1Department of Chemistry, Faculty of Science, Mahidol University, Bangkok, 10400 Thailand2Bioresources Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC)Thailand Science Park, Klong luang, Pathumthani, 12120 Thailand

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Selective and Sensitive Sensing of L-Aspatic Acid and L-Glutamic Acid

Employing Dansyl Functionalized Dextran

Weerachai Nasomphan1, Srung Smanmoo2 and Pramuan Tangboriboonrat1*

Advantages of L-Asp & L-Glu

Biosynthesis precursor

Amino acid metabolism

Human nutrition

Identifying diseases

Biological roles

2Okamoto H. et al. J Sep Sci 2005; 28: 522. Yen K. et al. Talanta 2007; 71: 2007.

H2N CH C

CH2

OH

O

CH2

C

OH

O

H2N CH C

CH2

OH

O

C

OH

O

(L-Asp) (L-Glu)

Acidic amino acid

High level in blood

Stroke Diabetic

L-Aspatic acid L-Glutamic acid

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Disadvantages: Expensive Complicate Skillful Time consuming

STANDARD METHOD

FluorescenceChemosensor

Advantages: High selectivity* High sensitivity** Low cost Simple-to-use Naked-eye diagnostic tools

*,** depend on chemical property of sensor

UV-Vis HPLC ICP-MS

Standard Method Vs Chemosensor

4Ramon M. et al. Chem Rev 2003; 103: 11. Suslick KS. et al. Tetrahedron 2004; 60: 11133.

Signaling subunit

Binding subunit “Ionophore”

Amino Acid

Amino Acid

Fluorescence quenching/enhancement Color change

Interactions

• Acid-base interaction • Hydrogen bonding• Metal-ligand coordination• Van der Waals and physical interaction

Fluorophore

Design of Chemosensor

OC6H13C6H13O

C6H13O

OC6H13

OC6H13C6H13O

C6H13O OC6H13

n

Molecular Vs Polymeric Chemosensors

Molecular chemosensor

Advantages • High sensitivity • Solubility enhancement

Polymeric chemosensorConjugated system

Non-Conjugated system

Long Y. et al. Macromolecules 2009; 42: 6501.Malashikhin S. et al. J Am Chem Soc 2008; 130: 12546.

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OMe

Signal Enhancement

OOH

OH OHO

OOH

OH OHO

Dextran (D)

Non-Conjugated Polymer: Dextran Probe

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Acidic amino acids (AAs)

H2N CH C

CH2

OH

O

CH2

C

OH

O

H2N CH C

CH2

OH

O

C

OH

O

L-Asp L-Glu

Polymeric template: Dextran

(Water soluble template)

OOH

OH OHO

OOH

OH OHO

Oxidation

OHCOHC

O

OOHC

OHC

O

O

Dextran (D)

Oxidized Dextran

BindingUnit

O

O

O

O

N

OH OHOH

HN

S OO

N

O

O

O

O

N

OH OHOH

HN

S OO

N Dansyl Dextran Conjugate (DD)

Fluorophore

Signaling UnitS OO

HNNH2

N

Dansyl Hydrazine(Acidic Sensitive)

Interaction of Dextran Probe & Amino Acid

“ Fluorescent on ”

Hydrogen bondingCharge-Charge interaction

“ Fluorescent off ”

Internal Charge Transfer (ICT) Process

ICT

7Ohtake K. et al. J Control Release 2002; 82: 263.Yen K. et al. Talanta 2007; 71: 2007.

DD

H2N CH C

CH2

OH

O

CH2

C

O

O

Objective

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

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O

O

O

O

N

OH OHOH

Dansyl Conjugated Dextran (DD)

HN

S OO

N

Non-Conjugated Polymer: DD

Synthetic scheme of dextran dansylhydrazine conjugate

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Molecular weight (kDa) = 2600Polydispersity index (Mw/Mn) = 1.33

OOH

OH OHO

OOH

OH OHO

NaIO4, 25 oC

Dextran (D)

O

O

O

O

N

OH OHOH

Dansyl Conjugated Dextran (DD)

S OO

HNNH2

N

HN

S OO

N

OHCOHC

O

OOHC

OHC

O

O

Oxidized Dextran

Dansyl Hydrazine

25 oC

OHCOHC

O

OOHC

OHC

O

O

Oxidized Dextran

Selectivity of DD

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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

FL change of DD in the presence of various concentrations of L-Asp

Conditions: • [DD] = 5 µM (1% PBS buffer)• [Amino Acids] = 2.5-25 mM

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Limit of detection (LOD)10 µM

DD + L-AspDD

DD Sensitivity to L-Glu

FL change of DD in the presence of various concentrations of L-Glu

Conditions: • [DD] = 5 µM (1% PBS buffer)• [Amino Acids] = 2.5-25 mM

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Limit of detection (LOD)50 µM

DD + L-GluDD

Conclusions

The FL quenching of DD exhibited excellent degree of selectivity towards L-Asp and L-Glu.

A new FL sensor, Dansyl Hydrazine dextran conjugate (DD) was prepared to use as a selective polymeric chemosensor for amino acids.

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DD is highly soluble in water, it prompts the application for the intracellular sensing of L-Asp and L-Glu.

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Acknowledgements

Prof. Dr. Pramuan Tangboriboonrat, Mahidol UniversityDr. Srung Smanmoo, BIOTEC

Faculty of Science, Mahidol UniversityBioresources Research Unit (BRU)

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Thank you foryour kind attention