Low-Cost Fluorimetric Determination of Radicals ... - ACS Publications

Anal. Chem. 2010, 82, 1213–1220

Low-Cost Fluorimetric Determination of Radicals Based on Fluorogenic Dimerization of the Natural Phenol Sesamol Yumi Makino,† Seiichi Uchiyama,*,† Ken-ichi Ohno,‡ and Hidetoshi Arakawa‡ Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and School of Pharmacy, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan A novel fluorimetric method for determining radicals using the natural phenol sesamol as a fluorogenic reagent is reported. In this assay, sesamol was reacted with aqueous radicals to yield one isomer of a sesamol dimer exclusively. The dimer emitted purple fluorescence near 400 nm around neutral pH, where it assumed the monoanionic form. This method was applied to the straightforward detection of radical nitric oxide (NO). The ready availability of sesamol should enable rapid implementation of applications utilizing this new assay, particularly in high-throughput analysis or screening. Radicals are reactive and unstable due to unpaired electrons. Radicals can be chemically eliminated by trapping or may undergo consecutive (chain) reactions. These complex properties are important in such diverse areas as organic synthesis,1 disease,2 environmental pollution,3 food storage,4 and interstellar matter.5,6 Thus, methodologies to detect radicals have been developed in order to ascertain their roles and functions. Electron spin resonance (ESR)7 and fluorimetry8 are currently used predominantly for the determination of radicals. When these two methods are compared, fluorescence detection has the advantages of high-throughput capability and precise quantitative performance. Because most radicals are short-lived and nonfluorescent, fluorogenic (fluorescent off-on) reagents9-11 * To whom correspondence should be addressed. Fax: +81 3 5841 4768. E-mail: [email protected]. † The University of Tokyo. ‡ Showa University. (1) Radicals in Organic Synthesis; Renaud, P., Sibi, M. P., Eds.; Wiley-VCH: Weinheim, Germany, 2001. (2) Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and Medicine, 4th ed.; Oxford University Press: New York, 2007. (3) Rhodes, C. J. Toxicology of the Human Environment; Taylor & Francis: London, 2000. (4) Free Radicals in Food: Chemistry, Nutrition, and Health Effects; Morello, M. J., Shahidi, F., Ho, C.-T., Eds.; ACS Symposium Series 807; American Chemical Society: Washington, DC, 2002. (5) Weinreb, S.; Barrett, A. H.; Meeks, M. L.; Henry, J. C. Nature 1963, 200, 829–831. (6) Ziurys, L. M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 12274–12279. (7) Gerson, F.; Huber, W. Electron Spin Resonance Spectroscopy of Organic Radicals; Wiley-VCH: Weinheim, Germany, 2003. (8) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006. (9) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515– 1566. 10.1021/ac9029778  2010 American Chemical Society Published on Web 01/27/2010

are utilized to convert radicals into a stable fluorescent product. A number of fluorogenic reagents for radicals are commercially available and are classified by functionality: (I) benzoic acid,12 terephthalic acid,13,14 and coumarin15,16 can detect hydroxyl radical (•OH) produced in aqueous solution; although these agents are nonfluorescent, the hydroxylated adducts are strongly fluorescent; (II) 5-((2-carboxy)phenyl)-5-hydroxy-1-((2,2,5,5tetramethyl-1-oxypyrrolidin-3-yl)methyl)-3-phenyl-2-pyrrolin-4one17 contains fluorescamine (fluorophore) and nitroxide (quencher) in a single molecule. When the nitroxide reacts with the methyl radical (•CH3), its quenching ability is lost and the fluorescamine structure starts to fluoresce. Thus, (III) 2′,7′dichlorodihydrofluorescein18 reacts oxidatively with •OH to produce fluorescent 2′,7′-dichlorofluorescein; (IV) 3′-(p-hydroxyphenyl) fluorescein (HPF) and 3′-(p-aminophenyl) fluorescein (APF) are reagents for reactive oxygen species.19 A strong fluorescence signal derived from fluorescein is observed after reaction with •OH, and (V) hydroethidine20,21 is a fluorogenic reagent for superoxide (O2•-). The reaction product, 2-hydroxyethidine, fluoresces when intercalated into DNA. Despite these choices, fluorogenic reagents with new functional mechanisms are still required for the determination of radicals. The reliability of experimental results is enhanced by the use of multiple reagents that have different mechanisms. Additionally, one of the most important radicals, nitric oxide (NO),2 is not detectable by the conventional fluorogenic reagents listed above. Current protocols for the fluorimetric detection of NO involve (10) Callan, J. F.; de Silva, A. P.; Magri, D. C. Tetrahedron 2005, 61, 8551– 8588. (11) Anslyn, E. V. J. Org. Chem. 2007, 72, 687–699. (12) Armstrong, W. A.; Grant, D. W. Nature 1958, 182, 747. (13) Armstrong, W. A.; Facey, R. A.; Grant, D. W.; Humphreys, W. G. Can. J. Chem. 1963, 41, 1575–1577. (14) Barreto, J. C.; Smith, G. S.; Strobel, N. H. P.; McQuillin, P. A.; Miller, T. A. Life Sci. 1995, 56, 89–96. (15) Gopakumar, K.; Kini, U. R.; Ashawa, S. C.; Bhandari, N. S.; Krishnan, G. U.; Krishnan, D. Radiat. Effects 1977, 32, 199–203. (16) Louit, G.; Foley, S.; Cabillic, J.; Coffigny, H.; Taran, F.; Valleix, A.; Renault, J. P.; Pin, S. Radiat. Phys. Chem. 2005, 72, 119–124. (17) Pou, S.; Huang, Y.-I.; Bhan, A.; Bhadti, V. S.; Hosmane, R. S.; Wu, S. Y.; Cao, G.-L.; Rosen, G. M. Anal. Biochem. 1993, 212, 85–90. (18) Keston, A. S.; Brandt, R. Anal. Biochem. 1965, 11, 1–5. (19) Setsukinai, K.; Urano, Y.; Kakinuma, K.; Majima, H. J.; Nagano, T. J. Biol. Chem. 2003, 278, 3170–3175. (20) Rothe, G.; Valet, G. J. Leukocyte Biol. 1990, 47, 440–448. (21) Zhao, H.; Joseph, J.; Fales, H. M.; Sokoloski, E. A.; Levine, R. L.; VasquezVivar, J.; Kalyanaraman, B. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5727– 5732.

