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Oxazole-based high resolution ratiometric fluorescent probes for hydrogen peroxide detection
Accepted Manuscript Title: Oxazole-based high resolution ratiometric fluorescent probes for hydrogen peroxide detection Authors: Xihui Li, Jiaming Li, Yuhan Tao, Zhixing Peng, Ping Lu, Yanguang Wang PII: DOI: Reference:
S0925-4005(17)30477-X http://dx.doi.org/doi:10.1016/j.snb.2017.03.060 SNB 21972
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
30-5-2016 3-3-2017 13-3-2017
Please cite this article as: Xihui Li, Jiaming Li, Yuhan Tao, Zhixing Peng, Ping Lu, Yanguang Wang, Oxazole-based high resolution ratiometric fluorescent probes for hydrogen peroxide detection, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.03.060 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Oxazole-based high resolution ratiometric fluorescent probes for hydrogen peroxide detection
Xihui Li, Jiaming Li, Yuhan Tao, Zhixing Peng, Ping Lu* and Yanguang Wang*
Department of Chemistry, Zhejiang University, Hangzhou 310027, China [email protected]; [email protected]
Graphical Abstract
Research Highlights
Two novel fluorescent probes, PNOBpin and B-BOBpin, were synthesized and characterized for their applications for rapid detection of H2O2. In the presence of base, the probes provide remarkable changes in optical properties with extremely fast response towards H2O2 based on the cleavage of C-O bond and the principle of excited state intramolecular proton transfer (ESIPT) or photo-induced electron transfer (PET). B-BOBpin is the first example of BODIPY derivative applied for H2O2 detection.
Abstract: Hydrogen peroxide (H2O2) has been associated as the most important member of reactive oxygen species (ROS) for a long time. Two novel fluorescent probes (P1 and P2) are synthesized and characterized for rapid detection of H2O2. In the presence of base, the probes provide remarkable changes in optical properties with extremely fast response towards H2O2 based on the cleavage of C-O bond and the principle of excited state intramolecular proton transfer (ESIPT) or photo-induced electron transfer (PET).
Keywords: fluorescent sensors, excited state intramolecular proton transfer, photo-induced electron transfer, hydrogen peroxide
1.
Introduction Reactive Oxygen Species (ROS) are generated during natural metabolism in living organisms and
play important roles in cell signaling and homeostasis. However, the abnormal ROS levels may result in significant damage to cells and cause numerous diseases [1]. This is a particular aspect of oxidative stress, which means an imbalance between the production of ROS and the ability of the system to counteract their harmful effects. As a major ROS in aerobic organism, hydrogen peroxide is involved in normal cellular signal transduction as a second messenger, which widely exists in many physiological and pathological processes [2]. The major endogenous source of H 2O2 is the process of O2 reduction by NADPH oxidase complex (Nox) with cellular stimulation in nearly all living cells [3]. It is believed that excessive H2O2 production is associated with aging and neurodegenerative diseases such as Parkinson’s and Alzheimer’s diseases [4]. Therefore, many efforts have been made to detect H2O2 accurately in cells. Compared with traditional approaches, fluorescent probes are among the most effective tools for imaging H2O2 with advantages of higher sensitivity, better selectivity, simpler manipulation and less invasiveness. The design strategies are based on specific reactions between recognition groups and H2O2 that give products with different photophysical properties such as oxidative hydrolysis of boronates [5], phenylphosphates [6] and sulfonates [7] and so on. The mechanisms usually involve efficient Förster resonance energy transfer (FRET), intramolecular charge transfer (ICT), photo-induced electron transfer (PET) and induce enormous change in fluorescence emission of fluorophore. In comparison with reported probes, fluorescent chemosensors based on excited-state intramolecular proton transfer (ESIPT) are leading a new trend owing to their unique photophysical property. The ESIPT molecules are usually oxazole, oxadiazole, and imidazole-based and normally more stable in enol form (E form) in the ground state. Once photo-excited, a fast proton transfer takes place from a hydroxyl (or amino) unit to a carbonyl oxygen (or imine nitrogen) to generate keto form (K form) [8]. Therefore, the absorption of E form and the emission of the excited K form (K* form) provide an extremely large Stokes shift that could effectively avoid self-absorption, giving an ideal solution for fluorescence sensing.
The key challenge of fluorescence bioimaging for H2O2 is to detect H2O2 specifically over other biologically relevant ROS. In recent years, a series of well-devised fluorescent chemosensors specific for H2O2 have been developed and help to make considerable progress in studying H 2O2 biology by using these probes [9]. Yet most of these probes are facing a common problem that they usually need a long reaction time to attain signal output which may have an influence on real-time monitoring and quantification. Here we present two novel fluorescent chemosensors (P1 and P2) with high sensitivity and rapid response towards H2O2. Motivated by the photophysical property and dramatic ESIPT phenomenon of 3-(4,5-diphenyloxazol-2-yl)- naphthalen-2-ol (HPNO) [10], we choose it as a fluorophore and protect the hydroxyl group with the boronated-based benzyl group in order to inhibit ESIPT process and detect H2O2 specifically. Thus, P1 is designed and synthesized (Scheme 1). Considering that a longer emission wavelength is desirable for bio-imaging in living cells [11], we combine 2-(2’-hydroxyphenyl) benzoxazole (HBO) as an ESIPT moiety, boron-dipyrromethene (BODIPY) dye as a red-shifted fluorophore [12], and phenylboronate as a cleavable group towards hydrogen peroxide and design a new probe, named as P2 (Scheme 2). Interestingly, unlike the emission peak shift of P1, P2 shows a turn-off fluorescence change towards hydrogen peroxide in the presence of base.
2.
Experimental Section
2.1 General All the reagents and deuterated solvents were purchased from Sigma-Aldrich, Acros Oganics and Alfa Aesar, and were used without further purification unless otherwise. HPNO and 3-(acetyloxy)-4(2-benzoxazolyl)-benzaldehyde (BOAc) were synthesized following literature protocol [13]. Organic solvents were purchased from Sinopharm and Tansoole then dried and purified before use as specified by standard procedures [14]. Analytical thin layer chromatography (TLC) was performed with silica gel HSGF254. Chromatography was performed on silica gel (300-400 mesh) by standard techniques eluting with solvents as indicated. All compounds were fully characterized. 1H NMR, 13C NMR spectra were obtained on a Bruker DMX-500 MHz or AVANCE 2B/400 MHz spectrometer operating in the FT mode. Unless otherwise noted, all the NMR spectra were recorded at room temperature. Coupling constants (J) were reported in hertz unit (Hz). Chemical shifts (in ppm) were referenced to tetramethylsilane (δ = 0 ppm) in CDCl 3. 13C NMR spectra were obtained on 100 MHz or 125 MHz and
chemical shifts were reported in ppm referenced to the central line of a triplet at 77.0 ppm of CDCl3. All high-resolution mass spectra (HRMS) were performed on a Bruker MALDI-TOF/TOF-MS. Fluorescence spectra were recorded by RF-5301pc spectrofluorometer (Shimadzu, Kyoto, Japan) equipped with a xenon lamp. UV spectra were recorded on Shimadzu UV-2450 spectrophotometer. pH measurements were performed using a Model PHS-3C meter.
2.2 Photophysical Measurements Twice-distilled water and spectroscopic grade MeOH were used for spectroscopic studies. Unless otherwise noted, all the absorption and emission spectra were recorded at room temperature. The slit width of fluorescence excitation is 3.0 nm and emission is 5.0 nm. Hydroxyl radicals and tert-butoxy radicals (•OtBu) were generated by reaction of Fe2+ with H2O2 or TBHP respectively. Singlet oxygen (1O2) was generated from ClO- and H2O2 [9c]. Various analytes represented by H2O2, TBHP, ClO-, OH•, 1
O2, and •OtBu were added to the solutions of P1 (20 μM) and P2 (5 μM), respectively. Slightly
variations in the pH of the solutions were achieved by adding minimal volumes of NaOH or HCl aqueous solution. And the total addition of H2O was less than 4% (v/v).
