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Detection of Enzymatically Generated Hydrogen Peroxide by Metal-Based Fluorescent Probe Yutaka Hitomi,* Toshiyuki Takeyasu, Takuzo Funabiki, and Masahito Kodera Department of Molecular Chemistry and Biochemistry, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan
bS Supporting Information ABSTRACT: We developed a metal-based fluorescent probe for H2O2 called MBFh1, which has an iron complex as a reaction site for H2O2 and a 3,7-dihydroxyphenoxazine derivative as the fluorescent reporter unit. The iron complex reacts quickly with H2O2 to form oxidants, and then the oxidants convert the closely appended nonfluorescent 3,7-dihydroxyphenoxazine moiety to resorufin in an intramolecular fashion. The quick response to H2O2 allows us to plot the enzymatic evolution of H2O2. A combination of N-acetyl-3,7-dihydroxyphenoxazine and horseradish peroxidase has been frequently used to detect enzymatically generated H2O2, but this method has interference with phenol derivatives. The use of MBFh1 overcomes this drawback.
H
ydrogen peroxide (H2O2) is generated as a result of substrate oxidations by some oxidoreductases.1 For example, glucose oxidase catalyzes the conversion of D-glucose to gluconolactone, which is accompanied by the generation of H2O2. By quantifying the amount of H2O2, interesting biological molecules such as specific enzymes can be indirectly quantified. A combination of N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) and horseradish peroxidase (HRP) is frequently used in assays of enzymes that produce H2O2 as well as their substrates such as D-glucose, uric acid, and acetylcholine.2,3 This method mainly relies on two characteristics of peroxidases: (1) specific and fast reaction with H2O2 and (2) subsequent generation of metal-based oxidants. H2O2-activated HRP rapidly converts nonfluorescent Amplex Red to highly fluorescent resorufin (jF = 0.754). However, Amplex Red cannot be oxidized by H2O2 itself. On the other hand, there are other types of fluorescent H2O2 probes that do not require the aid of peroxidases. In general, these fluorescent probes are small organic molecules and are converted from a weakly or nonfluorescent form to a strongly fluorescent form through the oxidation of probes by H2O2 itself.2,515 One exception is the Eutetracycline complex that exhibits strong luminescence when the water ligand is replaced with H2O2 without the occurrence of a redox reaction.16 Wolfbeis et al. successfully applied the Eu-based H2O2 probe to detect enzymatically produced H2O2.16,17 Recently, Chang and co-workers succeeded in developing a new class of fluorescent probes triggered by the oxidative conversion of phenyl boronic esters to phenol derivatives by H2O2.8 It has been shown that boronate-based fluorescent probes respond to H2O2 with high selectivity, but the response is rather slow (t1/2 ∼ 30 min). Very recently, Nagano et al. reported a fluorescent r 2011 American Chemical Society
probe having benzil as a reactive moiety for H2O2; the reactivity of the fluorescent probe is comparable to that of boronate-based probes.5 Herein, we present the synthesis and properties of a metalbased fluorescent probe for H2O2 called MBFh1, which has an iron complex as a reaction site for H2O2 and a 3,7-dihydroxyphenoxazine derivative as the fluorescent reporter unit. Scheme 1 shows the synthesis of MBFh1 and our strategy for H2O2 detection, wherein the iron complex reacts quickly with H2O2 to form oxidants and then the oxidants convert the closely appended nonfluorescent 3,7-dihydroxyphenoxazine moiety to resorufin in an intramolecular fashion. MBFh1 was synthesized using the resazurin sodium salt as the starting material. Reduction of the resazurin sodium salt was followed by selective O-acetylation, and subsequent treatment with bromoacetyl bromide afforded N-bromoacetyl-3,7-diacetoxyphenoxazine 1. Further reaction of 1 with N,N,N0 -tris (2-pyridyl-methyl)propane-1,3-diamine followed by mild hydrolysis yielded a 3,7-dihydroxyphenoxazine-appended ligand 2. Finally, the desired iron complex, MBFh1, was prepared by a simple metalation of 2 with Fe(OTf)2. The elemental analysis and UVvis spectroscopy indicate that MBFh1 contains an iron(III) center (Supporting Information). A rapid addition of H2O2 to an aqueous solution of MBFh1 caused the solution to turn pink and simultaneously caused bright red fluorescence (Figure 1). The absorption and fluorescence spectra of the reaction mixture were identical to those Received: September 24, 2011 Accepted: November 16, 2011 Published: November 16, 2011 9213
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Scheme 1. Synthetic Scheme of MBFh1 and the Formation of Resorufin by the Reaction of MBFh1 with H2O2a
Figure 2. (a) HPLC chromatograms of the reaction mixture (upper) and resorufin (below). (b) ESI-TOF MS spectrum for the reaction mixture (upper). The below panel shows the simulated mass peaks for the hydrolyzed iron complex, [FeIII(3)]2+ whose structure is shown in Scheme 1, (m/z 460.1 {[FeIII(3)]2+ + e}+).