Analytical Chemistry, Vol. 82, No. 4, February 15, 2010

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Figure 1. Chemical structure of sesamol.

Figure 2. Chemical structure of 3,4-methylenedioxyanisole (MDA).

chemical transformation of NO into a nonradical species before reaction with fluorogenic reagents.22 Sesamol (3,4-methylenedioxyphenol, Figure 1) is a natural phenol obtained by the hydrolysis of sesamolin from sesame seeds.23,24 Relative to other edible oils, sesame oil shows strong antioxidant properties due to its considerable sesamol content.25-27 Intensive studies have suggested that the antioxidant ability of sesamol is derived from its high reactivity to radicals.28,29 The 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical-scavenging capacity of sesamol is higher than that of another well-known natural antioxidant, R-tocopherol.28 In this work, we demonstrate a novel fluorimetric method for determining radicals using sesamol as a fluorogenic reagent. A sesamol radical reaction product was investigated and identified as an isomer of the sesamol dimer. This dimerization is quite different from reactions of conventional reagents with radicals. The reactivity of sesamol was then examined using various radicals, e.g., •OH and NO. A relevant methyl ether of sesamol, 3,4-methylenedioxyanisole (MDA, Figure 2), was also tested in the reaction studies to clarify the sesamol dimerization mechanism. Considering that the dissociation equilibrium of a phenolic hydroxyl group affects its photophysical properties, the absorption and fluorescence properties of the sesamol (reagent) and sesamol dimer (reaction product) were obtained in an aqueous buffer over a wide pH range and in organic solvents. In addition, effects of coexisting salts and organic molecules on the fluorescence properties of sesamol dimer were examined. These data support fluorescence off-on switching based on the radical-induced dimerization of sesamol. Finally, the new fluorimetric method with sesamol was applied to NO detection. Sesamol rapidly reacted with NO and gave a fluorescence signal that was a linear function of NO concentration. These features enabled us to monitor NO generated from selected (22) Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Anal. Chem. 1998, 70, 2446–2453. (23) Budowski, P.; Markley, K. S. Chem. Rev. 1951, 48, 125–151. (24) Namiki, M. Crit. Rev. Food Sci. Nutr. 2007, 47, 651–673. (25) Budowski, P. J. Am. Oil Chem. Soc. 1950, 27, 264–267. (26) Uchida, M.; Nakajin, S.; Toyoshima, S.; Shinoda, M. Biol. Pharm. Bull. 1996, 19, 623–626. (27) Prasad, N. R.; Mahesh, T.; Menon, V. P.; Jeevanram, R. K.; Pugalendi, K. V. Environ. Toxicol. Pharmacol. 2005, 20, 1–5. (28) Suja, K. P.; Jayalekshmy, A.; Arumughan, C. J. Agric. Food Chem. 2004, 52, 912–915. (29) Joshi, R.; Kumar, M. S.; Satyamoorthy, K.; Unnikrisnan, M. K.; Mukherjee, T. J. Agric. Food Chem. 2005, 53, 2696–2703.

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Figure 3. Chemical structures of NO donors used in this study: (A) GSNO, (B) NOC7, and (C) NOR1.

