Quantitative Fluorine NMR to Determine Carbohydrate Density on Glyconanomaterials Synthesized from Perfluorophenyl Azide-Functionalized Silica Nanoparticles by Click Reaction

A quantitative fluorine NMR (¹⁹F qNMR) method was developed to determine the carbohydrate density on glyconanomaterials. Mannose (Man)- and galactose (Gal)-conjugated silica nanoparticles (SNPs) were synthesized from perfluorophenyl azide (PFPA)-functionalized SNPs and propargylated Man or Gal by copper-catalyzed azide-alkyne cycloaddition (click reaction). After treating PFPA-SNPs or Man-SNPs with hydrofluoric acid followed by lyophilization, the remaining residues were directly subjected to ¹⁹F NMR analysis. The density of PFPA on PFPA-SNP was determined to be 7.7 ± 0.2 × 10^-16 nmol/nm² and Man on Man-SNP to be 6.4 ± 0.2 × 10^-16 nmol/nm² giving a yield of ∼83% for the click coupling reaction. The apparent dissociation constant (K_d) of Man-SNPs with fluorescein isothiocyanate (FITC)-concanavalin A (Con A) was determined using a fluorescence competition assay to be 0.289 ± 0.003 μM, which represents more than 3 orders of magnitude affinity increase compared to free Man with Con A.