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

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.

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

References and Notes

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9

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.

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11a The 2PA cross-sections measurement followed the procedure described in: Hernandez FE. Belfield KD. Cohanoschi I. Chem. Phys. Lett.  2004,  391:  22 

11b

interestingly, two-photon cross-section of compound 1 is 3500 GM measured by picosecond laser sources, about one order of magnitude higher than that measured by femtosecond laser source. Similar phenomena have been reported in results from nanosecond laser sources by Prasad and co-workers; for examples, see:

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11f

More detailed experimental investigation is underway to understand the mechanism.

12 The sample preparation and multiphoton fluorescence imaging followed the procedure described in: Schafer-Hales KJ. Belfield KD. Yao S. Frederiksen PK. Hales JM. Kolattukudy PE. J. Biomed. Opt.  2005,  10:  051402