4‑nitrophenyl‑α‑D‑galactopyranoside example): http://shengtuoda.com/en/index.php?a=show&m=Article&id=10
• α‑Gal‑related review (overview of α‑galactosidase and α‑galactosyl‑substrate relevance in glycan biology): https://pmc.ncbi.nlm.nih.gov/articles/PMC8601398/
• α‑Gal syndrome and α‑galactosidase‑context review (background to α‑galactosyl‑enriched enzyme systems): https://pmc.ncbi.nlm.nih.gov/articles/PMC8974695/
• X‑α‑Gal certificate‑of‑analysis (example of α‑galactosidase‑substrate handling and storage conditions): https://www.takarabio.com/documents/Certificate%20of%20Analysis/630463/630463-PA124280.pdf
• General glycosidase‑substrate reference (PNP‑glycoside assay principles applicable to ONP‑type substrates): https://www.sciencedirect.com/science/article/abs/pii/S0141813016320669
• 2‑nitrophenyl‑α‑D‑galactopyranoside entry in chemical‑supplier lists (CAS 19887‑85‑5 product‑name and technical data): https://www.chemicalbook.com/ (search for “19887‑85‑5”)
• Microbial‑contamination‑guideline context (for handling non‑sterile biochemical‑powders): https://www.ipc.gov.in/images/Revised_Microbial_Contamination_in_Non_sterile_Products_2.2.9.pdf