a (a) Zn, AcOH; (b) Ac2O, DMAP; (c) bromoacetyl bromide, K2CO3; (d) N,N,N0 -tris(2-pyridylmethyl)propane-1,3-diamine, K2CO3, MeCN; (e) Na2SO3, dioxaneH2O (1:1); (f) Fe(OTf)2, MeOH, MeCN.
Figure 1. (a) Fluorescence and UVvis spectra of MBFh1 (dashed line) and the products upon addition of H2O2 (100 equiv) (solid line). (b) Photographs of the cuvettes containing MBFh1 before (left) and after (right) addition of H2O2.
observed for authentic resorufin, which suggests the reaction proceeded according to our strategy in Scheme 1. The generation of both resorufin and a hydrolyzed iron complex, [FeIII(3)]2+, was confirmed by high-performance liquid chromatography and electrospray ionizationtandem mass spectrometry analysis, respectively (Figure 2 and Figure S2 in the Supporting Information). A possible mechanism is proposed in Scheme 2, on the basis of a proposed mechanism for the oxidative conversion of Amplex Red to resorufin18 and recent mechanistic studies of the oxidative chemistry of the mononuclear iron complex with H2O2.19 First, MBFh1 reacts with H2O2 to form [(2)FeIII(OOH)]2+. There are three possible reaction pathways proposed for the reactivity of Fe(III)OOH species in oxidation reactions: (a) homolytic cleavage of the OO bond, leading to an Fe(IV)oxo species and a hydroxyl radical; (b) heterolytic OO cleavage, generating a Fe(V)oxo species; and (c) a direct H-atom abstraction by Fe(III)OOH species. The direct H-atom abstraction has been proposed for DNA cleavage by Fe(III)OOH species of
bleomycins.20 In the transition state of the direct H-atom abstraction, the OO bond should be aligned with the H-atom to be abstracted. In the case of MBFh1, however, such an alignment is likely impossible due to the geometry between the H-atoms of two phenolic OH groups in 3,7-dihydroxyphenoxazine, which are the most suspicious H-atoms to be abstracted, and the distal O-atom of the hydroperoxo group. In either the homolytic or heterolytic pathway, the generated oxidants can oxidize the 3,7-dihydroxyphenoxazine moiety by two electrons in an intramolecular fashion due to the linker between the iron(III) complex and 3,7-dihydroxyphenoxazine,18 yielding a cationic amide group. Hydrolysis of the cationic amide group by an iron(III)-bound hydroxo group or an unbound water molecule finally gives resorufin and a hydrolyzed iron complex, [FeIII(3)]2+. The reaction rate of MBFh1 with H2O2 was evaluated by fluorescence spectral changes in the reaction of MBFh1 using varied concentrations of H2O2 (Figure 3). The reaction rate of 38.1 ( 0.2 M1 s1 is still below than that for HRP of 0.85 107 M1 s121 but greatly improved above the values reported for fluorescent H2O2 probes.5,8 Next, we examined the increase in fluorescence intensity in the reaction of MBFh1 using various reactive oxygen species (ROS) including H2O2, t-butyl peroxide, hypochlorite anion, and superoxide anion radical (Figure S3 in the Supporting Information). Although Amplex Red showed a negligible response to all ROS we studied, MBFh1 displayed a significant increase in the fluorescence intensity in response to H2O2 and t-butyl peroxide and a moderate increase with the superoxide anion radical. At this point, we cannot give a detailed mechanism for the reactivity