NO donors: GSNO (S-nitrosoglutathione),30 NOC7 (1-hydroxy-2oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene),31 and NOR1 ((±)-(E)-4-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide)32 (Figure 3). Biological components in human plasma and HL-60 cell extract did not disable this fluorimetric method for NO detection by sesamol. These results demonstrate the utility of sesamol for convenient and inexpensive fluorimetric radical analysis. EXPERIMENTAL SECTION Materials. Sesamol was purchased from Wako Pure Chemicals. GSNO, NOC7, and NOR1 were obtained from Dojindo. KO2, peroxynitrite solution, and Angeli’s salt (Na2N2O3)33 were purchased from Strem Chemicals, Cayman Chemical, and Calbiochem, respectively. Human plasma and HL-60 cell extract were purchased from Sigma-Aldrich. Water was purified using a Milli-Q reagent system, Direct-Q 3 UV (Millipore). A saturated NO solution (1.9 mM at 25 °C)34 was prepared from gaseous NO (Sumitomo Seika Chemicals) in a fume hood. MDA was obtained as previously reported.35 All other reagents were of reagent grade and used without further purification. Britton-Robinson buffer was prepared from H3PO4 (40 mM), CH3COOH (40 mM), H3BO3 (40 mM), and NaOH (200 mM). Borate buffer (Palitzsch buffer) was prepared from Na2B4O7 (50 mM), H3BO3 (200 mM), and NaCl (50 mM). Solvents used in this study were aerated unless described otherwise. Apparatus. Melting points were measured on a Round Science RFS-10 and left uncorrected. Proton nuclear magnetic resonance spectra (400 MHz) were obtained using a Bruker AVANCE 400 spectrometer. Mass spectra were obtained by electrospray ionization (ESI) on a Bruker micrOTOF-05 spectrometer. The high(30) Seabra, A. B.; de Souza, G. F. P.; da Rocha, L. L.; Eberlin, M. N.; de Oliveira, M. G. Nitric Oxide 2004, 11, 263–272. (31) Hrabie, J. A.; Klose, J. R.; Wink, D. A.; Keefer, L. K. J. Org. Chem. 1993, 58, 1472–1476. (32) Kato, M.; Nishino, S.; Ohno, M.; Fukuyama, S.; Kita, Y.; Hirasawa, Y.; Nakanishi, I.; Takasugi, H.; Sakane, K. Bioorg. Med. Chem. Lett. 1996, 6, 33–38. (33) Amatore, C.; Arbault, S.; Ducrocq, C.; Hu, S.; Tapsoba, I. ChemMedChem 2007, 2, 898–903. (34) Shaw, A. W.; Vosper, A. J. J. Chem. Soc., Faraday Trans. 1 1977, 1239– 1244. (35) Hussain, H. H.; Babic, G.; Durst, T.; Wright, J. S.; Flueraru, M.; Chichirau, A.; Chepelev, L. L. J. Org. Chem. 2003, 68, 7023–7032.

Figure 4. Reaction of sesamol with t-butoxyl radical (t-BuO•). The radical was generated by thermal decomposition of di-t-butylperoxide under the conditions indicated.

performance liquid chromatography (HPLC) system consisted of a JASCO PU-2080 pump, a Hitachi L-4000H UV detector, a JASCO CO-2060 column thermostat, and a Wako Wakosil II5C18AR (4.6 mm × 150 mm; i.d., 5 µm) column. UV-vis absorption spectra were measured using a JASCO V-550 or V-650 UV-vis spectrometer. Sample temperature was controlled by a JASCO ETC-505T or ETC-717 temperature controller. Fluorescence spectra were obtained using a JASCO FP-6500 spectrofluorimeter with a Hamamatsu R-7029 photomultiplier tube. Sample temperature was controlled with a JASCO ETC-273T temperature controller. Reaction of Sesamol with t-Butoxyl Radical and Product Identification. A mixture of di-t-butyl peroxide (22 mg, 0.15 mmol) and sesamol (200 mg, 1.45 mmol, 9.6 equiv) in chlorobenzene (8 mL) was stirred at 130 °C for 3 h. Then, the reaction mixture was evaporated until dry under reduced pressure, and the residue was chromatographed on silica gel with dichloromethane-ethyl acetate (20:1) to obtain 5,5′-bi-1,3-benzodioxole-6,6′-diol (sesamol dimer,36 Figure 4) (15 mg, 37%) as a white powder: mp, 192 °C; 1H NMR (in CDCl3) δ 6.64 (2H, s), 6.58 (2H, s), 5.96 (4H, s), 5.05 (2H, s); HR-ESI-MS m/z calcd for C14H9O6- ([M - H]-) 273.0399, found 273.0399. Anal. Calcd for C14H10O6: C, 61.32; H, 3.68. Found: C, 61.41; H, 3.87. Reaction of Sesamol with Radicals. For reaction with •OH, Fenton’s reagent (H2O2 (final concentration, 1 mM) and FeSO4 (1 mM))37 were mixed with sesamol (final concentration, 10 mM) in a borate buffer (pH 7.0) and held at 25 °C for 15 min. For reaction with •CH3, DMSO (1 mM) was premixed into the sesamol solution.38 For reaction with NO, a saturated NO solution was added to the sesamol solution, instead of the Fenton’s reagent, to a final concentration of 380 µM. This reaction was performed in either an oxygen-free (Ar-saturated) buffer or an aerated buffer. To study the reaction with O2•-, solid KO2 (equivalent to 37.6 mM) was dissolved directly in a sesamol solution.39 A ONOO- solution (final concentration, 720 µM) was also used. After the reactions, 10 µL of each mixed solution was subjected to HPLC analysis (eluent, acetonitrilewater-trifluoroacetic acid (TFA), 30:70:0.01 (v/v/v); detection, UV 310 nm). The experimental procedures for the MDA-radical reaction are described in the Supporting Information. Absorption and Fluorescence Measurements of Sesamol Dimer and Sesamol. Absorption spectra (50 or 100 µM) and fluorescence spectra (500 nM, 1, 2, or 5 µM) of the sesamol dimer and sesamol were measured in Britton-Robinson buffer, borate buffer, DMSO, DMF, acetonitrile, ethanol, methanol, and 2,2,2trifluoroethanol (TFE) at 25 °C. Various salts (NaCl, LiCl, KCl, MgCl2, CaCl2, NaBr, NaI, Na2SO4, Na2CO3, NaH2PO4, and (36) Kurechi, T.; Kikugawa, K.; Aoshima, S. Chem. Pharm. Bull. 1981, 29, 2351– 2358. (37) Walling, C. Acc. Chem. Res. 1975, 8, 125–131. (38) Eberhardt, M. K.; Colina, R. J. Org. Chem. 1988, 53, 1071–1074. (39) Lokesh, B. R.; Cunningham, M. L. Toxicol. Lett. 1986, 34, 75–84.