Determination of the detection limit The detection limit was calculated based on the fluorescence titration curve of P1 and P2 in the presence of H2O2 (Figure 4b and Figure 5b, respectively). The fluorescence intensity of P1 and P2 were measured by five times and the standard deviation of blank measurement were achieved. The detection limit was calculated with the following equation [15]: Detection limit = 3σ/k Where σ is the standard deviation of blank measurement, k is the slop between the fluorescence intensity versus H2O2 concentrations (Figure S8).
2.3 Calculations Density functional theory (DFT) calculations were carried out in the gas phase using the Gaussian 09 [16] quantum-chemical package. The geometry optimizations for the ground state of E form of B-HBO, P2 and B-BO-Na+ were performed using B3LYP function with the 6-31G basis set. Vibrational frequency calculations at the same level were performed for the obtained structures to confirm the
global minimum.
2.4 Synthesis P1: HPNO (69 mg, 0.19 mmol), potassium carbonate (79 mg, 0.57 mmol) and tetrabutylammonium iodide (21 mg, 0.06 mmol) were dissolved into 10 mL DMF under N2. 4-Bromomethylphenylboronic acid pinacol ester (79 mg, 0.27 mmol), dissolved in 10 mL DMF, was added into the solution dropwisely. The mixture was stirred at room temperature for 16 h. The crude product was collected by filtration, washed with water, dried by suction. The residue was purified through column chromatography on silica gel column (hexane: ethyl acetate: 20:1-5:1 v/v as eluent) to afford 53 mg P1 in 48% yield. White solid, m.p. 166-169 oC. 1H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H), 7.80-7.76 (m, 3H), 7.69-7.64 (m, 3H), 7.57 (d, 2H, J = 7.6 Hz), 7.41-7.38 (m, 3H), 7.35-7.25 (m, 5H), 7.20-7.18 (m, 3H), 5.22 (s, 2H), 1.28(s, 12H) ppm; 13C NMR (100 MHz, CDCl3) δ 159.1, 154.2, 145.9, 139.7, 136.4, 135.3, 135.1,
132.8, 131.2, 129.0, 128.64, 128.56, 128.4, 128.3, 128.2, 127.8, 127.2, 126.5, 124.5,
118.1, 107.9, 83.9, 70.7, 25.0. HRMS: Found: 579.2581; Calcd for C 38H34BNO4: 579.2584. B-BOAc: 2,4-Dimethylpyrrole (215 mg, 2.2 mmol) and BOAc (281 mg, 1 mmol) were dissolved in CH2Cl2 with a catalytic amount of trifluoroacetic acid (TFA) (1-2 drops). The mixture was stirred for 16 h at room temperature. Then a solution of 2,3- dichloro-5,6-dicyanobenzoquinone (DDQ) (227 mg, 1 mmol) in CH2Cl2 (30 mL) was added dropwisely, and the mixture was stirred for 15 min. Finally, BF3-OEt2 (3 mL) and triethylamine (3 mL) were added, and the mixture was stirred for 3 h at room temperature. The crude mixture was diluted with CH2Cl2 and washed with H2O. The organic extracts were dried over MgSO4, filtered and evaporated under reduced pressure. Flash chromatography (hexane: ethyl acetate: 5:1, v/v) afforded 179 mg (0.36 mmol) of B-BOAc as a red solid in 36% yield. m.p. > 250 oC. 1H NMR (400 MHz, CDCl3) δ 8.44 (d, 1H, J = 8.0 Hz), 7.81 – 7.75 (m, 1H), 7.59 (dd, 1H, J = 6.0, 3.1 Hz), 7.43 – 7.36 (m, 3H), 7.22 (d, 1H, J = 1.5 Hz), 6.01 (s, 2H), 2.57 (s, 6H), 2.50 (s, 3H), 1.53 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 169.7, 159.1, 156.3, 150.2, 150.1, 143.1, 141.9, 139.3, 138.6, 131.0, 130.9, 126.3, 125.9, 124.9, 124.6, 121.6, 121.2, 120.5, 119.4, 110.8, 110.6, 21.3, 14.7, 14.7, 14.6. HRMS: Found: 499.1886; Calcd for C28H24BF2N3O3: 499.1879.
B-HBO: B-BOAc (179 mg, 0.36 mmol) and K2CO3 (149 mg, 1.08 mmol) was disolved in mixed solution of CH2Cl2 and EtOH (1:1 v/v, 10 mL). The mixture was refluxed for 2 hours, then filtered and evaporated under reduced pressure. Flash chromatography (hexane: ethyl acetate 5:1, v/v) afforded 156 mg (0.34 mmol) of B-HBO as red solid in 94% yield. m.p. > 250 oC. 1H NMR (500 MHz, CDCl3) δ 11.68 (s, 1H), 8.17 (d, 1H, J = 8.0 Hz), 7.78 (dd, 1H, J = 6.2, 2.8 Hz), 7.65 (dd, 1H, J = 6.4, 2.8 Hz), 7.47 – 7.39 (m, 2H), 7.11 (s, 1H), 6.97 (d, 1H, J = 8.0 Hz), 6.00 (s, 2H), 2.57 (s, 6H), 1.55 (s, 6H). 13C NMR (125 MHz, CDCl3) δ 162.2, 159.2, 155.9, 149.2, 143.0, 140.3, 140.1, 139.9, 130.8, 128.0, 125.9, 125.3, 121.4, 119.5, 119.4, 117.4, 111.1, 110.8, 14.7, 14.6. HRMS: Found: 457.1775. Calcd for C26H22BF2N3O2: 457.1773. P2: A mixture of compound B-HBO (156 mg, 0.34 mmol), 4-(bromomethyl)benzene boronic acid pinacol ester (118 mg, 0.4 mmol), and K2CO3 (140 mg, 1.02 mmol) in dry N,N-dimethylformamide (DMF, 5 mL) was heated at 80 oC for 16 h under N2. After cooling, the mixture was filtered to remove salts. Then cold water (30 mL) was added to the mixture and extracted with DCM (10 mL×3). The organic solutions were combined, washed with water and brine, and dried with Na 2SO4. The solvents were evaporated to generate the crude product, which was purified by flash chromatography (silica gel, hexane/EA = 1:5) to give the desired product as red solid (92 mg, 40%). m.p. > 250 oC. 1H NMR (400 MHz, CDCl3) δ 8.33 (d, 1H, J = 8.4 Hz), 7.86 (dd, 1H, J = 6.1, 2.9 Hz), 7.81 (d, 2H, J = 8.0 Hz,), 7.67 – 7.61 (m, 1H), 7.57 (d, 2H, J = 8.0 Hz), 7.43 – 7.38 (m, 2H), 7.07 (d, 2H, J = 7.0 Hz), 5.98 (s, 2H), 5.34 (s, 2H), 2.57 (s, 6H), 1.41 (s, 6H), 1.35 (s, 12H).
C NMR (100 MHz, CDCl3) δ 161.1, 157.8,
13
156.1, 150.7, 143.0, 141.8, 139.8, 139.5, 139.1, 135.1, 132.3, 130.9, 128.5, 126.5, 125.9, 125.4, 124.7, 121.4, 121.0, 120.2, 117.5, 113.8, 110.6, 110.0, 83.9, 70.7, 24.9, 14.7, 14.4. HRMS Found: 674.2892. Calcd for [M+H]+ C39H40B2F2N3O4: 674.3168.