of MBFh1 toward the superoxide anion radical, but we speculate that superoxide-derived metal-based oxidants should be involved because Amplex Red is not oxidized by the superoxide anion radical. The limit of detection (LOD) was evaluated to be 3.2 μM from the data at 20 s in Figure 3 using the 3-sigma method (Figure S4 in the Supporting Information). Thus, the LOD value is comparable to those reported for the colorimetric method using N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt and N-methyl-N-phenyl-3-sulfophenylenediamine sodium salt (LOD = 1.0 μM),22 and the europium-ion-based luminescent probe (LOD = 1.8 μM),16 although it was reported that the Amplex Red/HRP coupling method can detect H2O2 at concentration as low as 50 nM.3 9214
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Scheme 2. Possible Mechanism of the Resorufin Generation by the Reaction of MBFh1 with H2O2
Figure 4. Inhibitory effect of phenolic compounds on the resorufin formation in the reaction of MBFh1 (a, right) and HRP/Amplex Red (b, left) with H2O2. The error bars indicate the standard deviation from three independent experiments. Conditions: 20 mM MOPS buffer (pH 7.2, 2.5% DMSO) at 25 °C, [phenol derivative] = 100 μM, [MBFh1] = 5 μM or [Amplex Red] = 5 μM, [HRP] = 22 nM.
Figure 3. Fluorescence response curve of MBFh1 upon addition of H2O2. Conditions: 20 mM MOPS buffer (pH 7.2, 2.5% DMSO) at 25 °C, [MBFh1] = 5 μM. Fluorescence intensity (λex = 570 nm) was followed at 590 nm.
Because HRP can oxidize a wide range of phenolic compounds, H2O2-activated HRP would oxidize phenolic compounds rather than Amplex Red.23 Therefore, we examined the H2O2-promoted fluorescence increase when it coexisted with some biorelevant phenolic compounds using our MBFh1 and the Amplex Red/HRP coupling method (Figure 4). The advantage of the MBFh1 method became evident from this experiment. The Amplex Red/HRP coupling method showed less fluorescence in the presence of a phenolic compound, such as tyramine, while the fluorescence from the MBFh1 coupling method was not affected or less affected. We then applied MBFh1 to detect H2O2 generated by glucose oxidase, which is an FAD-dependent H2O2 generating enzyme. Addition of glucose oxidase to a D-glucose solution containing MBFh1 quickly displayed a fluorescence increase. Addition of catalase in the middle of the reaction resulted in complete suppression of the fluorescence increase, which clearly demonstrated the ability of MBFh1 to detect enzymatically generated H2O2 (Figure 5a). The fluorescence intensity depends on the D-glucose concentration. Our MBFh1 is sensitive enough to detect very low concentrations of D-glucose (down to 2.5 μM) (Figure 5b). Moreover, the response curve of MBFh1 did not change even in the presence of tyramine, while a significant
Figure 5. (a) Fluorogenic detection of enzymatically generated H2O2 by MBFh1. Red trace: 5 μM MBFh1, 125 μM D-glucose, 8 μg/mL glucose oxidase. Blue trace: addition of catalase after 100 s. (b) Plot of the fluorescence intensities as a function of glucose concentration. Conditions: 20 mM MOPS buffer (pH 7.2, 2.5% DMSO) at 25 °C.
decrease in the fluorescence intensity was observed in the case of the HRP/Amplex Red method (Figure S5).