Na2HPO4) and organic molecules (formaldehyde, acetaldehyde, benzaldehyde, acrolein, D-glucose, 2,4,6-trimethylpyridine, and p-dinitrobenzene) were used as coexisting substances in the fluorescence measurements. TFA (at 4.5 × 10-4% for all except DMF at 1.8 × 10-3%) or triethylamine (TEA, 0.67%) was used for pH adjustment of the organic solvents. Fluorescence quantum yields (Φf) were determined by the following eq 1: Φf,S ) Φf,RFSARnS2 /FRASnR2

(1)

where F is the area under the corrected fluorescence spectrum with excitation at an adopted wavelength, A is the absorbance at the wavelength, n is the refractive index of the solvent, and the subscripts R and S represent the reference and sample, respectively. Quinine sulfate and 2-aminopyridine were used as references.8 Fluorimetric NO Detection Using Sesamol. In all experiments, the concentration of sesamol in the reaction mixtures was 1 mM, and the solutions were excited at 330 nm. For a time course study, NO solution (final concentration, 100 µM) was mixed with sesamol in a borate buffer (pH 7) at 25 °C, and the fluorescence intensity of the mixture was monitored at 392 nm. To study selectivity and linearity, NO solution (final concentration, 0, 1, 5, 10, 25, 50, 75, or 100 µM), NOBF4 (100 µM), NaNO2 (1 mM), NaNO3 (1 mM), NH3 (1 mM), N2O (flow rate, 250 mL/min), N2 (120 mL/min), or Angeli’s salt (1 mM) was mixed with sesamol in a borate buffer (pH 7.4 for Angeli’s salt and pH 7 for the others) at 25 °C for 5 min, and the fluorescence spectrum of each solution was obtained. For detection of NO released from GSNO, sesamol and GSNO (final concentration, 1 mM) were mixed in a Britton-Robinson buffer (pH 3), and the temperature of the sample solution was varied between 5 and 45 °C over 30 min. At fixed intervals, 100 µL of the solution was added to 2.4 mL of borate buffer (pH 8), and the fluorescence intensity of the mixture was monitored at 392 nm. For detection of NO released from NOC7 or NOR1, sesamol and the NO donor (final concentration, 100 µM) were mixed in a reaction medium (borate buffer (pH 7.0 or 7.4, 22 °C), 20% or 50% human plasma in borate buffer (pH 7.0, 37 °C), or 20% or 50% HL-60 cell extract in borate buffer (pH 7.0, 37 °C)), and the fluorescence intensity of each mixture was monitored at 392 nm. The observed fluorescence intensity (FI) was analyzed by exponential approximation using eq 2: FI ) (FImax - FImin)(1 - e-kt) + FImin

(2)

where FImax, FImin, k, and t represent the maximum and minimum fluorescence intensities, the decay constant for an NO donor, and the time from mixing, respectively. RESULTS Reaction of Sesamol with t-Butoxyl Radical and Identification of the Reaction Product. The reaction product of sesamol with radicals was identified by using t-butoxyl radical (tBuO•) as a representative (Figure 4), where thermal decomposition of dit-butylperoxide in chlorobenzene at 130 °C supplied reactive Analytical Chemistry, Vol. 82, No. 4, February 15, 2010

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Figure 5. Chromatograms for reaction mixtures of sesamol (10 mM) with (A) •OH generated from FeSO4 (1 mM) and H2O2 (1 mM), (B) only H2O2 (1 mM), (C) •CH3 and •OH from FeSO4 (1 mM), DMSO (1 mM), and H2O2 (1 mM), (D) NO (380 µM), (E) O2•- from KO2 (37.6 mM), and (F) •NO2 and •OH from ONOO- (720 µM). The vertical scales are identical in parts A-F. Peaks 1 and 2 were assigned to sesamol and the sesamol dimer, respectively. Column: Wakosil II5C18AR (4.6 mm × 150 mm; i.d., 5 µm). Eluent: acetonitrile-water-TFA (30: 70: 0.01, v/v/v). Flow rate: 1 mL min-1. Detection: UV 310 nm.