3
Result and discussion Compounds P1 and P2 were synthesized by the procedures as shown in Schemes 1 and 2. Their
structures along with HPNO, BOAc, B-BOAc, and B-HBO were comfirmed by 1H NMR, 13C NMR, and HRMS (see SI). Absorption and emission spectra of HPNO and P1 in different solvents are shown in Figure S1. Absorption spectra of HPNO and P1 are essentially the same no matter whether HPNO is alkylated or not, while emission spectra of P1 are also identical. The siginificant alteration was observed for the
emission of HPNO in different solvents. In dichloromethane (DCM), dual emission is presented (Figure S1b). HPNO was derived from the backbone of 2-(oxazol-2-yl)phenol. The principle of ESIPT process is displayed in Scheme 3. HPNO remains stable in enol form (E Form) in the ground state. Once photoexcited, tautoumerism occurs and creates keto form in excited state (K* form). Due to the increment of the efficient conjugation length in the K form, a narrower energy gap between HOMO and LUMO is observed for K-form. As shown in Figure 1, the emission of HPNO is highly impacted by the solvent properties. In polar solvents, such as methanol (MeOH) and N,N-dimethyl foramide (DMF), the emission of E form at 409 nm is dominant. Emission of K form at 602 nm is not shown in these solvents since the hydrogen bonding between the E form of HPNO and the solvent molecules helps to stabilize the E form and inhibits the process of intramolecular proton transfer. Thereofore, emission of K form in MeOH and DMF is vanished. In other solvents like dichloromethane (DCM), the interaction between the E-form of HPNO and the solvent is neglectable. Thus, a full ESIPT process is triggerd by the photoexcitation via intramolecular hydrogen bonding. As a result, dual emission of HPNO in DCM, respectively from E* form and K* form, is observed. In the solid state [17], the restriction of the intramolecular rotation helps in maintaining optimal geometry for intramolecular H-bonds which is a key factor for an efficient ESIPT process; meanwhile, the omission of intermolecular H-bonding with solvent molecules also causes the enhancement of K* emission. Both of the two mechanisms are considered responsible for solid state emission of HPNO in K* form resulting in red-light as photoed in Figure S1e. When it comes to B-HBO, the UV-spectrum show two independant absorption bands around 250-400 nm and 500 nm. As the solvent polarity increases, the absorption band for the larger energy gap is enhanced significantly (Figure S2a). Previous work inspires us that the absorption band at 250-400 nm is assigned to the π–π∗ band of HBO moiety in E form [18] whereas the absorption band for the lower energy gap at 500 nm is the characteristic absorption of BODIPY [12a]. The enhancement of the absorption band for the larger energy gap could be ascribed to the intermolecular hydrogen bonding between HBO and solvent. Meanwhile, the absorption of BODIPY is hardly impacted by the solvent polarity. This phenomenon implies the conjugatively uncoupling between HBO and BODIPY. The two moieties are twisted owning to the methyl groups at positions C-1 and C-7 of BODIPY. This angulation could be supported by the X-ray analysis of a structurally similar
compound [19] and the calculated result (Figure S3). The calculated dihedral angle for the plane of HBO and the plane of BODIPY is 90.03°, saying that they are held perpendicular to one another and decoupled. Although absorption of B-HBO is strongly influenced by solvent polarity, emissions of B-HBO in different solvents are almost the same and emit light at 514 nm which is the typical emission of the BODIPY. When B-HBO is alkylated and derived into P2, no hydroxyl group exists. As a net result, the absorption and emission spectra of P2 are essentially the same in all kinds of the solvents (Figure S2). For a clear discussion in the following sections, the absorption and emission spectra of the pairs of HPNO/P1 and B-HBO/P2 in methanol are shown in Figure 2. Then we studied the deprotonation of HPNO and B-HBO in methanol. HPNO absorbed light at 330 nm with molar absorption coefficient of 2.17×10 4 cm-1 M-1. Upon the addition of NaOH, the absorbance around 330 nm became weak and a concomitant increment of a new band at 425 nm appeared, with two isosbestic points at 307 nm and 388 nm. The titration of NaOH induced an increase in fluorescence intensity at 546 nm as well. The apparent pKa value of HPNO in methanol was determined to be 10.5 ± 0.4, representing the deprotonation capability of the subunits (Figure S4). Figure 3 shows the absorbance and fluorescence titration of B-HBO dissolved in MeOH by gradual addition of NaOH. Free B-HBO absorbs light at 295 nm, 321 nm and 500 nm with molar absorption coefficient of 4.76×104 cm-1 M-1, 5.16×104 cm-1 M-1 and 1.23×105 cm-1 M-1, respectively. As the gradual addition of NaOH increases to an end concentration of 10 mM, a new absorption band at 361 nm appears whereas the original ones at 295 nm and 321 nm decrease. Two distinct isosbestic points are found to be 269 nm and 341 nm. Emission intensity of B-HBO at 514 nm decreases significantly and a new peak arises at 440 nm triggered by adding NaOH, while the emission intensity at 481 nm remain the same. Notably the colorimetric change under basic condition can even be observed by naked eye which implies that the B-HBO might be used as a promising chemosensor for pH detection. Through fluorescence titration experiments we also determine the apparent pKa* value of B-HBO in methanol to be 10.9 ± 0.3 (Figure 3b inset). The absorption and emission spectra of P1 and P2 are quite simple without notable change in different solvent as we noted above (Figures S1c, d and S2c, d). Along with the gradul addition of NaOH, absorption and emission spectra of these two probes kept the same without any variation (Figure S5). It indicats that both P1 and P2 are stable in the alkaline environment. Further, we test the
response of P1 and P2 to H2O2 under neutral condition. Both of them are consistent too (Figure S6). As we further combine these two factors (NaOH and H2O2) to irritate the probes, fast and apparent alterations in absorption and emission spectra are observed. Both probes present the character of AND logic gate with two inputs, NaOH and H2O2. Methanol solution of P1 with 20 mM NaOH absorbed light at 311 nm with the absorption molar coefficient being 1.14×104 cm-1 M-1. The band decreased quickly by the addtion of H2O2, and a new band at 425 nm arised. The emission at 408 nm quenched concurrently as a new peak at 546 nm came into being, with unchanged emission intensity at 512 nm (Figure 4). Meanwhile the solution color under UV irritation changed from violet blue into yellow-green. And the ratiometric fluorescence (I546/I408) showed that the oxidation process almost finished when the concentration of H2O2 achieved 5 equivalence (Figure 4 inset). The detection limit of P1 was calculated to be 34 nM based on a signal-to-noise ratio of 3 (S/N = 3). The possible mechanism for this change is shown in Scheme 4. At the beginning, P1 is oxidized in the presence of hydrogen peroxide and results in the formation of borate intermediate A. With further titration of hydroxide anion, intermediate A hydrolyzes into phenolate anion B. An electron pushing from phenolate anion leads to the cleavge of C-O bond and finally generates PNO- species which emits light at 546 nm. The H2O2 titration experiment of P2 showed a quenching process instead of peak shift like P1. Methanol solution of P2 with 5 mM NaOH obsorbed at 310 nm and 500 nm with absorption molar coefficients being 2.10×104 cm-1 M-1 and 7.38×104 cm-1 M-1, respectively. As H2O2 was gradually added into the solution, a new absorption peak at 357 nm appeared, whereas the absorption band at 500 nm kept the same. At the same time the original emission peak at 512 nm quenched tremendously (Figure 5). The detection limit of P2 was calculated to be 13 nM (S/N = 3). The chemical process responsible for the sensory mechanismis is similar to P1 as shown in Scheme 4. However, once phenolate is formed, a photo-induced electron process (PET) takes place and causes the turn-off fluorescence due to the matched HOMO and LUMO levels of PNO-Na+ and BODIPY (Figure 6) [20]. For the detailed photophysical properties including band gap, Stokes shift, Φ FL and fluorescence lifetime data are listed in Table 1. As expected, both P1 and P2 behaved a rapid response towards H2O2 under alkaline condition within 20 min (Figure S7). Using pseudo-first-order kinetcs (P1: 20 μM, 20 mM NaOH, 2 mM H2O2; P2: 5 μM, 5 mM NaOH, 0.4 mM H2O2), the observed rate constant k for H2O2 detection using P1 and P2
were determined to be 8.18×10-2 s-1 and 1.68×10-1 s-1, respectively, which are conspicuously greater than most of previously reported H2O2 probes. Thus these two probes have the potential to make a contribution to the real-time detection. Last but not least we examined the selectivity of P1 and P2. The probes were incubated with various reactive oxygen species in dilute MeOH solution with NaOH, represented by hydrogen peroxide (H2O2), tert-butyl hydroperoxide (TBHP), hypochlorite (ClO -), peroxyacetic acid (CH3CO3H), tert-butoxylradical (•OBut), hydroxyl radical (•OH) and singlet oxygen (1O2) (Figure 7). Other species triggers a small or minimar fluorecent viarations while H2O2 induces enormous change of fluorescence emission. The magnitude contrast indicates that the two probes are selective to H2O2 as boronate-based reaction site is pertinently cleaved by H2O2.
