Carbohydrate Polymers 90 (2012) 1273–1280 Contents lists available at SciVerse ScienceDirect Carbohydrate Polymers jo u rn al hom epa ge: www.elsevier.com/locate/carbpol Regioselective fluorescent labeling of N,N,N-trimethyl chitosan via oxime formation Berglind E. Benediktsdóttir a , Kasper K. Sørensen b , Mikkel B. Thygesen b , Knud J. Jensen b , Thórarinn Gudjónsson c , Ólafur Baldursson d , Már Másson a,∗ a Faculty of Pharmaceutical Sciences, School of Health Sciences, University of Iceland, Hofsvallagata 53, IS-107 Reykjavik, Iceland b Center for Carbohydrate Recognition and Signaling, Faculty of Science, Department of Chemistry, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Fredriksberg C, Copenhagen, Denmark c Biomedical Center, School of Health Sciences, University of Iceland, Vatnsm´ yrarvegur 16, IS-101 Reykjavik, Iceland d Department of Pulmonary Medicine, Landspitali – The National University Hospital of Iceland, Eiríksgata 5, IS-101 Reykjavik, Iceland a r t i c l e i n f o Article history: Received 13 April 2012 Received in revised form 14 June 2012 Accepted 23 June 2012 Available online 1 July 2012 Keywords: N,N,N-trimethyl chitosan Fluorescence Polysaccharide Molecular weight Oxime a b s t r a c t Fluorescent labeling of chitosan and its derivatives is widely used for in vitro visualization and is accom- plished 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-NH 2 ) fluorophore to TMC to form TMC-oxime-EDANS (f-TMC) was confirmed by 1 H NMR and fluorescence spec- troscopy. Average molecular weight calculations of f-TMC with 1 H NMR and fluorescence spectroscopy gave similar results or ∼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. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Chitosan has been studied as a paracellular permeation enhancer for hydrophilic molecules (Artursson, Lindmark, Davis, & Illum, 1994) and macromolecules (Illum, Farraj, & Davis, 1994; S ¸ enel et al., 2000) by altering tight junction expression (Schipper et al., 1997; Yeh et al., 2011). Various other applications have also been investigated which include stimulation of cell differ- entiation and growth (Mathews, Gupta, Bhonde, & Totey, 2011; Muzzarelli, 2009; Muzzarelli, Greco, Busilacchi, Sollazzo, & Gigante, 2012) and use as an antimicrobial agent (Rabea, Badawy, Stevens, Smagghe, & Steurbaut, 2003). However, practical use of chitosan is limited due to poor aqueous solubility at physiological pH val- ues. Consequently, quaternized chitosan derivatives, such as fully N,N,N-trimethylated chitosan (TMC) (Benediktsdóttir et al., 2011) and trimethylated-6-amino-6-deoxy chitosan (Sadeghi et al., 2008) have been synthesized in order to enhance aqueous solubility and biological activity. Since early reports of synthesis of fluorescein isothiocyanate (FITC) labeled chitosan (Onishi & Machida, 1999; Qaqish & Amiji, ∗ Corresponding author. Tel.: +354 525 4463; fax: +354 525 4071. E-mail address: mmasson@hi.is (M. Másson). 1999), studies focused on visualizing the FITC-labeled chitosan in vitro have revealed that chitosan in solution usually adhere to the cellular membrane and chitosan nanoparticles are taken up by the cells (Huang, Ma, Khor, & Lim, 2002; Jia, Chen, Xu, Han, & Xu, 2009). In addition to FITC, other fluorophores have been randomly introduced to the amino group of chitosan, such as lissamine–rhodamine (Schipper et al., 1997) and Cy5.5 conjugated to glycol modified chitosan (Nam et al., 2010). Reports of structure characterization of these fluorescently labeled chitosan derivatives have been scarce. Incorporating these fluorophores randomly into the chitosan backbone has become a widely used application to visualize the localization of the polymer in vitro. However, this introduction could have some effects on the spatial structure of the polymer thereby possibly altering its biological properties (Fei & Gu, 2009). The size distribution of fluorescent chitosan nanopar- ticles formed can also be affected (Huang, Khor, & Lim, 2004). Furthermore, these random labeling techniques utilize the free amino group on the polymer backbone and are therefore not appli- cable to chitosan derivatives, such as TMC, where the amino group is blocked by methylation. In contrast, the reducing end of TMC pro- vides a unique chemical functionality for appending a fluorophore with high regioselectivity, provided that reagents and conditions can be developed for reactions at this masked aldehyde moiety. The aminooxy group is highly reactive towards carbonyl groups, 0144-8617/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carbpol.2012.06.070