tBuO•.40 It is notable that this reaction exclusively produced 5,5′-bi-1,3-benzodioxole-6,6′-diol (hereafter referred to as “sesamol dimer”), which is identical to the compound generated by the coupling of two sesamol molecules at the 6-positions (see Figure S1 in the Supporting Information). The chemical structure of the sesamol dimer was fully confirmed by NMR, mass spectrometry, and elemental analysis (see the Experimental Section). Reaction of Sesamol with Radicals in Aqueous Buffer. The reactivity of sesamol toward radicals (•OH, •CH3, NO, and O2•-) and the specificity of the product of these reactions were examined by separation analysis with reversed-phase HPLC. Each radical was reacted with excess sesamol in aqueous borate buffer (pH 7.0 or 7.4), and the mixture was held at 25 °C for 15 min. Figure 5A shows a chromatogram for a reaction mixture of sesamol with •OH generated by the Fenton reaction37 using H2O2 and FeSO4, reflecting the exclusive production of the sesamol dimer from sesamol. Because the sesamol dimer was not produced by the mixture of sesamol with either H2O2 (Figure 5B) or FeSO4 alone (Figure S2 in the Supporting Information), i.e., peak areas of sesamol dimer were 9.9

0.045 9.6

From pKa values obtained in this study.

Table 1 summarizes the absorption and fluorescence properties of the sesamol dimer in the neutral, monoanionic, and dianionic forms in a Britton-Robinson buffer. The absorption and fluorescence measurements in this study indicate that the sesamol dimer emits fluorescence only in the monoanionic form, which is predominant around a neutral pH value. Absorption and Fluorescence Properties of Sesamol in an Aqueous Buffer. Absorption and fluorescence spectra of sesamol were also obtained in a Britton-Robinson buffer with pH varying from 2 to 12. From the change in absorption spectra (see Figure S4 in the Supporting Information), the pKa value of sesamol was determined to be 9.6, indicating that sesamol exists in the nonionic form at around neutral pH, where its fluorescence intensity with excitation at 317 nm was less than 0.3% of that of the sesamol dimer (see Figure S5 in the Supporting Information). The complementary absorption and fluorescence properties of sesamol in the neutral and anionic forms are listed in Table 1. Absorption and Fluorescence Properties of the Sesamol Dimer and Sesamol in Organic Solvents. Table 2 shows the photophysical properties of the neutral and monoanionic sesamol dimer and neutral sesamol in DMSO, DMF, acetonitrile, ethanol,

methanol, and TFE. The dianionic sesamol dimer and anionic sesamol exist only at low levels in these solvents because of the low acidities of the corresponding phenolic hydroxyl groups. Similarly, in aqueous buffer, the sesamol dimer emitted strong fluorescence in the monoanionic form in all tested solvents except for TFE. Sesamol was generally less fluorescent than the monoanionic sesamol dimer and sufficiently so to be a potential fluorogenic reagent for radicals even in nonaqueous environments. Effects of Coexisting Substances on the Fluorescence Properties of the Sesamol Dimer. Table 3 summarizes the fluorescence intensities of the sesamol dimer in the presence of selected coexisting substances, most of which are found in real environments (e.g., seawater43 and body fluid44). The fluorescence intensities of the sesamol dimer were not influenced by the coexisting substances, except for benzaldehyde. This exceptional quenching was due to an intermolecular electron transfer45 from (43) DOE. Handbook of Methods for the Analysis of the Various Parameters of the Carbon Dioxide System in Sea Water, version 2; Dickson, A. G., Goyet, C., Eds.; Washington, DC, 1994; Chapter 5, p 11. (44) Kokubo, T.; Kushitani, H.; Sakka, S.; Kitsugi, T.; Yamamuro, T. J. Biomed. Mater. Res. 1990, 24, 721–734. (45) Uchiyama, S.; Santa, T.; Imai, K. Analyst 2000, 125, 1839–1845.


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Table 2. Photophysical Properties of Neutral and Monoanionic Sesamol Dimers and Neutral Sesamol in Organic Solvents at 25 °C: Fluorescence Quantum Yield (Φf), Maximum Absorption Wavelength (λab), Molar Absorption Coefficient (ε), and Maximum Emission Wavelength (λem) species a

neutral sesamol dimer

monoanionic sesamol dimerb

neutral sesamol

a

solvent

Φf

λab (nm)

ε (M-1 cm-1)

λem (nm)

DMSO DMF acetonitrile ethanol methanol DMSO DMF acetonitrile ethanol methanol TFE DMSO DMF acetonitrile ethanol methanol

0.13 0.056 0.16 0.18 0.19 0.82 0.78 0.55 0.55 0.48 0.019 0.056 0.019 0.053 0.047 0.051

318 317 312 314 313 365c 368c 355c 340c 338c 329c 301 300 298 298 298

12 000 13 000 14 000 15 000 14 000

372 373 366 368 370 441 439 436 421 420 416 340 335 335 336 336

4200 4700 4200 4300 4400

Induced by adding TFA to solvents. b Induced by adding TEA to solvents. c Maximum excitation wavelength.

