p‑Nitrophenyl‑α‑D‑maltoside (PNP‑α‑D‑Maltoside)

p‑Nitrophenyl‑α‑D‑maltoside (PNP‑α‑D‑Maltoside, CAS 17400‑77‑0) is a synthetic α‑glycoside substrate in which a 4‑nitrophenyl aglycone is linked via an α‑glycosidic bond to the reducing‑end glucose of α‑D‑maltose. When hydrolyzed by α‑glucosidases, α‑amylases, or other α‑glycoside‑cleaving enzymes, the glycosidic bond is cleaved to release 4‑nitrophenol along with α‑D‑maltose or its hydrolysis products (e.g., glucose units). 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 monitored continuously, providing a quantitative readout of enzyme velocity. PNP‑α‑D‑Maltoside is particularly useful for assaying α‑amylase activity (e.g., salivary or pancreatic α‑amylase) and for characterizing α‑glucosidase kinetics and selectivity. The compound is water‑soluble and is supplied as a high‑purity, crystalline powder suitable for routine biochemical and enzymatic research. Its disaccharide structure distinguishes it from monosaccharide‑type PNP‑glycosides such as PNP‑α‑Glc, making it suitable for studying enzymes that prefer α‑linked maltose‑derived substrates.

Appearance

• Off‑white to pale yellow crystalline solid.
• 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‑maltose, followed by purification from water or aqueous alcohols.
• Commercially manufactured by biochemical‑reagent suppliers (e.g., Merck/Calbiochem, Sigma‑Aldrich, GoldBio, Chem‑Impex, Echemi) as a research‑grade substrate for α‑amylase and α‑glucosidase assays.

Molecular weight and structure

• Molecular formula: C18H25NO13C18​H25​NO13​.
• Molecular weight: 463.39 g/mol.
• Structure: 4‑nitrophenyl α‑D‑maltopyranoside, with an α‑glycosidic linkage between the aromatic 4‑nitrophenyl group and the anomeric oxygen of the reducing glucose in an α‑D‑maltose unit (Glc(α1→4)Glc).

Sugar specificity

• Primarily hydrolyzed by α‑glucosidases and α‑amylases that cleave α‑linked maltoside or maltosyl‑containing structures.
• Also used to study α‑glycosidase substrate‑specificity patterns and to distinguish α‑amylase‑type hydrolysis from simple monosaccharide‑α‑glucosidase activity.

Biological activity

• Serves as a chromogenic substrate whose rate of 4‑nitrophenol release under defined pH and temperature conditions reflects the velocity of α‑amylase or α‑glucosidase activity.
• Enables determination of kinetic parameters (Km, Vmax), inhibitor‑IC₅₀, and comparative activity profiling between different α‑glycosidase or amylase preparations.

Purity and microbial contamination

• Typically supplied at ≥97–99% purity (e.g., ≥99% or ≥99.5% as indicated by supplier data sheets), verified by HPLC or elemental analysis.
• Described as non‑sterile, research‑grade material 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 (4‑nitrophenol‑type absorbance), and NMR‑type data.
• Quality‑control includes checks for melting point, appearance, solubility, and performance in a defined α‑amylase or α‑glucosidase activity assay (e.g., salivary α‑amylase using PNP‑maltoside).

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 should be prepared fresh or kept at 4 °C for short periods, as alkaline pH and elevated temperature increase background hydrolysis and absorbance.

Application

• Colorimetric assay of α‑amylase activity (e.g., salivary or pancreatic α‑amylase) in clinical, diagnostic, and food‑industry applications.
• Enzyme‑kinetic studies and inhibitor‑screening assays for α‑glucosidases and related α‑glycosidases.
• Use in glycosidase‑profiling panels and enzyme‑mechanism‑elucidation experiments (e.g., maltosidase vs glucosidase activity).

Key characteristics

• Colorimetric readout based on 4‑nitrophenol release, allowing spectrophotometric quantification of α‑amylase and α‑glucosidase activity.
• Defined 4‑nitrophenyl‑α‑D‑maltoside linkage providing selectivity for α‑amylase and maltoside‑preferring α‑glycosidases over simple monosaccharide‑specific enzymes.
• 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‑maltoside (CAS 17400‑77‑0, C₁₈H₂₅NO₁₃, MW 463.39): https://www.sigmaaldrich.com/US/en/product/sigma/n5885
• GoldBio – p‑Nitrophenyl‑α‑D‑maltopyranoside (PNP‑α‑D‑Maltoside) product and certificate‑of‑analysis page (CAT N‑340): https://www.goldbio.com/pages/coa/011701n340
• Echemi – P‑Nitrophenyl‑α‑D‑maltoside (CAS 17400‑77‑0) technical overview (MF C₁₈H₂₅NO₁₃, MW 463.39): https://www.echemi.com/products/pd180521121811-p-nitrophenyl-alpha-d-maltoside.html
• Chongqing Chemdad – P‑Nitrophenyl‑α‑D‑maltoside product information (CAS 17400‑77‑0, substrate for α‑amylase and α‑glucosidase assays): https://chemdad.com/index.php?c=article&id=3684
• Merck/Calbiochem – p‑Nitrophenyl‑α‑D‑maltoside (discontinued, but historical data available): https://www.merckmillipore.com/INTL/en/product/p-Nitrophenyl-D-maltoside,EMD_BIO-487542
• Benchchem – p‑Nitrophenyl‑α‑D‑maltoside product listing (CAS 17400‑77‑0, PNP‑α‑D‑maltoside for glycosidase assays): https://www.benchchem.com/product/b2634642
• PubChem – p‑Nitrophenyl‑α‑D‑maltoside entry (CAS 17400‑77‑0, CID 24897757): https://pubchem.ncbi.nlm.nih.gov/substance/24897757
• PubMed – Method using p‑nitrophenyl‑β‑maltosides for α‑glucosidase assay (context for PNP‑maltoside use in glycosidase‑activity profiling): https://pubmed.ncbi.nlm.nih.gov/8714432/
• Technical‑guide‑style document on 4‑Nitrophenyl‑α‑D‑glucopyranoside usage (methodology readily transferable to PNP‑α‑D‑Maltoside assays): https://www.benchchem.com/pdf/The_Use_of_4_Nitrophenyl_D_glucopyranoside_in_Glucosidase_Activity_Assays_A_Technical_Guide.pdf
• General glycosidase‑substrate catalog (PNP‑α‑D‑Maltoside and other PNP‑maltosides overview): https://www.chemicalbook.com/ (search for “17400‑77‑0”)