p‑Nitrophenyl‑α‑D‑xylopyranoside (PNP‑α‑Xyloside)

p‑Nitrophenyl‑α‑D‑xylopyranoside (PNP‑α‑Xyloside, CAS 10238‑28‑5) is a synthetic α‑xylosidase‑specific chromogenic substrate in which a 4‑nitrophenyl aglycone is linked via an α‑glycosidic bond to the anomeric carbon of D‑xylose. Upon hydrolysis by α‑D‑xylosidases or other α‑xyloside‑cleaving enzymes, the glycosidic bond is cleaved to release 4‑nitrophenol and α‑D‑xylose. In mildly alkaline buffer (pH ≥8–8.5), 4‑nitrophenol ionizes to form a yellow‑colored phenolate anion, whose absorbance at 400–410 nm can be quantified, providing a continuous, spectrophotometric readout of enzyme activity. PNP‑α‑Xyloside is widely used in enzymology to characterize α‑xylosidase kinetics (Km, Vmax), to screen α‑glycosidase inhibitors, and to study glycosidase‑specificity profiles in microbial, plant, and mammalian systems. The compound is water‑soluble and is supplied as a high‑purity, crystalline powder suitable for routine biochemical and enzymatic research.

Appearance

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

Source / synthesis

• Synthetically produced by glycosylation of 4‑nitrophenol with α‑D‑xylose (or α‑D‑xylopyranose) under controlled conditions, followed by crystallization from water or alcohol.
• Commercially manufactured by biochemical‑reagent suppliers (e.g., Sigma‑Aldrich, ChemicalBook‑linked vendors) as a research‑grade substrate for α‑xylosidase assays.

Molecular weight and structure

• Molecular formula: C11H13NO7C11​H13​NO7​.
• Molecular weight: 271.22 g/mol.
• Structure: 4‑nitrophenyl α‑D‑xylopyranoside, with an α‑glycosidic linkage between the aromatic 4‑nitrophenyl group and the anomeric oxygen of the D‑xylose ring.

Sugar specificity

• Specifically hydrolyzed by α‑D‑xylosidases and other α‑xyloside‑cleaving enzymes.
• Used as a selective substrate to distinguish α‑xylosidase activity from β‑glycosidases and other α‑glycosidases, especially in glycosidase‑specificity screens.

Biological activity

• Serves as a chromogenic/UV‑absorbing substrate that reports α‑xylosidase activity via the release of 4‑nitrophenol.
• The rate of 4‑nitrophenol formation under defined pH and temperature conditions reflects enzyme velocity, enabling kinetic analysis, determination of Ki/IC₅₀, and comparative activity profiling of different glycosidases.

Purity and microbial contamination

• Typically supplied at ≥97–99% purity as determined by HPLC, NMR, and elemental analysis, with product data sheets from major suppliers.
• Described as non‑sterile, research‑grade material suitable for use in standard enzymatic‑assay conditions when handled under clean laboratory practices.

Identity and quality control

• Identity confirmed by molecular formula, molecular weight, UV‑Vis spectral profile (4‑nitrophenol‑type absorbance), and NMR‑type data.
• Quality‑control includes checks for melting point, appearance, solubility, and performance in a defined α‑xylosidase or α‑glycosidase activity assay.

Shelf life and storage

• Shelf life is generally 12–24 months when stored as a dry powder at –20 °C in a tightly sealed, dark container, protected from moisture and light.
• Aqueous working solutions are preferably prepared fresh or kept at 4 °C for short periods, as alkaline pH and elevated temperature increase background hydrolysis and absorbance.

Application

• Enzyme‑kinetic assays and specificity studies of α‑xylosidases from microbial, fungal, and plant sources.
• High‑throughput screening of glycosidase inhibitors and characterization of α‑glycosidase inhibitor selectivity.
• Use in glycosidase‑profiling panels and enzyme‑mechanism‑elucidation studies (e.g., penofuranoside‑type PNP‑glycosides context).

Key characteristics

• Colorimetric readout based on 4‑nitrophenol release, allowing spectrophotometric quantification of α‑xylosidase activity.
• Defined α‑D‑xylopyranoside linkage providing selectivity for α‑xylosidases over many β‑glycosidases.
• Crystalline, stable powder with a known molecular formula and weight, suitable for quantitative enzyme assays and standardized protocols.

Citations

• Sigma‑Aldrich – 4‑Nitrophenyl‑α‑D‑xylopyranoside (PNP‑α‑Xyloside, CAS 10238‑28‑5, MF C₁₁H₁₃NO₇, MW 271.22): https://www.sigmaaldrich.com/AW/en/product/sigma/n1895
• PubChem – p‑Nitrophenyl‑α‑D‑xylopyranoside entry (CAS 10238‑28‑5, CID 82482): https://pubchem.ncbi.nlm.nih.gov/compound/82482
• ChemicalBook – p‑Nitrophenyl‑α‑D‑xylopyranoside (CAS 10238‑28‑5) product information: https://www.chemicalbook.com/ChemicalProductProperty_EN_CB4415037.htm
• DrugBank – 4‑Nitrophenyl‑α‑D‑xyloside relevant entry (for glycosidase‑substrate context): https://go.drugbank.com/drugs/DB04807
• Review/Synthesis of PNP‑α‑D‑xylofuranoside and related PNP‑glycosides (general use of chromogenic p‑nitrophenyl glycosides in glycosidase assays): https://www.sciencedirect.com/science/article/pii/S0008621525004288
• Enzymatic‑hydrolysis‑methodology paper (using p‑nitrophenyl‑glycosides for glycosidase‑activity profiling): https://pubmed.ncbi.nlm.nih.gov/1741775/
• Technical‑guide‑style document on 4‑Nitrophenyl‑α‑D‑glucopyranoside assays (methodology readily transferable to PNP‑α‑Xyloside assays): https://www.benchchem.com/pdf/The_Use_of_4_Nitrophenyl_D_glucopyranoside_in_Glucosidase_Activity_Assays_A_Technical_Guide.pdf
• 4‑Nitrophenyl‑β‑D‑xyloside product page (for xylosidase‑substrate background and handling reference): http://chemsynlab.com/en/product/2001-96-9.html
• General α‑glycosidase‑substrate catalog (including PNP‑α‑xyloside and related PNP‑glycosides): https://www.chemicalbook.com/ (search for “10238‑28‑5”)
• Enzymology review on glycosidase‑substrate use (PNP‑glycosides as standard chromogenic tools): https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/glycosidase