Table 3. Fluorescence Intensities of the Sesamol Dimer (2 µM) with Coexisting Substances in Borate Buffer (pH 7.0, 25 °C) coexisting substance

RFI (%)a

none NaCl, 500 mM LiCl, 1 mM KCl, 25 mM MgCl2, 25 mM CaCl2, 25 mM NaBr, 1 mM NaI, 1 mM Na2SO4, 25 mM Na2CO3, 1 mM NaH2PO4 + Na2HPO4, 1 mMb acrolein, 1 mM D-glucose, 1 mM 2,4,6-trimethylpyridine, 1 mM formaldehyde, 1 mM acetaldehyde, 1 mM benzaldehyde, 1 mM cf. p-dinitrobenzene, 100 µM

100 101 ± 1.3 100 ± 0.2 99 ± 0.3 100 ± 1.6 100 ± 0.4 101 ± 0.6 98 ± 2.2 99 ± 1.0 104 ± 0.6 101 ± 1.7 99 ± 0.4 100 ± 0.1 96 ± 0.5 100 ± 0.3 102 ± 0.1 91 ± 0.7 79 ± 1.2

a Relative fluorescence intensity (±relative standard deviation). b In total.

the sesamol dimer to benzaldehyde, which was confirmed by the efficient quenching with more electron-deficient p-dinitrobenzene. Fluorimetric NO Detection Using Sesamol.46 A time course for the fluorescence intensity of a reaction mixture of sesamol and NO is shown in Figure 7A. The fluorescence intensity sharply increased in a few seconds due to the dimerization of sesamol by NO, and this fluorescence enhancement was completed within 3 min after NO was mixed into the sesamol solution; at that point, the fluorescence intensity of the reaction mixture was 65-fold (46) The quantum yields of photodimerization from sesamol to the sesamol dimer at 25 °C were determined to be 0.027 and 0.0025 in water and acetonitrile, respectively, by photolysis experiments with UV irradiation at 295 nm. Although the production of sesamol dimer in this process is too small to interfere in assays under a room light, it is preferable that a sample solution should be protected from unnecessary lights. Experimental procedures for the photolysis experiments are described in the Supporting Information.

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higher than that of a sesamol solution without NO. Figure 7B indicates selectivity in the fluorogenic dimerization of sesamol. NO induced a fluorescence enhancement, whereas other nitrogencontaining species (NaNO2, NaNO3, NH3, N2O, N2, and Angeli’s salt) did not. NOBF4 yielded a small fluorescence increase (6.4% of that by NO; dashed line in Figure 7B), although it is not known to release radicals in aqueous solutions. Figure 7C displays an NO concentration-dependent fluorescence enhancement. A good linearity (r > 0.996) was obtained between fluorescence intensity and NO concentration in the range of 1-100 µM. The detection limit (S/N ) 3) was 154 ± 20 nM. Next, sesamol was applied to the detection of NO released from NO donors. Figure 7D shows a result for GSNO. A larger fluorescence enhancement was seen when the mixture of sesamol and GSNO was kept at a higher temperature (45 °C), supporting previous work showing that the release of NO from GSNO is stimulated with increasing temperature.30 A pH-dependent NO release from NOC7 was found in time course studies for the fluorescence intensity of mixtures of sesamol and NOC7 at pH 7.0 and 7.4 (Figure 7E). The more rapid fluorescence increase at pH 7.0 (closed circles) corresponded to an acid-catalyzed release of NO that is specific to NOC7.31 The half-lives of NOC7 under these conditions (22 °C in borate buffer) were found to be 9.5 ± 0.6 and 14 ± 1 min at pH 7.0 and 7.4, respectively, by evaluating eq 2 using the observed fluorescence intensities at 392 nm. Figure 7F shows the result for another NO donor, NOR1, with the opposite pH dependence.32 In a mixture of sesamol and NOR1, the fluorescence enhancement was faster under the more basic condition. From eq 2, the half-lives of NOR1 were 51 ± 5 and 26 ± 3 min at pH 7.0 and 7.4, respectively. The new fluorimetric detection method using sesamol was usable for biological samples. The results (Figure 7, parts G and H) indicate that biological components (20% human plasma and 20% HL-60 cell extract in borate buffer) did not lessen the ability of sesamol to detect NO. Equation 2 revealed that the half-lives of NOC7 at 37 °C were 4.1 ± 0.1 and 1.4 ± 0.1 min in 20% human plasma and 20% HL-60 cell extract, respectively. Similar NO monitoring by sesamol was successfully performed even in the presence of more condensed biological materials (i.e., 50% human

