Magenta‑GalNAc

Magenta‑GalNAc, 5‑bromo‑6‑chloro‑3‑indolyl N‑acetyl‑β‑D‑galactosaminide, is a synthetic β‑N‑acetyl‑D‑galactosaminidase‑specific chromogenic substrate in which a 5‑bromo‑6‑chloro‑3‑indoxyl aglycone is linked via a β‑glycosidic bond to N‑acetyl‑D‑galactosamine (GalNAc). When hydrolyzed by β‑N‑acetyl‑D‑galactosaminidase or related β‑GalNAc‑cleaving enzymes, the indoxyl moiety is released and undergoes aerial oxidation to form an insoluble magenta‑colored indigo‑type precipitate, enabling clear visual detection of enzyme activity. This substrate is used in selective culture media to screen β‑GalNAcase‑producing organisms, in enzyme‑kinetic assays for recombinant or purified β‑N‑acetyl‑D‑galactosaminidases, and in histochemical staining for GalNAcase localization in glycoproteins and glycolipids. The magenta color provides improved contrast over traditional colorimetric or fluorogenic GalNAc substrates, making it suitable for phenotypic profiling and multiplex assays. The analogue Magenta‑GlcNAc (for GlcNAc‑specific enzymes) has an established CAS, but the 5‑bromo‑6‑chloro‑3‑indolyl N‑acetyl‑β‑D‑galactosaminide variant is often listed under similar “Magenta‑GalNAc” or “X‑GalNAc‑type” naming without a universally reported CAS; it is sourced from specialty carbohydrate and enzyme‑substrate catalogs.

Appearance

• Off‑white to pale yellow crystalline or lyophilized powder.
• Fine, free‑flowing texture suitable for accurate weighing and dissolution in aqueous or mixed aqueous‑organic solvents.

Source / synthesis

• Synthetically produced by coupling 5‑bromo‑6‑chloro‑3‑indoxyl with N‑acetyl‑β‑D‑galactosamine under glycosylation conditions to form the N‑acetyl‑β‑D‑galactosaminide derivative.
• Commercially prepared by specialty enzyme‑substrate and biochemical suppliers as a high‑purity, research‑grade reagent, often marketed alongside X‑GalNAc or other N‑acetyl‑β‑hexosaminidase substrates.

Molecular weight and structure

• Molecular formula: C16H18BrClN2O6C16​H18​BrClN2​O6​ (same elemental formula as the GlcNAc‑analogue, differing only in sugar stereochemistry).
• Molecular weight: 449.68 g/mol.
• Structure: 5‑bromo‑6‑chloro‑1H‑indol‑3‑yl 2‑acetamido‑2‑deoxy‑β‑D‑galactopyranoside, with a β‑(1→4)‑type linkage between the indole C3‑O and the N‑acetyl‑D‑galactosamine ring (GalNAc in the 4‑epimer configuration of GlcNAc).

Sugar specificity

• Specifically hydrolyzed by β‑N‑acetyl‑D‑galactosaminidases (β‑GalNAcase, EC 3.2.1.49) and related β‑GalNAc‑cleaving glycosidases.
• Shows high selectivity over other β‑glycosidases and minimal cross‑reactivity when used under optimized assay conditions, making it suitable for probing GalNAc‑specific pathways.

Biological activity

• Acts as a chromogenic reporter for β‑N‑acetyl‑D‑galactosaminidase‑mediated hydrolysis of terminal N‑acetyl‑β‑D‑galactosaminyl linkages in glycoproteins, glycolipids, and oligosaccharides.
• Enzymatic cleavage yields 5‑bromo‑6‑chloro‑3‑indoxyl, which oxidizes and dimerizes into a magenta‑colored, water‑insoluble dye, providing a sensitive, low‑background readout for solid‑medium and solution‑based assays.

Purity and microbial contamination

• Typically supplied at ≥98% purity as determined by HPLC or similar analytical methods.
• Certificates of analysis from major vendors generally confirm low residual solvents, acceptable heavy‑metal content, and non‑sterile, low‑microbial‑contamination status suitable for microbiological and biochemical use.

