p‑Nitrophenyl‑β‑D‑mannopyranoside (PNP‑β‑D‑Man)

p‑Nitrophenyl‑β‑D‑mannopyranoside (PNP‑β‑D‑Man, CAS 35599‑02‑1) is a synthetic β‑glycoside substrate in which a 4‑nitrophenyl aglycone is linked via a β‑glycosidic bond to the anomeric carbon of β‑D‑mannose in the pyranose form. When hydrolyzed by β‑D‑mannopyranosidases (EC 3.2.1.25) or other β‑mannoside‑cleaving enzymes, the glycosidic bond is cleaved to release 4‑nitrophenol and β‑D‑mannose. In mildly alkaline buffer (pH ~8–8.5), 4‑nitrophenol ionizes to form a yellow‑colored phenolate anion whose absorbance at 400–420 nm can be monitored continuously, providing a convenient readout of enzyme velocity. PNP‑β‑D‑Man is widely used to characterize β‑mannosidase activity from microbial, fungal, and mammalian sources, to study enzyme‑substrate specificity among PNP‑glycosides, and to quantify activity in kinetic experiments (Km, Vmax). The compound is water‑soluble, supplied as a high‑purity, crystalline powder, and is suitable for routine biochemical and glycosidase‑related research. It is a key reagent for studying β‑mannosidases involved in N‑linked glycoprotein processing and lysosomal glycan‑catabolism pathways.

Appearance

• White to off‑white crystalline solid.
• Forms a clear, colorless solution in water; develops a yellow color only after enzymatic hydrolysis and alkaline adjustment.

Source / synthesis

• Synthetically produced by glycosylation of 4‑nitrophenol with β‑D‑mannopyranose or a protected β‑D‑mannoside derivative, followed by deprotection and crystallization from water or aqueous alcohols.
• Commercially manufactured by biochemical‑reagent suppliers (e.g., Sigma‑Aldrich, G‑Biosciences, various ChemicalBook‑linked vendors) as a high‑purity substrate for β‑D‑mannopyranosidase assays.

Molecular weight and structure

• Molecular formula: C12H15NO8C12H15NO8.
• Molecular weight: 301.25 g/mol.
• Structure: 4‑nitrophenyl β‑D‑mannopyranoside, with a β‑glycosidic linkage between the aromatic 4‑nitrophenyl group and the anomeric oxygen of the D‑mannose ring in the pyranose form.

Sugar specificity

• Specifically hydrolyzed by β‑D‑mannopyranosidases and related β‑glycosidases acting on β‑(1→4)‑ or β‑linked mannosyl linkages.
• Used to distinguish β‑mannosidase activity from other β‑glycosidases and α‑glycosidases in enzyme‑profiling panels.

Biological activity

• Serves as a chromogenic reporter whose rate of 4‑nitrophenol release under defined pH and temperature reflects β‑D‑mannopyranosidase activity.
• Enables determination of kinetic parameters (Km, Vmax), inhibitor‑IC₅₀, and comparative activity profiling of different glycosidases, especially in glycoprotein and lysosomal‑glycan‑metabolism studies.

Purity and microbial contamination

• Typically supplied at ≥97–99% purity (e.g., ≥98% by HPLC) as indicated by supplier data sheets and MSDS‑style documents.
• 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‑like absorbance), and NMR‑type data consistent with 4‑nitrophenyl‑β‑D‑mannopyranoside.
• Quality‑control includes checks for melting point (~207–209 °C), appearance, solubility, and performance in a defined β‑mannopyranosidase activity assay (e.g., Aspergillus niger or mammalian β‑mannosidase).

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, shielded from moisture.
• Aqueous working solutions are best prepared fresh or stored 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 β‑D‑mannopyranosidases from microbial, fungal, and mammalian sources.
• Use in glycosidase‑profiling panels and glycoprotein‑remodeling studies focusing on N‑linked glycan β‑mannose linkages.
• Inhibitor‑screening and enzyme‑mechanism‑elucidation experiments for β‑mannosidases, including lysosomal enzymes linked to glycosphingolipid and glycoprotein catabolism.

Key characteristics

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

Citations

• ChemicalBook – 4‑Nitrophenyl‑β‑D‑mannopyranoside (PNP‑β‑D‑Man, CAS 35599‑02‑1, C₁₂H₁₅NO₈, MW 301.25): https://www.chemicalbook.com/ChemicalProductProperty_EN_CB8230285.htm
• Sigma‑Aldrich – 4‑Nitrophenyl‑β‑D‑mannopyranoside (≥98%, CAS 35599‑02‑1, substrate for β‑D‑mannopyranosidase): https://www.sigmaaldrich.com/US/en/product/sigma/n1268
• G‑Biosciences – 4‑Nitrophenyl‑β‑D‑mannopyranoside (molecular‑biology grade, PNP‑β‑D‑Man): https://www.gbiosciences.com/Bioassays/Enzyme-Substrates/4-Nitrophenyl-beta-D-mannopyranoside
• CymitQuimica – p‑Nitrophenyl‑β‑D‑mannopyranoside, CAS 35599‑02‑1 (physical and assay‑use data): https://cymitquimica.com/cas/35599-02-1/
• BuyersGuideChem – p‑Nitrophenyl‑β‑D‑mannoside product information (CAS 35599‑02‑1, melting point, solubility, etc.): https://buyersguidechem.com/chemical_supplier/p-Nitrophenyl-beta-D-mannoside
• PubMed – Properties of a β‑D‑mannosidase from Aspergillus niger using p‑nitrophenyl‑β‑D‑mannopyranoside as substrate: https://pubmed.ncbi.nlm.nih.gov/623772/
• PubMed – Structural study of mammalian β‑mannosidase providing insight into β‑glycosidase‑substrate specificity (context for PNP‑β‑glycoside use): https://pubmed.ncbi.nlm.nih.gov/30552791/
• G‑Biosciences – Mannosidase‑substrate overview (including 4‑methylumbelliferyl‑β‑D‑mannoside, for fluorogenic‑counterpart context): https://www.gbiosciences.com/Mannosidase_Substrate
• Organic‑synthesis and biochemical‑use database (quick‑lookup for PNP‑β‑D‑Man CAS and formula): https://webbook.nist.gov/ (search for “35599‑02‑1”)
• General glycosidase‑substrate catalog (PNP‑β‑D‑Man and related PNP‑glycosides overview): https://www.chemicalbook.com/ (search for “35599‑02‑1”)