2,6-Dansyl Azide as a Fluorescent Probe for Hydrogen Sulfide

Ke Wang,‡ Hanjing Peng,‡ Nanting Ni, Chaofeng Dai and Binghe Wang*

Department of Chemistry, Center for Diagnostics and Therapeutics, and Center for Biotechnology and Drug Design, Georgia State University, P.O. Box 4098, Atlanta, GA, USA 30302-4098, Fax: (+1) 404-413-5543; Tel: : (+1) 404-413-5545;E-mail:

‡ These authors contributed equally to this work

Supporting Information

Table of Contents:

  1. General information
  2. Synthesis and characterization
  3. Reaction time profile of 2,6-DNS-Az with sulfide
  4. Selectivity of 2,6-DNS-Az for sulfide over reducing agents and nucleophilic amino acids.
  5. Measurement of limit-of-detection (LOD) based on 3:1 signal/noise ratio.
  6. Relative quantum yield determinationof 2,6-DNS-Azand 2,6-DNS-NH2
  7. Spectralinformation
  8. Reference
  1. General information

Solvents and reagents were purchased from VWR International, Oakwood Product Inc., or Sigma-Aldrich Co., and used without purification unless specified otherwise. When necessary, solid reagents were dried under high vacuum. Reactions with compounds sensitive to air or moisture were performed under argon. Solvent mixtures are indicated as volume/volume ratios. Thin layer chromatography (TLC) was run on Sorbtech W/UV254 plates (0.25 mm thick), and visualized under UV-light or by a Ce-Mo staining solution (phosphomolybdate, 25 g; Ce(SO4)2.4H2O, 10 g; conc. H2SO4, 60 mL; H2O, 940 mL) with heating. Flash chromatography was performed using Fluka silica gel 60 (mesh size: 0.040-0.063 mm) using a weight ratio of ca. 30:1 for silica gel over crude compound. 1H and 13C-NMR spectra were recorded on a Bruker 400 spectrometer (400 and 100MHz, respectively) in deuterated chloroform (CDCl3), methanol-d4 (CD3OD), or DMSO-d6 with either tetramethylsilane (TMS) (0.00 ppm) or the NMR solvent as the internal reference. UV-Vis absorption spectra were recorded on a Shimadzu PharmaSpec UV-1700 UV-Visible spectrophotometer. Fluorescence spectra were recorded on a Shimadzu RF-5310PC spectrofluorophotometer. 96-Well plates were read and recorded on a PerkinElmer 1420 multilabel counter.

  1. Synthesis and characterization

6-(Dimethylamino)naphthalene-2-sulfonic acid4:To a solution of compound 3(220mg, 0.99mmol)in acetonitrile(3mL), formaldehyde (1mL)was added dropwise. After the reaction mixture was stirred for 10 min, NaCNBH3(450mg, 7.2mmol)was added in 2 portions. Then, AcOH (1mL) was added in 4 portions during 4h. The reaction was stirred at r.t. for an additional 2h. The product precipitated out from the reaction solution.The white precipitate was filtered out, washed with H2O and MeOH, and dried (225 mg, yield: 91%). 1H NMR (CD3OD): δ 8.17 (1H, s), 7.79 (1H,d, J = 8.8 Hz), 7.75 (1H, d, J = 8.8 Hz), 7.69 (1H, d, J = 8.4 Hz), 7.28 (1H, d, J = 8.8 Hz), 7.00 (1H, s), 3.08 (6H, s); MS (ES+): 252.1 (M+1)+.

6-(Dimethylamino)naphthalene-2-sulfonyl azide 1: To an Ar-protected solution of 4(225 mg, 0.9mmol) in POCl3 (1.1 mL) in an ice bath, PCl5 (832mg, 4mmol) was added in one portion. The reaction mixture was stirred at 0oC for 2h. Then,the reaction was stirred at r.t. for an additional 2 h. The reaction mixture was poured into 100g ice and the product was extracted with EtOAc (100mL×3). The organic phase was dried over Na2SO4, and concentrated under vacuum to yieldcrudeproduct 5. The crude product 5was dissolved in THF (5mL) and added into a stirred solution of NaN3 (580 mg, 9 mmol) in a 1:1 mixture of THF/H2O (10 mL). The reaction mixture was stirred at room temperature for 2 h. Organic solvent was evaporated to obtain light yellow solid. The crude product was purified by flash chromatography purification (Hex:EtOAc, 20:1) to givecompound 1 as light yellow solid (134 mg, 39% in two steps).1H NMR (CDCl3): 8.33 (1H, s), 7.82 (1H, d, J = 9.2 Hz), 7.72 (2H, s), 7.24 (1H, t, J = 9.2 Hz), 6.88 (1H, s), 3.14 (6H, s); 13C NMR (CDCl3): 151.0, 138.0, 130.7, 129.6, 127.5, 124.2, 122.5, 117.2, 104.9, 40.3; IR:2133, 1614, 1507, 1361, 1069, 840, 812, 739cm-1;MS (ES+):277.1 (M+1)+.

