PNP‑α‑NeuNAc Sodium Salt

PNP‑α‑NeuNAc Sodium Salt (CAS 123549‑14‑4) 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) in the form of its sodium salt. When hydrolyzed by sialidases (neuraminidases), the glycosidic bond is cleaved to release 4‑nitrophenol and N‑acetylneuraminic acid (typically as the sodium salt in aqueous buffer). 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 Sodium Salt 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, hygroscopic powder or lyophilized solid, and is suitable for glycobiology, virology, and diagnostic‑kit development where sialic‑acid‑cleaving activity is a key readout.

Appearance

• White to off‑white or 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 introduction of the sodium counterion and crystallization from aqueous or mixed‑solvent systems.
• Commercially supplied by glycobiology and enzyme‑substrate vendors (e.g., Sigma‑Aldrich, Biosynth, Chem‑Synlab, ChemicalBook‑linked suppliers) as a high‑purity sialidase substrate.

Molecular weight and structure

• Molecular formula: C17H21N2NaO11C17H21N2NaO11.
• Molecular weight: ~452.35 g/mol (for the sodium‑salt form).
• 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 a sodium salt.

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 (e.g., influenza neuraminidase, bacterial sialidases) where α‑NeuNAc linkages predominate.

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 as a high‑purity, “research‑use‑only” material (often ≥95–98% by HPLC or similar methods) by glycobiology‑chemistry suppliers.
• Described as a non‑sterile, hygroscopic powder 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 related to 2‑O‑(p‑nitrophenyl)‑α‑D‑N‑acetylneuraminic acid (CAS 26112‑88‑9).
• 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 (e.g., anti‑influenza drug candidates) and sialidase‑specificity profiling.
• Use in glycobiology and virology studies where sialic‑acid removal from glycoconjugates is under investigation.

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.
• Sodium‑salt form enhancing water solubility and compatibility with physiological‑pH buffer systems commonly used for neuraminidase and bacterial sialidase assays.

Citations

• Biosynth – 2‑O‑(4‑Nitrophenyl)‑α‑D‑N‑acetylneuraminic acid sodium salt (chromogenic substrate for neuraminidase/sialidase): https://www.biosynth.com/p/EN07314/123549-14-4‑2‑o‑4‑nitrophenyl‑a‑d‑n‑acetylneuram
• Chem‑Synlab – 2‑O‑(4‑Nitrophenyl)‑α‑D‑N‑acetylneuraminic acid sodium salt (Neu5Ac‑α‑PNP·Na; CAS 123549‑14‑4): http://chemsynlab.com/en/product/123549-14-4.html
• ChemWhat – 2‑O‑(p‑Nitrophenyl)‑α‑D‑N‑acetylneuraminic acid, sodium salt, x hydrate (CAS 123549‑14‑4, supplier and property data): https://www.chemwhat.com/2‑o‑p‑nitrophenyl‑α‑d‑n‑acetylneuraminic‑acid‑sodium‑salt‑x‑hydrate‑cas‑123549‑14‑4/
• ChemicalBook – 2‑O‑(p‑Nitrophenyl)‑α‑D‑N‑acetylneuraminic acid, sodium salt, x hydrate (CAS 123549‑14‑4; MF C₁₇H₂₁N₂NaO₁₁, MW 452.35): https://m.chemicalbook.com/ChemicalProductProperty_EN_CB91564832.htm
• American Chemical Suppliers – 2‑O‑(p‑nitrophenyl)‑α‑D‑N‑acetylneuraminic acid sodium salt listing (CAS 123549‑14‑4, Neu5Ac‑α‑PNP): https://www.americanchemicalsuppliers.com/list/search?search=2‑o‑%284‑nitrophenyl%29‑a‑d‑n‑acetylneuraminic+acid
• Sigma‑Aldrich – 2‑O‑(p‑Nitrophenyl)‑α‑D‑N‑acetylneuraminic acid (neutral parent form, CAS 26112‑88‑9, structural basis for the sodium‑salt substrate): https://www.sigmaaldrich.com/US/en/product/sigma/n1516
• PubMed – Purification and characterization of sialidase L using sialyl‑donor substrates (methodology relevant to PNP‑α‑NeuNAc‑type assays): https://pubmed.ncbi.nlm.nih.gov/8034634/
• TCI Chemicals – Sialidase substrate overview (including sialidase use in glycan‑modification studies, applicable to PNP‑NeuNAc substrates): https://www.tcichemicals.com/OP/en/product/tci‑topics/ProductHighlights_20250630
• General glycosidase‑substrate catalog (PNP‑α‑NeuNAc‑type sialosides overview): https://www.chemicalbook.com/ (search for “123549‑14‑4” or “2‑O‑(p‑nitrophenyl)‑α‑D‑N‑acetylneuraminic acid”).
• Review or methods paper on neuraminidase‑activity assays using 4‑nitrophenyl‑tagged sialic acid derivatives (e.g., IC₅₀ screening or microplate‑based sialidase assays): search on PubMed via https://pubmed.ncbi.nlm.nih.gov/ for “4‑nitrophenyl‑α‑N‑acetylneuraminic acid sialidase assay”