Conclusion In summary, we have developed two fluorescent probes P1 and P2 for rapid detection of H2O2 in alkaline environment based on the principles of ESIPT and PET. P1 responds to H2O2 under basic conditions with a fluorescence signal shift, while the fluorescence of P2 is turned-off. Both probes exhibitted excellent sensitivity and selectivity towards H2O2. Moreover the apparently accelerated reaction rate may provide a new solution for real-time sensing of H2O2. Comparing with P1, P2 shows a much better sensitivity. To the best of our knowledge, compound P2 is the first example of BODIPY derivative applied for H2O2 detection.
Acknowledgment We are grateful to the National Natural Science Foundation of China (Nos. 21472173 and J1210042).
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Biographies Xihui Li received her BSc degree in chemistry from Zhejiang University, China, in 2011. She is currently working toward a PhD in organic chemistry in Ping Lu’s group in the Department of Chemistry at the same University. Her research interest includes design, synthesis of fluorescent materials and their applications as chemosensors and biosensors. Jiaming Li received his BSc degree in chemistry in 2014. He used to do undergraduate research in Ping Lu’s group. His research interest includes design and synthesis of fluorescent probes for sensing reactive oxygen species and bio-imaging. Ping Lu was born in Zhejiang, China in 1964. She graduated with a BSc degree in chemistry from Hangzhou University, currently called Zhejiang University, in 1984. After she received her PhD degree in 1990, she became a faculty member in Department of Chemistry at the same University. Since 1995, she has worked for 3 years as a visiting scholar in Professor William P. Weber’s group in University of Southern California. Her main research interest is in the organic synthesis of fluorescent compounds and their application in optoelectronics and in sensors as well. Yanguang Wang was born in 1964 in Shanxi Provence, China. He received his B.S. (1985), M.S. (1988) and Ph.D. (1993) degrees from Lanzhou University. He did the post-doctoral research with Professor Henry N. C. Wong at the Chinese University of Hong Kong during 1997~1998. Since 1994, he joined Zhejiang University, where he was promoted to be a full professor in 1998. His major research interests include new synthetic methodologies of heterocyclic and carbocyclic molecules as well as total synthesis of natural products and their analogues.
Figure Captions Figure 1. Normalized emission spectra of HPNO in DCM, MeCN, MeOH and DMF, excited at 343 nm. Figure 2. Normalized absorption and emission spectra of (a) HPNO/P1, excited at 343 nm; (b) B-HBO/P2 in MeOH (all of the following), excited at 336 nm and 329 nm, respectively. Figure 3. (a) Absorption and (b) emission spectra of 5 μM B-HBO in the presence of 0-10 mM NaOH. Inset: (a) Color changes of B-HBO before (left) and after (right) the addition of NaOH (10 mM); (b) Fluorescence intensity–pH of B-HBO (pKa 10.9) in MeOH: (■) measured value, (-) Boltzmann Fit; λex = 336 nm, λem = 514 nm. Figure 4. (a) Absorption and (b) emission spectra of 20 μM P1 with 20 mM NaOH in the presence of different concentrations of H2O2 (0.05-10 equiv.). Inset: Ratiometric fluorescence I546/I408 as a function of the concentration of H2O2 and the color changes of P1 under a 365-nm hand-held UV lamp. Figure 5. (a) Absorption and (b) emission spectra of 5 μM P2 with 5 mM NaOH in the presence of different concentrations of H2O2 (0.1 – 80 equiv.). Inset: Fluorescence changes of P2 under a 365-nm hand-held UV lamp. Figure 6. Calculated HOMO and LUMO energy levels (eV) of PNO-Na+ and BODIPY (DFT, B3LYP/6-31G) and the possible photoinduced electron transfer process of P2 towards H2O2 in the presence of NaOH excited at 329 nm. Figure 7. (a) I546/I408 of 20 μM P1 (20 mM NaOH, excited at 343 nm) and (b) relative fluorescence intensity at 512 nm of 5 μM P2 (5 mM NaOH, excited at 329 nm) with various ROS (100 equiv.): H2O2, TBHP, ClO-, CH3CO3H, ·OtBu, 1O2 and ·OH. All spectra are measured 20 min after adding ROS, respectively.
DCM MeCN MeOH DMF
Fl. Intensity (a.u.)
1.0 0.8 0.6 0.4 0.2 0.0 400
450
500
550
600
650
700
Wavelength (nm) Figure 1
(b)
Absorbance (a.u.)
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0 300
400
0.8
1.0 0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0 300
500
400
500
Fl. Intensity (a.u.)
0.8
Abs. of B-HBO Abs. of P2 Em. of B-HBO Em. of P2
1.0
1.0
Fl. Intensity (a.u.)
Abs. of HPNO Abs. of P1 Em. of HPNO Em. of P1
1.0
Absorbance (a.u.)
(a)
600
Wavelength (nm)
Wavelength (nm)
Figure 2
(b) 1000 0.6
Fl. Intensity
Absorbance
800 0.4
0.2
0.0 250
400
800 600 400 200 0 7
8
9
10
11
12
13
14
pH
200 0
300
350
400
450
500
Wavelength (nm)
Figure 3
600
1000
Fl. Intensity at 514nm
(a)
550
600
350
400
450
500
550
Wavelength (nm)
600
(b)
0.6
I546/I408
700
20
I546/I408
(a)
Fl. Intensity
Absorbance
600
0.4
0.2
500 400
10
0 0
25
50
75
100
125
150
175
200
c[H2O2] / M
300 200 100
0.0
0
300
350
400
450
500
350
550
Wavelength (nm)
400
450
500
550
600
650
Wavelength (nm)
Figure 4
(a) 0.5
(b) 1000 800
Fl. Intensity
Absorbance
0.4 0.3 0.2 0.1 0.0 300
Figure 6
400 200 0
350
400
450
500
Wavelength (nm)
Figure 5
600
550
600
450
500
550
600
Wavelength (nm)
650
20
Blank 20 min
I546/I408
16 12 8 4 0
O 2 BHP H2 T
Figure 7
3H t u ClO H 3CO ·O B C
1 O2
·OH
Blank 20 min
(b)
Rel. Fl. Intensity at 512 nm
(a)
1.0 0.8 0.6 0.4 0.2 0.0 O 2 BHP 3H t u H2 T ClO H 3CO ·O B 1 O 2 C
·OH
Scheme Captions Scheme 1. Synthetic route of P1. Scheme 2. Synthetic route of P2. Scheme 3. The principle of ESIPT photophysical process of HPNO and B-HBO. Scheme 4. The response mechanism of P1 towards H2O2 under basic conditions
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Table 1 Photophysical properties of HPNO, P1, B-HBO, P2 in MeOH. Abs. λmax,
Ems. λmax,
Stokes shift
Lifetime
(nm)
(nm)
(nm)
ΦFLa
HPNO
330
409
79
0.004
-b
P1
311
408
97
0.324
-b
B-HBO
500
514
14
0.165
2.16
P2
500
512
12
0.278
3.18
(ns)
a
Referring to the standard quinine sulfate (0.1 M H2SO4)
b
The average lifetime was very small and beyond the detection limit of the instrument.