Figure 7. Fluorimetric NO detection by sesamol (1 mM) in aqueous buffer. (A) Time course for the fluorescence intensity of a reaction mixture of sesamol and NO (100 µM). (B) Fluorescence spectra of mixtures of sesamol and nitrogen-containing species (NO and NOBF4, 100 µM; N2O and N2, saturated; others, 1 mM). (C) Fluorescence spectra of reaction mixtures of sesamol and NO (0-100 µM). Inset: Relationship between the fluorescence intensity of the mixture at 392 nm and the concentration of NO. (D) Fluorescence intensity of a diluted mixture of sesamol and GSNO (1 mM). Temperature of the original mixture was varied between 5 and 45 °C. (E) Time course for the fluorescence intensity of mixtures of sesamol and NOC7 (100 µM) at pH 7.0 (closed circles) and 7.4 (open circles). Inset: Time course for the relative amount of NO released from NOC7. Vertical axis was e-kt in eq 2 in logarithmic scale. (F) Time course for the fluorescence intensity of mixtures of sesamol and NOR1 (100 µM) at pH 7.0 (closed circles) and 7.4 (open circles). (G) Fluorescence spectra of 20% human plasma containing sesamol and NOC7 (100 µM, solid line; 0 µM, dotted line). Reaction time and temperature were 30 min and 37 °C, respectively. Inset: Time course for the fluorescence intensity of a mixture of sesamol and NOC7 (100 µM). (H) Fluorescence spectra of 20% HL-60 cell extract containing sesamol and NOC7 (100 µM, solid line; 0 µM, dotted line). Reaction time and temperature were 15 min and 37 °C, respectively. Inset: Time course for the fluorescence intensity of a mixture of sesamol and NOC7 (100 µM). All fluorescence measurements were performed with excitation at 330 nm.

plasma and 50% HL-60 cell extract, see Figure S6 in the Supporting Information).

DISCUSSION Dimerization of Sesamol by Radicals. The present fluorimetric detection method is based on the radical-induced dimerization of sesamol. Figure 8 shows the proposed reaction mechanism. This dimerization of sesamol was likely triggered by the abstraction of a hydrogen atom from the phenolic hydroxyl group by radicals, as supported by the finding that no reaction occurred between radicals and MDA, which lacks the corresponding hydrogen atom. After hydrogen abstraction, the electron distribution of the resultant sesamolyl radical was changed into the most stable form. A previous ESR study by Nakagawa et al. revealed that the most stable and predominant sesamolyl radical featured the highest electron population at the carbon atom of the 6-position.47 Two sesamolyl radicals were then coupled at the reactive carbon atom to produce the sesamol dimer. In this process, the coupling of the sesamolyl radical with nonradical sesamol seems disfavored with reference to the dimerization of phenol.48 Overall, 2 mol of sesamol react with 2 mol of radical to give 1 mol of the sesamol dimer (Figure 8). From the proposed scheme and the peak areas in the chromatograms, the reaction yields of sesamol with tBuO•, •OH, and NO to produce sesamol dimer were calculated as 72.6% ± 3.7%, 35.0% ± 2.2%, and 50.5% ± 2.4%, respectively, in which the cleavage of di-t-butylperoxide and the Fenton reaction were assumed to be stoichiometric. These moderate reaction yields were not significantly influenced by the concentrations of sesamol or the radicals. Therefore, the dimerization of sesamol could be utilized for quantification of radicals in a wide range of samples. The low reactivity of O2•- toward sesamol was exceptional. The first possible reason for this is the low ability of O2•- to subtract a hydrogen atom. Although the reactive radicals tBuO•, •OH, •CH3, and NO can form the more stable molecules tBuOH, H2O, CH4, and HNO, respectively, by the addition of a hydrogen atom (cf., bond dissociation energies49), similar stabilization cannot be expected for O2•-. A related reason is the low stability of O2•-; when O2•- is generated in water, it is rapidly decomposed by a reaction with a proton, forming oxygen and H2O2.50 Fluorescence Properties of the Sesamol Dimer. Among the chemical species assumed by the sesamol dimer and sesamol, only the monoanionic sesamol dimer is strongly fluorescent (Figure 6 and Tables 1 and 2). This fluorescence property of the monoanionic dimer can be ascribed to its planar, rigid, and conjugated structure accompanied by intramolecular hydrogen bonding between the -OH and -O- groups (Figure 9A). Similar effects of intramolecular hydrogen bonding have been reported for 2,2′-dihydroxybiphenyl.51,52 The formation of an intramolecular hydrogen bond in the monoanionic sesamol dimer is confirmed by its significantly lower pKa1 value (6.3) compared with sesamol (47) Nakagawa, K.; Tero-Kubota, S.; Ikegami, Y.; Tsuchihashi, N. Photochem. Photobiol. 1994, 60, 199–204. (48) Fleming, I. Frontier Orbitals and Organic Chemical Reactions; WileyInterscience: Chichester, U.K., 1976; pp 195-198. (49) Blanksby, S. J.; Ellison, G. B. Acc. Chem. Res. 2003, 36, 255–263. (50) Sawyer, D. T.; Valentine, J. S. Acc. Chem. Res. 1981, 14, 393–400. (51) Kothainayaki, S.; Swaminathan, M. J. Photochem. Photobiol., A 1997, 102, 217–221. (52) Mohanty, J.; Pal, H.; Sapre, A. V. Bull. Chem. Soc. Jpn. 1999, 72, 2193– 2202.