Identity and quality control

• Identity inferred from NMR, HPLC, MS, and elemental analysis of analogous 5‑bromo‑6‑choloro‑N‑acetyl‑β‑hexosaminides; full characterization mirrors that of the GlcNAc‑analogue (Magenta‑GlcNAc, CAS 5609‑91‑6).
• Quality‑control procedures include checks for appearance, solubility, spectral characteristics, and performance in a defined β‑GalNAcase activity assay system.

Shelf life and storage

• Shelf life is generally 12–24 months when stored as a dry powder at –20 °C in a tightly sealed container, protected from moisture and light.
• Aqueous or stock solutions should be prepared immediately before use or stored frozen for short periods to minimize degradation and nonspecific oxidation of the indoxyl moiety.

Application

• Screening and phenotypic differentiation of β‑N‑acetyl‑D‑galactosaminidase‑producing microorganisms on selective agar media.
• Enzyme‑activity assays for recombinant or purified β‑GalNAcases in solution or immobilized formats.
• Histochemical or cytochemical localization of β‑GalNAcase activity in tissue sections and glycan‑remodeling studies.

Key characteristics

• High sensitivity and low background due to formation of an insoluble magenta‑colored dye upon β‑N‑acetyl‑D‑galactosaminidase hydrolysis.
• Defined N‑acetyl‑β‑D‑galactosaminide linkage providing selectivity for β‑GalNAcase over other glycosidases.
• Crystalline, stable powder with a known molecular formula and weight, suitable for quantitative enzyme assays and standardized media formulations.

Citation 

• Apollo Scientific – 5‑Bromo‑4‑chloro‑3‑indolyl N‑acetyl‑β‑D‑galactosaminide (X‑GalNAc‑type product, structure and handling reference): https://store.apolloscientific.co.uk/product/5-bromo-4-chloro-3-indolyl-n-acetyl-beta-d-galactosaminide
• PubChem – 5‑Bromo‑6‑chloro‑3‑indolyl N‑acetyl‑β‑D‑glucosaminide (CAS 5609‑91‑6; close analogue for structural and assay context): https://pubchem.ncbi.nlm.nih.gov/compound/2733911
• ChemicalBook – 5‑Bromo‑6‑chloro‑3‑indolyl‑N‑acetyl‑β‑D‑glucosaminide (CAS 5609‑91‑6) data page (for N‑acetyl‑β‑hexosaminide‑substrate properties): https://m.chemicalbook.com/ChemicalProductProperty_EN_CB4228846.htm
• Nature Communications – genetic and functional diversity of β‑N‑acetylgalactosaminidases (enzyme context): https://www.nature.com/articles/s41467-024-47653-2
• GBiosciences – enzyme‑substrate catalog (β‑Gal/β‑GalNAc and magenta‑type substrates overview): https://www.gbiosciences.com/Enzyme-Substrates
• Apollo Scientific / ChemWhat – N‑acetyl‑β‑D‑glucosaminide supplier pages (for synthetic and QC‑methodology reference): https://store.apolloscientific.co.uk/product/5-bromo-6-chloro-3-indolyl-n-acetyl-beta-d-glucosaminide, https://www.chemwhat.com/5-bromo-4-chloro-3-indolyl-n-acetyl-beta-d-glucosaminide-cas-5609-91-6/
• NextSSDS – chemical‑data entry for 5‑Bromo‑6‑chloro‑3‑indolyl‑N‑acetyl‑β‑D‑glucosaminide (spectroscopic and safety context): https://nextsds.com/substances/5609-91-6-5-bromo-6-chloro-3-indolyl-n-acetyl-beta-d-glucosaminide
• Glycosidase‑substrate overview (chromogenic and fluorogenic β‑hexosaminidase substrates): https://www.medchemexpress.com/ (search for “β‑N‑acetyl‑D‑galactosaminidase substrate”)
• BenchChem / supplier catalog – 5‑Bromo‑6‑chloro‑3‑indolyl‑β‑D‑galactoside (Magenta‑Gal) page (for dye‑color and indolyl‑β‑glycoside handling context): https://www.benchchem.com/product/b1333544
• General life‑science‑enzyme‑substrate catalog (Magenta‑butyrate, Magenta‑caprylate, and related magenta‑type substrates): https://www.gbiosciences.com/Enzyme-Substrates (for formulation and assay‑format reference)