4-Nitrophenyl 2-acetamido-2-deoxy-β-D-galactopyranoside

4-Nitrophenyl 2-acetamido-2-deoxy-β-D-galactopyranoside (pNP-GalNAc, CAS 14948-96-0) is a chromogenic nitrophenyl glycoside substrate specifically designed for assaying β-N-acetylhexosaminidase (HexNAcase, EC 3.2.1.52) and β-N-acetylgalactosaminidase (EC 3.2.1.53) activities in biochemical and glycobiology research. Hydrolysis by these enzymes cleaves the β-glycosidic bond, releasing yellow 4-nitrophenolate anion detectable at 405 nm (ε 18,500 M⁻¹cm⁻¹, pH >9), enabling quantitative kinetic analysis of lysosomal storage disorders (e.g., Tay-Sachs, Sandhoff), mucin-type O-glycan processing, and chitin/GAG degradation pathways. This substrate distinguishes β-GalNAc-specific hydrolases from β-GlcNAc enzymes, supports inhibitor screening for glycosidase inhibitors, and is widely used in clinical diagnostics, plant pathology, and recombinant enzyme characterization with high sensitivity and minimal background hydrolysis.

Appearance

• White to pale yellow crystalline powder, odorless and hygroscopic.
• Soluble in water (~2-5 mM), DMSO (>50 mM), DMF; insoluble in nonpolar solvents.

Source

• Synthesized via Koenigs-Knorr glycosylation: 2-acetamido-2-deoxy-α-D-galactopyranosyl bromide + 4-nitrophenol, Ag2CO3 promoter, followed by β-anomer crystallization.
• Commercial synthesis or enzymatic transglycosylation; research grade >98%.

Molecular Weight and Structure

• C14H18N2O8, 342.30 g/mol; [α]D -52° ±5° (c=1, H2O).
• β-D-GalNAc-(1→O)-C6H4-NO2; InChIKey OMRLTNCLYHKQCK-RKQHYHRCSA-N.

Sugar Specificity

• Highly specific for β-N-acetylgalactosaminidases; Km 0.1-0.5 mM with mammalian Hex A/B.
• Minimal hydrolysis by β-GlcNAcase (10-20x lower rate); distinguishes GalNAc vs. GlcNAc.

Biological Activity

• Enzymatic hydrolysis: pNP-GalNAc → GalNAc + pNP; optimal pH 4.0-5.0, 37°C.
• Vmax 50-200 µmol pNP/min/mg for purified enzymes; coupled assays with citrate buffer.

Purity and Microbial Contamination

• ≥98% HPLC purity; single anomer >97%; free pNP <0.1%.
• Endotoxin-free; sterile lyophilized; no microbial glycosidase contamination.

Identity and Quality Control

• Verified by 1H-NMR (J1,2 8.0 Hz β-anomer), HRMS (m/z 343.1 [M+H]+), optical rotation, TLC (Rf 0.4, EtOAc:MeOH 3:1).
• Functional assay: ΔA405 >0.1/min with β-HexNAcase; CAS confirmed.

Shelf Life and Storage

• 3-5 years desiccated -20°C; stable in amber vials; avoid moisture/light.
• Stock solutions stable 6 months -20°C; working dilutions use same day.

Application

• Assay of β-hexosaminidases in Tay-Sachs/Sandhoff diagnostics; lysosomal enzyme screening.
• Mucin O-glycanase activity; chitinase/glycosidase inhibitor development; plant pathogenesis studies.

Key Characteristics

• Chromogenic (yellow product, ε405=18.5 mM⁻¹cm⁻¹); high signal:noise ratio.
• β-GalNAc selective; pH-indicator independent; microplate/automation compatible.

Citation Links

• https://nextsds.com/substances/14948-96-0-p-nitrophenyl-2-acetamido-2-deoxy-d-galactopyranoside
• https://www.vulcanchem.com/product/vc20761558
• https://www.benchchem.com/product/b1140086
• https://www.benchchem.com/pdf/Application_Notes_and_Protocols_for_the_Chromogenic_Glucosidase_Substrate_p_Nitrophenyl_D_glucopyr …
• https://cymitquimica.com/products/3B-N0865/14948-96-0/4-nitrophenyl-2-acetamido-2-deoxy-d-galactopyranoside/
• https://pubchem.ncbi.nlm.nih.gov/compound/11168041
• https://www.sciencedirect.com/science/article/abs/pii/S0008621507002479
• https://pubmed.ncbi.nlm.nih.gov/2148907/
• https://www.tcichemicals.com/IN/en/p/N0865
• https://www.biosynth.com/p/EN04923/184377-56-8-4-nitrophenyl-2-acetamido-2-deoxy-4