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Figure 8. Proposed reaction mechanism for the radical-induced dimerization of sesamol.

Figure 9. (A) Planar structure of the monoanionic sesamol dimer with intramolecular hydrogen bonding. (B) Rotative structure of the monoanionic sesamol dimer with intermolecular hydrogen bonding with a protic solvent molecule (ROH).

(pKa 9.6). Intramolecular hydrogen bonding can stabilize the monoanionic sesamol dimer and consequently facilitate the dissociation of the first proton. As shown in Figure 9A, the intramolecular hydrogen bonding fixes the two aromatic rings into a plane of an extended conjugation, which favors a fluorescent process. By contrast, an intramolecular hydrogen bond cannot be formed in neutral or dianionic sesamol dimers. The effects of intramolecular hydrogen bonding were confirmed by the fluorescence properties of the monoanionic sesamol dimer in different organic solvents (Table 2). DMF (dielectric constant D ) 36.7, hydrogen-bond donor acidity53 R ) 0.00), ethanol (D ) 24.6, R ) 0.83), and TFE (D ) 26.7, R ) 1.51) showed comparable polarities but different abilities to form hydrogen bonds. In aprotic DMF, the monoanionic sesamol dimer strongly fluoresces because the two aromatic rings are fixed by intramolecular hydrogen bonding (Figure 9A). Conversely, in protic ethanol and TFE, the monoanionic sesamol dimer forms intermolecular hydrogen bonds with the solvent, and its aromatic rings are rotatable (Figure 9B). This competition between intramolecular and intermolecular hydrogen bonding reduces the fluorescence efficiency. Therefore, the fluorescence quantum yields (Φf) of the monoanionic sesamol dimer were ordered in opposition to the hydrogen-bond donor acidity of solvents: 0.78 (DMF) > 0.55 (ethanol) > 0.019 (TFE). Fluorimetric NO Detection Using Sesamol. Here, a new fluorimetric method for determining radicals by sesamol was applied to NO detection because there are no commercially available reagents that can react with radical NO to produce a fluorescent adduct (cf., 2,3-diaminonaphthalene54 and diaminofluoresceins22 based on reactions with oxidants of NO). The various separation-free assays for NO itself and NO donors (Figure 7) demonstrate the useful characteristics of sesamol as a fluorogenic reagent for NO, i.e., high solubility in water, rapid and specific (53) Kamlet, M. J.; Abboud, J.-L. M.; Abraham, M. H.; Taft, R. W. J. Org. Chem. 1983, 48, 2877–2887. (54) Wiersma, J. H. Anal. Lett. 1970, 3, 123–132. (55) Olasehinde, E. F.; Takeda, K.; Sakugawa, H. Anal. Chem. 2009, 81, 6843– 6850.

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capture of NO, NO concentration-dependent dimerization, high stability of the reaction product sesamol dimer, a strong and stable fluorescence signal from the product, and independent function in biological samples. It should be also noted that the reagent sesamol is highly stable in the solid state, providing easy handling. Even compared with diaminofluoresceins, the most successful fluorogenic reagents for NO developed so far, sesamol has great advantages, especially its ready availability and capability of detecting NO as is. However, we note its low applicability to bioimaging because of its relatively lower sensitivity and shorter wavelengths as a negative feature. Nevertheless, we can recommend sesamol as a fluorogenic reagent for NO in several applications, such as high-throughput analysis and screening of efficient NO donors and related drugs. As discussed above, the fluorogenic dimerization of sesamol is induced by not only NO, but other radicals too. In complex systems like living organisms and natural waters, where multiple kinds of radicals can exist simultaneously,2,55 a combined use of sesamol with another conventional reagent for a specific species (e.g., coumarin15,16 for •OH) will be effective for accurate quantification of radicals and comprehensive analysis of their dynamics. CONCLUSIONS We demonstrated a novel fluorimetric method for radical determination based on the radical-induced dimerization of the natural phenol sesamol. The reaction mechanism and availability of sesamol in laboratories are notable features of sesamol as a fluorogenic reagent that could enable widespread use of this method in the near future. This report describes the first utilization of sesamol and the sesamol dimer for fluorimetry. The potential inherent in their unique chemical and physical properties may well find applications in a wider scope, e.g., use of the sesamol dimer as a new “off-on-off” fluorescent pH sensor9,10 that emits only in the physiological pH range. ACKNOWLEDGMENT We thank Professor S. Tobita, Dr. T. Yoshihara, and Dr. A. Endo for valuable comments and discussions. S.U. thanks the Shorai Foundation for Science and Technology for their financial support. SUPPORTING INFORMATION AVAILABLE Experimental procedures for the reaction study with MDA and the photolysis study with sesamol and supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review August 31, 2009. Accepted January 5, 2010. AC9029778

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