Structural and Mechanistic Insights into the Formation of Xanthylium Chromophores in Uronic Acid Quantification

The Blumenkrantz-Asboe-Hansen assay is the most widely used colorimetric method for quantifying uronic acids (UAs), key components of pectins, and plant cell wall polysaccharides. However, the molecular origin of the assay's pink coloration remained unresolved. Here, we elucidate the chromophore structure and formation mechanism by integrating mass spectrometry, NMR, and quantum-chemical analyses. Under strongly acidic conditions, UAs or their degradation product, 5-formyl-2-furancarboxylic acid, condense with 3-phenylphenol to form a triarylmethane intermediate that produces the chromophore in an anhydrous acidic environment. Experimental and theoretical data show that color formation arises from acid-catalyzed condensation followed by oxidation-induced aromatization within the triarylmethane scaffold, generating a xanthylium-type structure. Using an analogous system based on 4-carboxybenzaldehyde, we developed a structurally related but more stable chromophore whose structure was confirmed by comprehensive experimental analysis. Thus, we provide a structural basis for the Blumenkrantz-Asboe-Hansen assay and establish a mechanistic framework for understanding and evaluating UA quantification.