6-(Dimethylamino)naphthalene-2-sulfonamide2:To a stirred solution of 2,6-DNS-Az (1, 19.8mg, 0.072mmol) in 1.44mL acetonitrile was added dropwise a solution of Na2S (51.6mg, 0.22mmol) in H2O. The solution was stirred at r.t. for 30 min and was dried under vacuum. The crude product was purified by column chromatography to give compound 2 as whitesolids (13mg, 72%).1H NMR (DMSO-d6): 8.18 (1H, s), 7.90 (1H, d, J = 9.2 Hz), 7.78 (1H, d, J = 8.8 Hz), 7.68 (1H, d, J = 8.8 Hz), 7.31 (1H, d, J = 9.2 Hz),7.24 (2H, s), 6.99 (1H, s), 2.50 (6H, s); MS (ES+):251.08 (M+1)+.

  1. Reaction time profile of 2,6-DNS-Az with sulfide

A stock solution of 2,6-DNS-Az (10 mM in acetonitrile, 2 µL, final concentration 20 µM) was added into 1.0 mL of a solution containing 10 µM of Na2S. Then it was mixed thoroughly and placed in a fluorometer for measurements with λex= 325 nm at a series of time points for 120 min (sample was not exposed to UV between these time points) (Figure S1). Fluorescence intensity at 450nm was plotted against time (Figure S2).

Figure S1. Time-dependent fluorescence response of 2,6-DNS-Az (20 µM) to hydrogen sulfide (10 µM) in 0.1 M phosphate buffer (pH =7.4)

Figure S2. Reaction time profile of 2,6-DNS-Az (20 µM) in the presence of hydrogen sulfide (10 µM) in 0.1 M phosphate buffer (pH =7.4)

  1. Selectivity of 2,6-DNS-Az for sulfide over reducing agents and nucleophilic amino acids.

Figure S3. Fluorescence response of 2,6-DNS-Az to sulfide in the presence and absence of reducing agents (cysteine 100µM, GSH 100µM) and nucleophilic amino acids (lysine 1mM, glycine 1mM) in 0.1 M phosphate buffer (pH =7.4).

  1. Measurement of limit-of-detection (LOD) based on 3:1 signal/noise ratio.

Figure S4. Measurement of LOD. Red line representsfluorescence response of 2,6-DNS-Az (20 µM) to sulfide (1.0 µM);blue line represents the background fluorescence of 2,6-DNS-Az (20 µM)in 0.1 M phosphate buffer (pH =7.4).

  1. Relative quantum yield determinationof 2,6-DNS-Az (1) and 2,6-DNS-NH2 (2)

1,5-DNS-NH2was used as the reference for relative quantum yield determination. Absorption and emission (λex= 325 nm) spectra were recorded for a series of concentrations (20 µM, 16 µM, 12 µM, 8 µM, 4 µMand 0 µM in deionized water) of 1,5-DNS-NH2, 2,6-DNS-NH2, and 2,6-DNS-Az. Integrated fluorescence intensity was plotted against the absorption values at each concentration (Figures S5 and S6) [1]. Relative quantum yield values can be calculated using slopes of each compound.

Figure S5.Quantum yield determination of2,6-DNS-Az (0-20 µM) in H2O

FigureS6. Quantum yield determination of1,5-DNS-NH2 (0-20 µM) in H2O

  1. Spectralinformation






  1. Reference

1. Williams ATR, Winfield SA, Miller JN (1983) Relative fluorescence quantum yields using a computer-controlled luminescence spectrometer. Analyst 108 (1290):1067-1071. doi:10.1039/an9830801067

2. Peng H, Cheng Y, Dai C, King A, Predmore B, Lefer D, Wang B (2011) A fluorescent probe for fast and quantitative detection of hydrogen sulfide in blood. Angew Chem Int Ed 50 (41):9672-9675. doi:10.1002/anie.201104236

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