PNP‑α‑NeuNAc

PNP‑α‑NeuNAc (CAS 26112‑88‑9) is a synthetic α‑sialoside substrate in which a 4‑nitrophenyl aglycone is linked via an α‑glycosidic bond to the 2‑OH position of α‑D‑N‑acetylneuraminic acid (NeuNAc, sialic acid). When hydrolyzed by sialidases or other sialic‑acid‑cleaving enzymes, the glycosidic bond is cleaved to release 4‑nitrophenol and free N‑acetylneuraminic acid. 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 convenient readout of enzyme velocity. PNP‑α‑NeuNAc is widely used to characterize sialidase activity from viral, bacterial, and mammalian sources, to study enzyme‑substrate specificity among sialyl‑glycosides, and to quantify activity in kinetic experiments (Km, Vmax) and high‑throughput inhibitor‑screening assays. The compound is water‑soluble, typically supplied as a high‑purity, lyophilized or crystalline solid, and is suitable for glycobiology, virology, and diagnostic‑kit development where sialic‑acid‑cleaving activity is a key readout. It serves as the parent neutral form of related salt‑type derivatives (e.g., PNP‑α‑NeuNAc Sodium Salt, CAS 123549‑14‑4).

Appearance

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

Source / synthesis

• Synthetically prepared by glycosylation of 4‑nitrophenol with α‑D‑N‑acetylneuraminic acid using protected intermediates, followed by deprotection and crystallization from aqueous or mixed‑solvent systems.
• Commercially supplied by biochemical‑reagent and glycobiology‑chemistry vendors (e.g., Sigma‑Aldrich, Hepattack, ChemicalBook‑linked suppliers) as a research‑grade sialidase substrate.

Molecular weight and structure

• Molecular formula: C17H22N2O11C17​H22​N2​O11​.
• Molecular weight: 430.36–430.37 g/mol.
• Structure: 4‑nitrophenyl bonded to the 2‑oxygen of α‑D‑N‑acetylneuraminic acid via an α‑glycosidic linkage, with the carboxylic acid group present as the free acid.

Sugar specificity

• Specifically hydrolyzed by sialidases (neuraminidases) that cleave α‑linked sialic‑acid residues from glycoproteins and glycolipids.
• Frequently used in microplate‑based substrate‑specificity screens for viral and bacterial sialidases, including influenza neuraminidase, where α‑NeuNAc linkages are natural substrates.

Biological activity

• Serves as a chromogenic reporter whose rate of 4‑nitrophenol release under defined pH and temperature conditions reflects sialidase activity.
• Enables determination of kinetic parameters (Km, Vmax), IC₅₀ of inhibitors, and comparative profiling of different sialidases (e.g., human, viral, bacterial) in 96‑ or 384‑well‑plate‑based assays.

Purity and microbial contamination

• Typically supplied at ≥95% purity (e.g., “≥95%” or “≥97%” by HPLC or similar methods) as indicated by supplier data sheets and SDS‑style documents.
• Described as a non‑sterile, research‑grade solid suitable for use in enzymatic assays under standard clean‑laboratory conditions.

Identity and quality control

• Identity inferred from molecular formula, molecular weight, UV‑Vis spectral profile (4‑nitrophenol‑type chromophore), and NMR‑type data; closely matches the neutral 2‑O‑(p‑nitrophenyl)‑α‑D‑N‑acetylneuraminic acid structure.
• Quality‑control generally includes checks for appearance, solubility, spectral properties, and performance in a defined sialidase assay (e.g., in microplate‑based neuraminidase‑activity kits).

Shelf life and storage

• Shelf life is generally 12–24 months when stored at −20 °C in a tightly sealed, light‑protected container, with protection from moisture and inert‑atmosphere storage recommended due to hygroscopicity.
• 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

• Colorimetric assay of sialidase (neuraminidase) activity from influenza virus, bacteria, and mammalian sources.
• High‑throughput screening of neuraminidase inhibitors and sialidase‑specificity profiling in 96‑ or 384‑well‑plate formats.
• Use in glycobiology and virology studies where sialic‑acid removal from glycoconjugates is under investigation (e.g., cell‑surface‑sialidase studies and glycan‑remodeling experiments).

Key characteristics

• Colorimetric readout based on 4‑nitrophenol release, enabling spectrophotometric quantification of sialidase activity in microplate formats.
• Defined α‑sialosidic linkage providing high selectivity for sialidases over many other glycosidases.
• Neutral‑acid form (free‑COOH), with a known molecular formula and weight, suitable for quantitative enzyme assays and standardized protocols.

Citations

• Sigma‑Aldrich – 2‑O‑(p‑Nitrophenyl)‑α‑D‑N‑acetylneuraminic acid (≥95%, CAS 26112‑88‑9): https://www.sigmaaldrich.com/MN/en/product/sigma/n1516
• Sigma‑Aldrich (alternate region) – Same product page (CAS 26112‑88‑9, formula C₁₇H₂₂N₂O₁₁, MW 430.36): https://www.sigmaaldrich.com/BB/en/product/sigma/n1516
• ChemicalBook – 2‑O‑(p‑Nitrophenyl)‑α‑D‑N‑acetylneuraminic acid (CAS 26112‑88‑9, formula C₁₇H₂₂N₂O₁₁, MW 430.36): https://www.chemicalbook.com/ChemicalProductProperty_EN_CB9315169.htm
• Hepattack – PNP‑α‑NeuNAc product listing (CAS 26112‑88‑9, high‑purity raw material for research): https://www.hepattack.com/en/product03_cid_217_pid_612.html
• Chemical‑supplier directory (PNP‑α‑NeuNAc CAS 26112‑88‑9, structure and property data): https://amp.chemicalbook.com/ChemicalProductProperty_EN_CB9315169.htm
• PNP‑α‑NeuNAc Sodium Salt supplier (CAS 123549‑14‑4; useful for cross‑reference as a closely related salt form): https://www.hepattack.com/en/product03_cid_217_pid_611.html
• Sigma‑Aldrich PubChem‑linked page (CAS 26112‑88‑9 substance view): https://pubchem.ncbi.nlm.nih.gov/substance/24897497
• Sialidase‑activity assay‑methods review (context for sialidase‑substrate use, including PNP‑α‑NeuNAc formats): https://www.ncbi.nlm.nih.gov/pubmed/ (search “sialidase substrate 2‑O‑(p‑nitrophenyl)‑α‑N‑acetylneuraminic acid”)
• Glycosidase‑substrate catalog (PNP‑α‑NeuNAc and related sialic‑acid substrates): https://www.chemicalbook.com/ (search for “26112‑88‑9”)
• Sialidase‑specific research article using PNP‑tagged sialic acid derivatives (e.g., high‑throughput sialidase‑specificity assays): search on PubMed via https://pubmed.ncbi.nlm.nih.gov/ for “4‑nitrophenyl‑α‑N‑acetylneuraminic acid sialidase”.