’ CONCLUSIONS We developed a metal-based fluorescent probe for the generation of H2O2. The new fluorescent H2O2 probe, MBFh1, causes a red fluorescence faster than organic molecule-based H2O2 probes because of the presence of 3,7-dihydroxyphenoxazine in the vicinity of the iron complex. MBFh1 can detect enzymatically generated H2O2 even in the presence of phenol derivatives. We are currently developing a family of MBFh with a higher specificity toward H2O2. 9215
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’ EXPERIMENTAL SECTION UVVisible and Fluorescence Measurements. UVvis spectra were taken on an Agilent 8543 UVvis spectrometer. Fluorescence spectra were taken with λex = 570 nm on a Hitachi F-7000 spectrophotometer. Conditions: 20 mM MOPS buffer (pH 7.2, 2.5% DMSO) at 25 °C, [MBFh1] = 50 and 5 μM for UVvis and fluorescence measurements, respectively. Fluorescence intensity was followed at 590 nm for the kinetic studies. Products Analysis. The products in the reaction of MBFh1 with H2O2 were analyzed on a Shimadzu LC-10AT highperformance liquid chromatography equipped with a reverse phase column (Wako Sil-II 5C18, 4.6 mm 250 mm) and a photodiode array detector Shimadzu SPD-M10AVP (eluent, MeCN/H2O = 90/10; flow rate, 5.0 mL/min; monitored at 570 nm) and on a JEOL JMS-T100CS ESI-TOF spectrometer. An aliquot of H2O2 (10 equiv) was added to a 5 μM solution of MBFh1 in MeCN/H2O (v/v = 1/9) under aerobic conditions. The mixture was stirred for 1 min at room temperature. Effect of Phenolic Compounds. An aliquot of H2O2 (1 equiv) was added to a 5 μM solution of MBFh1 or to a 5 μM solution of Amplex Red containing HRP (22 nM) in the presence or absence of phenol derivatives (100 μM). The percentage of resorufin is expressed relative to the percentage without the phenolic compounds. Fluorogenic Detection of H2O2 Generated by Glucose Oxidase. An aliquot of glucose oxidase (8 μg/mL) was added to a 5 μM solution of MBFh1 containing D-glucose (2125 μM) in 20 mM MOPS (pH 7.2, 2.5% DMSO) at 25 °C. In order to decompose H2O2, an aliquot of catalase (80 μg/mL) was added to the above solution after 100 s. Fluorescence intensities were followed at 590 nm (λex = 570 nm).
’ ASSOCIATED CONTENT
bS
Supporting Information. Synthesis procedure for MBFh1; computationally optimized structure of MBFh1; reactivity of MBFh1 and AmplexRed against oxidants; and inhibitory effect of tyramine on the fluorometirc detection of enzymatically generated H2O2 by using the MBFh1 and HRP/Amplex Red methods. This material is available free of charge via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Fax: +81-774-65-6801. Phone: +81-774-65-7437.
’ ACKNOWLEDGMENT This work was supporting by “Creating Research Center for Advanced Molecular Biochemistry”, Strategic Development of Research Infrastructure for Private Universities, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. ’ REFERENCES (1) Massey, V.; Strickland, S.; Mayhew, S. G.; Howell, L. G.; Engel, P. C.; Matthews, R. G.; Schuman, M.; Sullivan, P. A. Biochem. Biophys. Res. Commun. 1969, 36, 891–897. (2) Haugland, R. P. Handbook of Fluorescent Probes and Research Products; Molecular Probes, Inc.: Eugene, OR, 2002. (3) Zhou, M.; Diwu, Z.; Panchuk-Voloshina, N.; Haugland, R. P. Anal. Biochem. 1997, 253, 162–168. 9216
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