Selective and Sensitive Sensing of L-Aspatic Acid and L-Glutamic Acid Employing Dansyl Functionalized Dextran Weerachai Nasomphan1, Srung Smanmoo2 and Pramuan Tangboriboonrat1* 1Department of Chemistry, Faculty of Science, Mahidol University, Bangkok, 10400 Thailand 2Bioresources Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC) Thailand Science Park, Klong luang, Pathumthani, 12120 Thailand

Advantages of L-Asp & L-Glu Biological roles Acidic amino acid Biosynthesis precursor Amino acid metabolism Human nutrition Identifying diseases L-Aspatic acid L-Glutamic acid (L-Asp) (L-Glu) High level in blood Diabetic Stroke Okamoto H. et al. J Sep Sci 2005; 28: 522. Yen K. et al. Talanta 2007; 71: 2007.

Standard Method Vs Chemosensor Disadvantages: Expensive Complicate Skillful Time consuming UV-Vis HPLC ICP-MS Fluorescence Chemosensor Advantages: High selectivity* High sensitivity** Low cost Simple-to-use Naked-eye diagnostic tools *,** depend on chemical property of sensor

Binding subunit “Ionophore” Fluorescence quenching/enhancement Design of Chemosensor Signaling subunit Binding subunit “Ionophore” Amino Acid Fluorescence quenching/enhancement Color change Fluorophore Acid-base interaction Hydrogen bonding Metal-ligand coordination Van der Waals and physical interaction Interactions Ramon M. et al. Chem Rev 2003; 103: 11. Suslick KS. et al. Tetrahedron 2004; 60: 11133.

Molecular Vs Polymeric Chemosensors Polymeric chemosensor Signal Enhancement Polymeric chemosensor Molecular chemosensor Conjugated system Advantages High sensitivity Solubility enhancement Non-Conjugated system Long Y. et al. Macromolecules 2009; 42: 6501. Malashikhin S. et al. J Am Chem Soc 2008; 130: 12546.

Non-Conjugated Polymer: Dextran Probe Polymeric template: Dextran (Water soluble template) Binding Unit Dansyl Dextran Conjugate (DD) Acidic amino acids (AAs) L-Asp L-Glu Fluorophore Signaling Unit (Acidic Sensitive)

Internal Charge Transfer (ICT) Process Interaction of Dextran Probe & Amino Acid Hydrogen bonding DD Charge-Charge interaction ICT “ Fluorescent on ” Internal Charge Transfer (ICT) Process “ Fluorescent off ” Ohtake K. et al. J Control Release 2002; 82: 263. Yen K. et al. Talanta 2007; 71: 2007.

Objective To synthesize a polymeric chemosensor for L-Asp and L-Glu based on dansyl hydrazine dextran conjugate (DD) via fluorescence quenching

Synthetic scheme of dextran dansylhydrazine conjugate Non-Conjugated Polymer: DD Synthetic scheme of dextran dansylhydrazine conjugate Molecular weight (kDa) = 2600 Polydispersity index (Mw/Mn) = 1.33

Fluorescence (FL) spectra of DD upon the addition of 20 amino acids Selectivity of DD Fluorescence (FL) spectra of DD upon the addition of 20 amino acids Conditions: [DD] = 5 × 10-6 mol L-1 [Amino Acids] = 25 × 10-3 mol L-1 1% PBS buffer Ex = 275 nm. (L-Asp) (L-Glu) DD Fluorescence image

DD Sensitivity to L-Asp DD + L-Asp DD FL change of DD in the presence of various concentrations of L-Asp Limit of detection (LOD) 10 µM Conditions: [DD] = 5 µM (1% PBS buffer) [Amino Acids] = 2.5-25 mM

DD Sensitivity to L-Glu DD + L-Glu DD FL change of DD in the presence of various concentrations of L-Glu Limit of detection (LOD) 50 µM Conditions: [DD] = 5 µM (1% PBS buffer) [Amino Acids] = 2.5-25 mM

Conclusions A new FL sensor, Dansyl Hydrazine dextran conjugate (DD) was prepared to use as a selective polymeric chemosensor for amino acids. The FL quenching of DD exhibited excellent degree of selectivity towards L-Asp and L-Glu. DD is highly soluble in water, it prompts the application for the intracellular sensing of L-Asp and L-Glu.

Acknowledgements Prof. Dr. Pramuan Tangboriboonrat, Mahidol University Dr. Srung Smanmoo, BIOTEC Faculty of Science, Mahidol University Bioresources Research Unit (BRU)

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