Technical Reference #279

279.

A tautomeric zinc sensor for ratiometric fluorescence imaging: Application to nitric oxide-induced release of intracellular zinc Christopher J. Chang; Jacek Jaworski; Elizabeth M. Nolan; Morgan Sheng; and Stephen J. Lipp, Howars Hughes Medical Institute, PNAS, 101(279), (2004)
Link To Paper

Abstract:
Zinc is an essential metal ion for human growth and developmentthe disruption of cellular Zn2 homeostasis being implicated inseveral major disorders including Alzheimer’s disease diabetesand cancer. The molecular mechanisms of Zn2 physiology an

Materials & Methods:
Synthetic Materials and Methods. Silica gel 60 (70–230 mesh Merck) and octadecyl-functionalized silica gel (RP18 Aldrich) were used for column chromatography. Analytical thin layer chromatography was performed by using Merck 60 F254 silica gel and Merck RP-18 F254S silica gel (precoated sheets 0.25mm thick). Solvents for synthesis were of reagent grade or better and were dried according to standard methods (37). 2-[Bis(2- pyridylmethyl)aminomethyl]aniline (6) was prepared as described (31). All other reagents for synthesis were purchased and used as received. 1H NMR spectra were collected in CDCl3 CD3OD or d7-dimethylformamide (Cambridge Isotope Laboratories CambridgeMA) at 25°C at the Massachusetts Institute of Technology Department of Chemistry Instrumentation Facility (DCIF) on either a Varian Inova 500 or a Varian Mercury 300 spectrometer. All chemical shifts are reported in the standard notation of parts per million; positive chemical shifts are to higher frequency from the given reference. High-resolution mass spectral analyses were carried out at the Massachusetts Institute of Technology DCIF. 2 -Carboxy-3-methyl-24-dihydroxybenzophenone (1). Under an argon atmosphere 2-methylresorcinol (10.0 g 80.6 mmol) and phthalic anhydride (11.2 g 75.6 mmol) were combined in dry nitrobenzene (250 ml). The mixture was cooled to 0°C and aluminum(III) chloride (23.5 g 176 mmol) was added in one portion. The resulting dark olive slurry was allowed to warm to room temperature and stirred for an additional 16 h under argon. The reaction was poured into a vigorously stirring mixture of hexanes (300 ml) and 1 M HCl (1 liter). The precipitate was filtered and recrystallized twice from methanol water to afford benzophenone 1 as a beige powder (16.0 g 78% yield). 1H NMR (CD3OD 500 MHz): 8.05 (1 H dd J1 8.0 Hz J2 1.0 Hz) 7.62 (2 H dt J1 33.0 Hz J2 9.0 Hz) 7.33 (1 H d J 7.5 Hz) 6.74 (1 H d J 8.5 Hz) 6.20 (1 H d J 8.5 Hz) 2.05 (3 H s). High-resolution MS (HRMS) (electrospray ionization ESI) calculated for [M-H] 271.0601 found 271.0601. 4-Methyl-310-dihydroxy-spiro[7H-benzo[c]xanthen-71 (3 H)-isobenzofuran]- 3 -one (2). 2 -Carboxy-3-methyl-24-dihydroxybenzophenone (1 8.16 g 30.0 mmol) and 16-dihydroxynaphthalene (4.81 g 30.0 mmol) were combined in methanesulfonic acid (120 ml) and sealed in a thick-walled glass tube. The resulting viscous mixture was stirred at 90°C for 24 h. The reaction was poured into ice-cold water (1 liter) and the precipitate was filtered and washed with water (3 200 ml). Purification by flash column chromatography (silica gel 9:1 dichloromethane methanol) furnished seminaphthofluorescein 2 as a brick red powder (9.40 g79% yield). 1H NMR (CD3OD 500 MHz): 8.41 (1 H d J 9.0 Hz) 8.00 (1 H d J 7.0 Hz) 7.69 (2 H m) 7.27 (1 H dd J1 8.5 Hz J2 3.5 Hz) 7.23 (1 H d J 10.0 Hz) 7.16 (1 H d J 7.5 Hz) 7.09 (1 H br s) 6.59 (2 H m) 6.46 (1 H d J 8.5 Hz) 2.48 (3 H s). HRMS (ESI) calculated for [M H] 397.1071 found 397.1057.

Microscopic Technique
Fluorescence Microscopy, Time lapse

Cell Type(s)
COS-7