S0925-4005(17)30477-X http://dx.doi.org/doi:10.1016/j.snb.2017.03.060 SNB 21972
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
30-5-2016 3-3-2017 13-3-2017
Please cite this article as: Xihui Li, Jiaming Li, Yuhan Tao, Zhixing Peng, Ping Lu, Yanguang Wang, Oxazole-based high resolution ratiometric fluorescent probes for hydrogen peroxide detection, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.03.060 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Oxazole-based high resolution ratiometric fluorescent probes for hydrogen peroxide detection
Xihui Li, Jiaming Li, Yuhan Tao, Zhixing Peng, Ping Lu* and Yanguang Wang*
Department of Chemistry, Zhejiang University, Hangzhou 310027, China [email protected]; [email protected]
Graphical Abstract
Research Highlights
Two novel fluorescent probes, PNOBpin and B-BOBpin, were synthesized and characterized for their applications for rapid detection of H2O2. In the presence of base, the probes provide remarkable changes in optical properties with extremely fast response towards H2O2 based on the cleavage of C-O bond and the principle of excited state intramolecular proton transfer (ESIPT) or photo-induced electron transfer (PET). B-BOBpin is the first example of BODIPY derivative applied for H2O2 detection.
Abstract: Hydrogen peroxide (H2O2) has been associated as the most important member of reactive oxygen species (ROS) for a long time. Two novel fluorescent probes (P1 and P2) are synthesized and characterized for rapid detection of H2O2. In the presence of base, the probes provide remarkable changes in optical properties with extremely fast response towards H2O2 based on the cleavage of C-O bond and the principle of excited state intramolecular proton transfer (ESIPT) or photo-induced electron transfer (PET).
Keywords: fluorescent sensors, excited state intramolecular proton transfer, photo-induced electron transfer, hydrogen peroxide
1.
Introduction Reactive Oxygen Species (ROS) are generated during natural metabolism in living organisms and
play important roles in cell signaling and homeostasis. However, the abnormal ROS levels may result in significant damage to cells and cause numerous diseases [1]. This is a particular aspect of oxidative stress, which means an imbalance between the production of ROS and the ability of the system to counteract their harmful effects. As a major ROS in aerobic organism, hydrogen peroxide is involved in normal cellular signal transduction as a second messenger, which widely exists in many physiological and pathological processes [2]. The major endogenous source of H 2O2 is the process of O2 reduction by NADPH oxidase complex (Nox) with cellular stimulation in nearly all living cells [3]. It is believed that excessive H2O2 production is associated with aging and neurodegenerative diseases such as Parkinson’s and Alzheimer’s diseases [4]. Therefore, many efforts have been made to detect H2O2 accurately in cells. Compared with traditional approaches, fluorescent probes are among the most effective tools for imaging H2O2 with advantages of higher sensitivity, better selectivity, simpler manipulation and less invasiveness. The design strategies are based on specific reactions between recognition groups and H2O2 that give products with different photophysical properties such as oxidative hydrolysis of boronates [5], phenylphosphates [6] and sulfonates [7] and so on. The mechanisms usually involve efficient Förster resonance energy transfer (FRET), intramolecular charge transfer (ICT), photo-induced electron transfer (PET) and induce enormous change in fluorescence emission of fluorophore. In comparison with reported probes, fluorescent chemosensors based on excited-state intramolecular proton transfer (ESIPT) are leading a new trend owing to their unique photophysical property. The ESIPT molecules are usually oxazole, oxadiazole, and imidazole-based and normally more stable in enol form (E form) in the ground state. Once photo-excited, a fast proton transfer takes place from a hydroxyl (or amino) unit to a carbonyl oxygen (or imine nitrogen) to generate keto form (K form) [8]. Therefore, the absorption of E form and the emission of the excited K form (K* form) provide an extremely large Stokes shift that could effectively avoid self-absorption, giving an ideal solution for fluorescence sensing.
The key challenge of fluorescence bioimaging for H2O2 is to detect H2O2 specifically over other biologically relevant ROS. In recent years, a series of well-devised fluorescent chemosensors specific for H2O2 have been developed and help to make considerable progress in studying H 2O2 biology by using these probes [9]. Yet most of these probes are facing a common problem that they usually need a long reaction time to attain signal output which may have an influence on real-time monitoring and quantification. Here we present two novel fluorescent chemosensors (P1 and P2) with high sensitivity and rapid response towards H2O2. Motivated by the photophysical property and dramatic ESIPT phenomenon of 3-(4,5-diphenyloxazol-2-yl)- naphthalen-2-ol (HPNO) [10], we choose it as a fluorophore and protect the hydroxyl group with the boronated-based benzyl group in order to inhibit ESIPT process and detect H2O2 specifically. Thus, P1 is designed and synthesized (Scheme 1). Considering that a longer emission wavelength is desirable for bio-imaging in living cells [11], we combine 2-(2’-hydroxyphenyl) benzoxazole (HBO) as an ESIPT moiety, boron-dipyrromethene (BODIPY) dye as a red-shifted fluorophore [12], and phenylboronate as a cleavable group towards hydrogen peroxide and design a new probe, named as P2 (Scheme 2). Interestingly, unlike the emission peak shift of P1, P2 shows a turn-off fluorescence change towards hydrogen peroxide in the presence of base.
2.
Experimental Section
2.1 General All the reagents and deuterated solvents were purchased from Sigma-Aldrich, Acros Oganics and Alfa Aesar, and were used without further purification unless otherwise. HPNO and 3-(acetyloxy)-4(2-benzoxazolyl)-benzaldehyde (BOAc) were synthesized following literature protocol [13]. Organic solvents were purchased from Sinopharm and Tansoole then dried and purified before use as specified by standard procedures [14]. Analytical thin layer chromatography (TLC) was performed with silica gel HSGF254. Chromatography was performed on silica gel (300-400 mesh) by standard techniques eluting with solvents as indicated. All compounds were fully characterized. 1H NMR, 13C NMR spectra were obtained on a Bruker DMX-500 MHz or AVANCE 2B/400 MHz spectrometer operating in the FT mode. Unless otherwise noted, all the NMR spectra were recorded at room temperature. Coupling constants (J) were reported in hertz unit (Hz). Chemical shifts (in ppm) were referenced to tetramethylsilane (δ = 0 ppm) in CDCl 3. 13C NMR spectra were obtained on 100 MHz or 125 MHz and
chemical shifts were reported in ppm referenced to the central line of a triplet at 77.0 ppm of CDCl3. All high-resolution mass spectra (HRMS) were performed on a Bruker MALDI-TOF/TOF-MS. Fluorescence spectra were recorded by RF-5301pc spectrofluorometer (Shimadzu, Kyoto, Japan) equipped with a xenon lamp. UV spectra were recorded on Shimadzu UV-2450 spectrophotometer. pH measurements were performed using a Model PHS-3C meter.
2.2 Photophysical Measurements Twice-distilled water and spectroscopic grade MeOH were used for spectroscopic studies. Unless otherwise noted, all the absorption and emission spectra were recorded at room temperature. The slit width of fluorescence excitation is 3.0 nm and emission is 5.0 nm. Hydroxyl radicals and tert-butoxy radicals (•OtBu) were generated by reaction of Fe2+ with H2O2 or TBHP respectively. Singlet oxygen (1O2) was generated from ClO- and H2O2 [9c]. Various analytes represented by H2O2, TBHP, ClO-, OH•, 1
O2, and •OtBu were added to the solutions of P1 (20 μM) and P2 (5 μM), respectively. Slightly
variations in the pH of the solutions were achieved by adding minimal volumes of NaOH or HCl aqueous solution. And the total addition of H2O was less than 4% (v/v).
