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Synlett 2006(12): 1863-1866  
DOI: 10.1055/s-2006-948165
LETTER
© Georg Thieme Verlag Stuttgart · New York

A Water-Soluble Diaminostilbene Derivative as a Two-Photon Fluorescent Probe

Sheng Yao, Katherine J. Schafer-Hales, Ion Cohanoschi, Florencio E. Hernández, Kevin D. Belfield*
Department of Chemistry and College of Optics and Photonics: CREOL & FPC, University of Central Florida, P.O. Box 162366, Orlando, FL 32816-2366, USA
Fax: +1(407)8232252; e-Mail: kbelfiel@mail.ucf.edu;
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Publication History

Received 23 March 2006
Publication Date:
24 July 2006 (online)

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Abstract

A hydrophilic trans-4,4′-diaminostilbene derivative was prepared by the introduction of amideamino peripheral substituents. Very high aqueous solubility was achieved (>15 mM), making it possible as a fluorescent probe for biological imaging. The relatively straightforward synthetic methodology has great potential to transform even large hydrophobic two-photon absorbing chromophores into hydrophilic derivatives. The fluorescence quantum yield of 0.27 for the diaminostilbene in water is sufficiently high for use as a fluorescent probe. In addition, two-photon fluorescence ­images of NT2 cells stained by this probe were also obtained.

Key words

two-photon - fluorescent probe - PAMAM: hydrophilic - Z-scan - two-photon microscopy

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Experimental Procedure.
1H NMR spectra were recorded on a Varian Mercury-300 NMR (300 MHz) spectrometer using TMS as the internal standard. Chemical shifts (δ) are reported in parts per million (ppm). Elemental analyses were performed by Atlantic Microlab, Inc. Norcross, GA. UV/visible spectra were recorded on an Agilent 8453 spectrophotometer using standard 1 cm path length cuvettes. Steady-state and fluorescence excitation anisotropy measurements were performed on a PTI Quantamaster spectrofluorometer. Fluorescence quantum yields were determined relative to 9,10-diphenylanthracone in cyclohexane.
Preparation of 3,3′,3′′,3′′′-[( E )-Ethene-1,2-diylbis(4,1-phenylenenitrilo)]tetrapropionic Acid Tetraethyl Ester ( 4). A mixture of 4,4′-diaminostilbene (6.3 g, 0.03 mol), freshly distilled ethyl acrylate (30 mL, 0.27 mol), and AcOH (4.5 mL) was refluxed for 48 h. The excess ethyl acrylate was removed under reduced pressure. The residue was purified by column chromatography using hexane-EtOAc (2:1) as eluent. Solvent removal and recrystallization from 95% EtOH afforded 7.2 g of pure product (39% yield). 1H NMR (300 MHz, CDCl3): δ = 7.35 (d, J = 7.8 Hz, 4 H, PhH), 6.82 (s, 2 H, CH=CH), 6.67 (d, J = 7.8 Hz, 4 H, PhH), 4.13 (q, J = 6.9 Hz, 8 H, OCH2), 3.67 (t, J = 7.1 Hz, 8 H, NCH2), 2.59 (t, J = 6.8 Hz, 8 H, C=OCH2), 1.26 (t, J = 7.2 Hz, 12 H, CH3). Anal. Calcd for C34H46N2O8 (610.74): C, 66.86; H, 7.59; N, 4.59. Found: C, 67.02; H, 7.62; N, 4.65.
Preparation of 3,3′,3′′,3′′′-[( E )-Ethene-1,2-diylbis(4,1-phenylenenitrilo)]tetrakis[ N -(2-aminoethyl)]propan-amide ( 1).
Diaminoethylene (6.67 g, 111 mmol) was added to a solution of 4 (0.61 g, 1.0 mmol) in MeOH (3.2 mL), and the mixture was reacted at r.t. under N2 for 60 h. The solvent and the remaining diaminoethylene were removed by distillation at 40 °C under reduced pressure. The yellow solid obtained was washed with Et2O and collected by suction filtration to afford 0.63 g of product (94% yield). NMR analysis indicated this product is a mixture of trans- and cis-isomers, which was recrystallized in EtOH-hexane for three times to give a pure trans-product; mp 180-181 °C. 1H NMR (300 MHz, D2O): δ = 7.25 (d, J = 5.1 Hz, 4 H, PhH), 6.76 (d, J = 3.9 Hz, 2 H, CH=CH), 6.64 (d, J = 5.4 Hz, 4 H, PhH), 3.46 (t, J = 3.5 Hz, 8 H, CH2), 2.95 (t, J = 3.6 Hz, 8 H, CH2), 2.40 (t, J = 3.6 Hz, 8 H, CH2), 2.29 (t, J = 3.8 Hz, 8 H, CH2). Anal. Calcd for C34H54N10O4·3H2O (720.90): C, 56.65; H, 8.40; N, 19.43. Found: C, 56.82; H, 8.25; N, 19.07.