Regioselective fluorescent labeling of N,N,N-trimethyl chitosan via oxime formation

Fluorescent labeling of chitosan and its derivatives is widely used for in vitro visualization and is accomplished by random introduction of the fluorophore to the polymer backbone, conceivably altering the bioactivity of the polymer. Here, we report for the first time the regioselective conjugation of a fluorophore to the reducing end of a fully N,N,N-trimethylated chitosan (TMC) by oxime formation. End-labeled conjugation of 5-(2-((aminooxyacetyl)amino)ethylamino)naphthalene-1-sulfonic acid (EDANS-O-NH2) fluorophore to TMC to form TMC-oxime-EDANS (f-TMC) was confirmed by H-1 NMR and fluorescence spectroscopy. Average molecular weight calculations of f-TMC with H-1 NMR and fluorescence spectroscopy gave similar results or similar to 7.7 kDa. f-TMC in human bronchial epithelial cells was both cell membrane bound as well as intracellularly localized. This demonstrates the proof-of-concept for selective oxime formation at the reducing end of a chitosan derivative, which can be used for tracking chitosan in gene and drug delivery purposes and gives rise to further modifications with other functional groups. (C) 2012 Elsevier Ltd. All rights reserved.