Determination of the detection limit The detection limit was calculated based on the fluorescence titration curve of P1 and P2 in the presence of H2O2 (Figure 4b and Figure 5b, respectively). The fluorescence intensity of P1 and P2 were measured by five times and the standard deviation of blank measurement were achieved. The detection limit was calculated with the following equation [15]: Detection limit = 3σ/k Where σ is the standard deviation of blank measurement, k is the slop between the fluorescence intensity versus H2O2 concentrations (Figure S8).
2.3 Calculations Density functional theory (DFT) calculations were carried out in the gas phase using the Gaussian 09 [16] quantum-chemical package. The geometry optimizations for the ground state of E form of B-HBO, P2 and B-BO-Na+ were performed using B3LYP function with the 6-31G basis set. Vibrational frequency calculations at the same level were performed for the obtained structures to confirm the
global minimum.
2.4 Synthesis P1: HPNO (69 mg, 0.19 mmol), potassium carbonate (79 mg, 0.57 mmol) and tetrabutylammonium iodide (21 mg, 0.06 mmol) were dissolved into 10 mL DMF under N2. 4-Bromomethylphenylboronic acid pinacol ester (79 mg, 0.27 mmol), dissolved in 10 mL DMF, was added into the solution dropwisely. The mixture was stirred at room temperature for 16 h. The crude product was collected by filtration, washed with water, dried by suction. The residue was purified through column chromatography on silica gel column (hexane: ethyl acetate: 20:1-5:1 v/v as eluent) to afford 53 mg P1 in 48% yield. White solid, m.p. 166-169 oC. 1H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H), 7.80-7.76 (m, 3H), 7.69-7.64 (m, 3H), 7.57 (d, 2H, J = 7.6 Hz), 7.41-7.38 (m, 3H), 7.35-7.25 (m, 5H), 7.20-7.18 (m, 3H), 5.22 (s, 2H), 1.28(s, 12H) ppm; 13C NMR (100 MHz, CDCl3) δ 159.1, 154.2, 145.9, 139.7, 136.4, 135.3, 135.1,
132.8, 131.2, 129.0, 128.64, 128.56, 128.4, 128.3, 128.2, 127.8, 127.2, 126.5, 124.5,
118.1, 107.9, 83.9, 70.7, 25.0. HRMS: Found: 579.2581; Calcd for C 38H34BNO4: 579.2584. B-BOAc: 2,4-Dimethylpyrrole (215 mg, 2.2 mmol) and BOAc (281 mg, 1 mmol) were dissolved in CH2Cl2 with a catalytic amount of trifluoroacetic acid (TFA) (1-2 drops). The mixture was stirred for 16 h at room temperature. Then a solution of 2,3- dichloro-5,6-dicyanobenzoquinone (DDQ) (227 mg, 1 mmol) in CH2Cl2 (30 mL) was added dropwisely, and the mixture was stirred for 15 min. Finally, BF3-OEt2 (3 mL) and triethylamine (3 mL) were added, and the mixture was stirred for 3 h at room temperature. The crude mixture was diluted with CH2Cl2 and washed with H2O. The organic extracts were dried over MgSO4, filtered and evaporated under reduced pressure. Flash chromatography (hexane: ethyl acetate: 5:1, v/v) afforded 179 mg (0.36 mmol) of B-BOAc as a red solid in 36% yield. m.p. > 250 oC. 1H NMR (400 MHz, CDCl3) δ 8.44 (d, 1H, J = 8.0 Hz), 7.81 – 7.75 (m, 1H), 7.59 (dd, 1H, J = 6.0, 3.1 Hz), 7.43 – 7.36 (m, 3H), 7.22 (d, 1H, J = 1.5 Hz), 6.01 (s, 2H), 2.57 (s, 6H), 2.50 (s, 3H), 1.53 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 169.7, 159.1, 156.3, 150.2, 150.1, 143.1, 141.9, 139.3, 138.6, 131.0, 130.9, 126.3, 125.9, 124.9, 124.6, 121.6, 121.2, 120.5, 119.4, 110.8, 110.6, 21.3, 14.7, 14.7, 14.6. HRMS: Found: 499.1886; Calcd for C28H24BF2N3O3: 499.1879.
B-HBO: B-BOAc (179 mg, 0.36 mmol) and K2CO3 (149 mg, 1.08 mmol) was disolved in mixed solution of CH2Cl2 and EtOH (1:1 v/v, 10 mL). The mixture was refluxed for 2 hours, then filtered and evaporated under reduced pressure. Flash chromatography (hexane: ethyl acetate 5:1, v/v) afforded 156 mg (0.34 mmol) of B-HBO as red solid in 94% yield. m.p. > 250 oC. 1H NMR (500 MHz, CDCl3) δ 11.68 (s, 1H), 8.17 (d, 1H, J = 8.0 Hz), 7.78 (dd, 1H, J = 6.2, 2.8 Hz), 7.65 (dd, 1H, J = 6.4, 2.8 Hz), 7.47 – 7.39 (m, 2H), 7.11 (s, 1H), 6.97 (d, 1H, J = 8.0 Hz), 6.00 (s, 2H), 2.57 (s, 6H), 1.55 (s, 6H). 13C NMR (125 MHz, CDCl3) δ 162.2, 159.2, 155.9, 149.2, 143.0, 140.3, 140.1, 139.9, 130.8, 128.0, 125.9, 125.3, 121.4, 119.5, 119.4, 117.4, 111.1, 110.8, 14.7, 14.6. HRMS: Found: 457.1775. Calcd for C26H22BF2N3O2: 457.1773. P2: A mixture of compound B-HBO (156 mg, 0.34 mmol), 4-(bromomethyl)benzene boronic acid pinacol ester (118 mg, 0.4 mmol), and K2CO3 (140 mg, 1.02 mmol) in dry N,N-dimethylformamide (DMF, 5 mL) was heated at 80 oC for 16 h under N2. After cooling, the mixture was filtered to remove salts. Then cold water (30 mL) was added to the mixture and extracted with DCM (10 mL×3). The organic solutions were combined, washed with water and brine, and dried with Na 2SO4. The solvents were evaporated to generate the crude product, which was purified by flash chromatography (silica gel, hexane/EA = 1:5) to give the desired product as red solid (92 mg, 40%). m.p. > 250 oC. 1H NMR (400 MHz, CDCl3) δ 8.33 (d, 1H, J = 8.4 Hz), 7.86 (dd, 1H, J = 6.1, 2.9 Hz), 7.81 (d, 2H, J = 8.0 Hz,), 7.67 – 7.61 (m, 1H), 7.57 (d, 2H, J = 8.0 Hz), 7.43 – 7.38 (m, 2H), 7.07 (d, 2H, J = 7.0 Hz), 5.98 (s, 2H), 5.34 (s, 2H), 2.57 (s, 6H), 1.41 (s, 6H), 1.35 (s, 12H).
C NMR (100 MHz, CDCl3) δ 161.1, 157.8,
13
156.1, 150.7, 143.0, 141.8, 139.8, 139.5, 139.1, 135.1, 132.3, 130.9, 128.5, 126.5, 125.9, 125.4, 124.7, 121.4, 121.0, 120.2, 117.5, 113.8, 110.6, 110.0, 83.9, 70.7, 24.9, 14.7, 14.4. HRMS Found: 674.2892. Calcd for [M+H]+ C39H40B2F2N3O4: 674.3168.
3
Result and discussion Compounds P1 and P2 were synthesized by the procedures as shown in Schemes 1 and 2. Their
structures along with HPNO, BOAc, B-BOAc, and B-HBO were comfirmed by 1H NMR, 13C NMR, and HRMS (see SI). Absorption and emission spectra of HPNO and P1 in different solvents are shown in Figure S1. Absorption spectra of HPNO and P1 are essentially the same no matter whether HPNO is alkylated or not, while emission spectra of P1 are also identical. The siginificant alteration was observed for the
emission of HPNO in different solvents. In dichloromethane (DCM), dual emission is presented (Figure S1b). HPNO was derived from the backbone of 2-(oxazol-2-yl)phenol. The principle of ESIPT process is displayed in Scheme 3. HPNO remains stable in enol form (E Form) in the ground state. Once photoexcited, tautoumerism occurs and creates keto form in excited state (K* form). Due to the increment of the efficient conjugation length in the K form, a narrower energy gap between HOMO and LUMO is observed for K-form. As shown in Figure 1, the emission of HPNO is highly impacted by the solvent properties. In polar solvents, such as methanol (MeOH) and N,N-dimethyl foramide (DMF), the emission of E form at 409 nm is dominant. Emission of K form at 602 nm is not shown in these solvents since the hydrogen bonding between the E form of HPNO and the solvent molecules helps to stabilize the E form and inhibits the process of intramolecular proton transfer. Thereofore, emission of K form in MeOH and DMF is vanished. In other solvents like dichloromethane (DCM), the interaction between the E-form of HPNO and the solvent is neglectable. Thus, a full ESIPT process is triggerd by the photoexcitation via intramolecular hydrogen bonding. As a result, dual emission of HPNO in DCM, respectively from E* form and K* form, is observed. In the solid state [17], the restriction of the intramolecular rotation helps in maintaining optimal geometry for intramolecular H-bonds which is a key factor for an efficient ESIPT process; meanwhile, the omission of intermolecular H-bonding with solvent molecules also causes the enhancement of K* emission. Both of the two mechanisms are considered responsible for solid state emission of HPNO in K* form resulting in red-light as photoed in Figure S1e. When it comes to B-HBO, the UV-spectrum show two independant absorption bands around 250-400 nm and 500 nm. As the solvent polarity increases, the absorption band for the larger energy gap is enhanced significantly (Figure S2a). Previous work inspires us that the absorption band at 250-400 nm is assigned to the π–π∗ band of HBO moiety in E form [18] whereas the absorption band for the lower energy gap at 500 nm is the characteristic absorption of BODIPY [12a]. The enhancement of the absorption band for the larger energy gap could be ascribed to the intermolecular hydrogen bonding between HBO and solvent. Meanwhile, the absorption of BODIPY is hardly impacted by the solvent polarity. This phenomenon implies the conjugatively uncoupling between HBO and BODIPY. The two moieties are twisted owning to the methyl groups at positions C-1 and C-7 of BODIPY. This angulation could be supported by the X-ray analysis of a structurally similar
compound [19] and the calculated result (Figure S3). The calculated dihedral angle for the plane of HBO and the plane of BODIPY is 90.03°, saying that they are held perpendicular to one another and decoupled. Although absorption of B-HBO is strongly influenced by solvent polarity, emissions of B-HBO in different solvents are almost the same and emit light at 514 nm which is the typical emission of the BODIPY. When B-HBO is alkylated and derived into P2, no hydroxyl group exists. As a net result, the absorption and emission spectra of P2 are essentially the same in all kinds of the solvents (Figure S2). For a clear discussion in the following sections, the absorption and emission spectra of the pairs of HPNO/P1 and B-HBO/P2 in methanol are shown in Figure 2. Then we studied the deprotonation of HPNO and B-HBO in methanol. HPNO absorbed light at 330 nm with molar absorption coefficient of 2.17×10 4 cm-1 M-1. Upon the addition of NaOH, the absorbance around 330 nm became weak and a concomitant increment of a new band at 425 nm appeared, with two isosbestic points at 307 nm and 388 nm. The titration of NaOH induced an increase in fluorescence intensity at 546 nm as well. The apparent pKa value of HPNO in methanol was determined to be 10.5 ± 0.4, representing the deprotonation capability of the subunits (Figure S4). Figure 3 shows the absorbance and fluorescence titration of B-HBO dissolved in MeOH by gradual addition of NaOH. Free B-HBO absorbs light at 295 nm, 321 nm and 500 nm with molar absorption coefficient of 4.76×104 cm-1 M-1, 5.16×104 cm-1 M-1 and 1.23×105 cm-1 M-1, respectively. As the gradual addition of NaOH increases to an end concentration of 10 mM, a new absorption band at 361 nm appears whereas the original ones at 295 nm and 321 nm decrease. Two distinct isosbestic points are found to be 269 nm and 341 nm. Emission intensity of B-HBO at 514 nm decreases significantly and a new peak arises at 440 nm triggered by adding NaOH, while the emission intensity at 481 nm remain the same. Notably the colorimetric change under basic condition can even be observed by naked eye which implies that the B-HBO might be used as a promising chemosensor for pH detection. Through fluorescence titration experiments we also determine the apparent pKa* value of B-HBO in methanol to be 10.9 ± 0.3 (Figure 3b inset). The absorption and emission spectra of P1 and P2 are quite simple without notable change in different solvent as we noted above (Figures S1c, d and S2c, d). Along with the gradul addition of NaOH, absorption and emission spectra of these two probes kept the same without any variation (Figure S5). It indicats that both P1 and P2 are stable in the alkaline environment. Further, we test the
response of P1 and P2 to H2O2 under neutral condition. Both of them are consistent too (Figure S6). As we further combine these two factors (NaOH and H2O2) to irritate the probes, fast and apparent alterations in absorption and emission spectra are observed. Both probes present the character of AND logic gate with two inputs, NaOH and H2O2. Methanol solution of P1 with 20 mM NaOH absorbed light at 311 nm with the absorption molar coefficient being 1.14×104 cm-1 M-1. The band decreased quickly by the addtion of H2O2, and a new band at 425 nm arised. The emission at 408 nm quenched concurrently as a new peak at 546 nm came into being, with unchanged emission intensity at 512 nm (Figure 4). Meanwhile the solution color under UV irritation changed from violet blue into yellow-green. And the ratiometric fluorescence (I546/I408) showed that the oxidation process almost finished when the concentration of H2O2 achieved 5 equivalence (Figure 4 inset). The detection limit of P1 was calculated to be 34 nM based on a signal-to-noise ratio of 3 (S/N = 3). The possible mechanism for this change is shown in Scheme 4. At the beginning, P1 is oxidized in the presence of hydrogen peroxide and results in the formation of borate intermediate A. With further titration of hydroxide anion, intermediate A hydrolyzes into phenolate anion B. An electron pushing from phenolate anion leads to the cleavge of C-O bond and finally generates PNO- species which emits light at 546 nm. The H2O2 titration experiment of P2 showed a quenching process instead of peak shift like P1. Methanol solution of P2 with 5 mM NaOH obsorbed at 310 nm and 500 nm with absorption molar coefficients being 2.10×104 cm-1 M-1 and 7.38×104 cm-1 M-1, respectively. As H2O2 was gradually added into the solution, a new absorption peak at 357 nm appeared, whereas the absorption band at 500 nm kept the same. At the same time the original emission peak at 512 nm quenched tremendously (Figure 5). The detection limit of P2 was calculated to be 13 nM (S/N = 3). The chemical process responsible for the sensory mechanismis is similar to P1 as shown in Scheme 4. However, once phenolate is formed, a photo-induced electron process (PET) takes place and causes the turn-off fluorescence due to the matched HOMO and LUMO levels of PNO-Na+ and BODIPY (Figure 6) [20]. For the detailed photophysical properties including band gap, Stokes shift, Φ FL and fluorescence lifetime data are listed in Table 1. As expected, both P1 and P2 behaved a rapid response towards H2O2 under alkaline condition within 20 min (Figure S7). Using pseudo-first-order kinetcs (P1: 20 μM, 20 mM NaOH, 2 mM H2O2; P2: 5 μM, 5 mM NaOH, 0.4 mM H2O2), the observed rate constant k for H2O2 detection using P1 and P2
were determined to be 8.18×10-2 s-1 and 1.68×10-1 s-1, respectively, which are conspicuously greater than most of previously reported H2O2 probes. Thus these two probes have the potential to make a contribution to the real-time detection. Last but not least we examined the selectivity of P1 and P2. The probes were incubated with various reactive oxygen species in dilute MeOH solution with NaOH, represented by hydrogen peroxide (H2O2), tert-butyl hydroperoxide (TBHP), hypochlorite (ClO -), peroxyacetic acid (CH3CO3H), tert-butoxylradical (•OBut), hydroxyl radical (•OH) and singlet oxygen (1O2) (Figure 7). Other species triggers a small or minimar fluorecent viarations while H2O2 induces enormous change of fluorescence emission. The magnitude contrast indicates that the two probes are selective to H2O2 as boronate-based reaction site is pertinently cleaved by H2O2.
Conclusion In summary, we have developed two fluorescent probes P1 and P2 for rapid detection of H2O2 in alkaline environment based on the principles of ESIPT and PET. P1 responds to H2O2 under basic conditions with a fluorescence signal shift, while the fluorescence of P2 is turned-off. Both probes exhibitted excellent sensitivity and selectivity towards H2O2. Moreover the apparently accelerated reaction rate may provide a new solution for real-time sensing of H2O2. Comparing with P1, P2 shows a much better sensitivity. To the best of our knowledge, compound P2 is the first example of BODIPY derivative applied for H2O2 detection.
Acknowledgment We are grateful to the National Natural Science Foundation of China (Nos. 21472173 and J1210042).
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Biographies Xihui Li received her BSc degree in chemistry from Zhejiang University, China, in 2011. She is currently working toward a PhD in organic chemistry in Ping Lu’s group in the Department of Chemistry at the same University. Her research interest includes design, synthesis of fluorescent materials and their applications as chemosensors and biosensors. Jiaming Li received his BSc degree in chemistry in 2014. He used to do undergraduate research in Ping Lu’s group. His research interest includes design and synthesis of fluorescent probes for sensing reactive oxygen species and bio-imaging. Ping Lu was born in Zhejiang, China in 1964. She graduated with a BSc degree in chemistry from Hangzhou University, currently called Zhejiang University, in 1984. After she received her PhD degree in 1990, she became a faculty member in Department of Chemistry at the same University. Since 1995, she has worked for 3 years as a visiting scholar in Professor William P. Weber’s group in University of Southern California. Her main research interest is in the organic synthesis of fluorescent compounds and their application in optoelectronics and in sensors as well. Yanguang Wang was born in 1964 in Shanxi Provence, China. He received his B.S. (1985), M.S. (1988) and Ph.D. (1993) degrees from Lanzhou University. He did the post-doctoral research with Professor Henry N. C. Wong at the Chinese University of Hong Kong during 1997~1998. Since 1994, he joined Zhejiang University, where he was promoted to be a full professor in 1998. His major research interests include new synthetic methodologies of heterocyclic and carbocyclic molecules as well as total synthesis of natural products and their analogues.
Figure Captions Figure 1. Normalized emission spectra of HPNO in DCM, MeCN, MeOH and DMF, excited at 343 nm. Figure 2. Normalized absorption and emission spectra of (a) HPNO/P1, excited at 343 nm; (b) B-HBO/P2 in MeOH (all of the following), excited at 336 nm and 329 nm, respectively. Figure 3. (a) Absorption and (b) emission spectra of 5 μM B-HBO in the presence of 0-10 mM NaOH. Inset: (a) Color changes of B-HBO before (left) and after (right) the addition of NaOH (10 mM); (b) Fluorescence intensity–pH of B-HBO (pKa 10.9) in MeOH: (■) measured value, (-) Boltzmann Fit; λex = 336 nm, λem = 514 nm. Figure 4. (a) Absorption and (b) emission spectra of 20 μM P1 with 20 mM NaOH in the presence of different concentrations of H2O2 (0.05-10 equiv.). Inset: Ratiometric fluorescence I546/I408 as a function of the concentration of H2O2 and the color changes of P1 under a 365-nm hand-held UV lamp. Figure 5. (a) Absorption and (b) emission spectra of 5 μM P2 with 5 mM NaOH in the presence of different concentrations of H2O2 (0.1 – 80 equiv.). Inset: Fluorescence changes of P2 under a 365-nm hand-held UV lamp. Figure 6. Calculated HOMO and LUMO energy levels (eV) of PNO-Na+ and BODIPY (DFT, B3LYP/6-31G) and the possible photoinduced electron transfer process of P2 towards H2O2 in the presence of NaOH excited at 329 nm. Figure 7. (a) I546/I408 of 20 μM P1 (20 mM NaOH, excited at 343 nm) and (b) relative fluorescence intensity at 512 nm of 5 μM P2 (5 mM NaOH, excited at 329 nm) with various ROS (100 equiv.): H2O2, TBHP, ClO-, CH3CO3H, ·OtBu, 1O2 and ·OH. All spectra are measured 20 min after adding ROS, respectively.
DCM MeCN MeOH DMF
Fl. Intensity (a.u.)
1.0 0.8 0.6 0.4 0.2 0.0 400
450
500
550
600
650
700
Wavelength (nm) Figure 1
(b)
Absorbance (a.u.)
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0 300
400
0.8
1.0 0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0 300
500
400
500
Fl. Intensity (a.u.)
0.8
Abs. of B-HBO Abs. of P2 Em. of B-HBO Em. of P2
1.0
1.0
Fl. Intensity (a.u.)
Abs. of HPNO Abs. of P1 Em. of HPNO Em. of P1
1.0
Absorbance (a.u.)
(a)
600
Wavelength (nm)
Wavelength (nm)
Figure 2
(b) 1000 0.6
Fl. Intensity
Absorbance
800 0.4
0.2
0.0 250
400
800 600 400 200 0 7
8
9
10
11
12
13
14
pH
200 0
300
350
400
450
500
Wavelength (nm)
Figure 3
600
1000
Fl. Intensity at 514nm
(a)
550
600
350
400
450
500
550
Wavelength (nm)
600
(b)
0.6
I546/I408
700
20
I546/I408
(a)
Fl. Intensity
Absorbance
600
0.4
0.2
500 400
10
0 0
25
50
75
100
125
150
175
200
c[H2O2] / M
300 200 100
0.0
0
300
350
400
450
500
350
550
Wavelength (nm)
400
450
500
550
600
650
Wavelength (nm)
Figure 4
(a) 0.5
(b) 1000 800
Fl. Intensity
Absorbance
0.4 0.3 0.2 0.1 0.0 300
Figure 6
400 200 0
350
400
450
500
Wavelength (nm)
Figure 5
600
550
600
450
500
550
600
Wavelength (nm)
650
20
Blank 20 min
I546/I408
16 12 8 4 0
O 2 BHP H2 T
Figure 7
3H t u ClO H 3CO ·O B C
1 O2
·OH
Blank 20 min
(b)
Rel. Fl. Intensity at 512 nm
(a)
1.0 0.8 0.6 0.4 0.2 0.0 O 2 BHP 3H t u H2 T ClO H 3CO ·O B 1 O 2 C
·OH
Scheme Captions Scheme 1. Synthetic route of P1. Scheme 2. Synthetic route of P2. Scheme 3. The principle of ESIPT photophysical process of HPNO and B-HBO. Scheme 4. The response mechanism of P1 towards H2O2 under basic conditions
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Table 1 Photophysical properties of HPNO, P1, B-HBO, P2 in MeOH. Abs. λmax,
Ems. λmax,
Stokes shift
Lifetime
(nm)
(nm)
(nm)
ΦFLa
HPNO
330
409
79
0.004
-b
P1
311
408
97
0.324
-b
B-HBO
500
514
14
0.165
2.16
P2
500
512
12
0.278
3.18
(ns)
a
Referring to the standard quinine sulfate (0.1 M H2SO4)
b
The average lifetime was very small and beyond the detection limit of the instrument.