A Novel Fluorescent Conjugate Applicable To Visualize the

Article pubs.acs.org/JAFC

A Novel Fluorescent Conjugate Applicable To Visualize the Translocation of Glucose−Fipronil Jie Wang,†,‡,§ Zhiwei Lei,†,‡,§ Yingjie Wen,†,‡ Genlin Mao,†,‡ Hanxiang Wu,†,‡ and Hanhong Xu*,†,‡ †

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources and ‡Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, 510642, Guangdong China S Supporting Information *

ABSTRACT: The ability to visualize the movement of glycosyl insecticides contributes to learning their translocation and distribution in plants. In our present work, a novel fluorescent glucose−fipronil conjugate N-[3-cyano-1-[2,6-dichloro-4(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl]-1-(2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2deoxy-β-D-glucopyranosyl)-1H-1,2,3-triazole-4-methanamine (2-NBDGTF), was obtained via click chemistry. Disk uptake experiments showed that an active carrier-mediated system was involved in the 2-NBDGTF uptake process. Meanwhile, 2NBDGTF exhibited comparable phloem mobility with GTF in castor bean seedlings. Visualization of 2-NBDGTF uptake and transport experiment showed that this fluorescent glucose−fipronil conjugate could be loaded into sieve tubes after transiting through epidermal cells and mesophyll cells and then translocate from cotyledon to hypocotyl via phloem in castor bean seedlings. The results above determined that it is a promising fluorescence tagging approach for revealing the activities of glycosyl insecticides and 2-NBDGTF is a reasonable and feasible fluorescent surrogate of GTF for tracing the distribution of glucose− fipronil conjugates. KEYWORDS: visualization, carrier-mediated, phloem mobility, fluorescence tagging, castor bean

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INTRODUCTION Insecticides with phloem mobility are desirable for controlling piercing and sucking insects as well as vascular insects in agriculture. Despite the positive implications of such insecticides, only a few phloem mobile or ambimobile insecticides are currently available. An optional strategy used in developing phloem-mobile pesticides is to chemically modify parent pesticides. Such modifications include sugar conjugation, acid functionalization, and formation of quaternary salts from the parent pesticides.1 The addition of carboxyl groups to immobile or poorly mobile pesticides has proven to be an efficient method of imparting phloem mobility to parent pesticides. 2,3 The conjugation of parent pesticides to endogenous substances, such as amino acids and monosaccharides, is another approach that is used for developing propesticides with phloem mobility.4,5 In our previous work, we demonstrated that the glucose− fipronil conjugate N-[3-cyano-1-[2,6-dichloro-4(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl]-1-(β-D-glucopyranosyl)-1H-1,2,3-triazole-4-methanamine (GTF) exhibits moderate phloem mobility in the phloem of castor bean seedlings.6 That the hydroxyl of the monosaccharide significantly affects the phloem mobility of the monosaccharide−fipronil conjugates was also demonstrated using castor bean seedlings.7 Additionally, the uptake and transport of GTF within castor bean seedlings demonstrated the involvement of an active carrier-mediated mechanism.8 Despite the progress made in this field, the uptake mechanisms of GTF analogues and the distribution within plants remain unknown. The ability to visualize GTF analogues within plants has great potential for addressing these questions. © 2014 American Chemical Society

Fluorescence tagging techniques are promising approaches to trace uptake pathways, transport routes, and distributions of xenobiotics within plants. Compared with radiolabeling techniques, which have been widely used to investigate the uptake and translocation of xenobiotics,9,10 fluorescence tagging offers both high spatial and temporal resolution. Moreover, the use of fluorescence tagging could provide dynamic and realtime information on xenobiotics at the cellular level. In order to visualize transport of xenobiotics within plants, several studies were performed using various fluorescent dyes with similar physicochemical properties.11,12 Recently, fluorescently labeled plant hormones were used to reveal the distribution and signaling receptor of hormones within plants.13,14 To visualize the uptake activity and translocation of GTF analogues in plants, a novel fluorescently labeled GTF derivative was synthesized with the modification of an NBD moiety at the C-2 position of the glucose−fipronil conjugate (Figure 1). The uptake mechanism and the phloem mobility of

Figure 1. Structures of GTF and 2-NBDGTF. Received: April 29, 2014 Accepted: August 18, 2014 Published: August 18, 2014 8791

dx.doi.org/10.1021/jf502838m | J. Agric. Food Chem. 2014, 62, 8791−8798

Journal of Agricultural and Food Chemistry

Article

Scheme 1. Synthesis of 2-NBDGTF; Reaction conditions: 1.1 CF3COOC2H5, Et3N, MeOH; 1.2 SnCl4, TMSN3, CH2Cl2; 1.3 CuSO4·5H2O, sodium ascorbate; 1.4 MeOH, NaOMe; 1.5 1M NaOH; 1.6 NBD-Cl, K2CO3, MeOH

2-NBDGTF were investigated to prove that the fluorescent glucose−fipronil derivative 2-NBDGTF is a reasonable fluorescent GTF surrogate. Then 2-NBDGTF was used to visualize the movement of GTF analogues within castor bean seedlings using confocal laser scanning microscopy.

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added dropwise. The resulting mixture was stirred at room temperature until TLC indicated the disappearance of starting material (about 2 h). The mixture was extracted with CH2Cl2 (3 × 20 mL) and then washed with NaHCO3 (3 × 20 mL) and water (20 mL). The organic layer was dried (Na2SO4) and filtered, and the solvent was evaporated under reduced pressure. The residue was purified by flash chromatography (petroleum ether:ethyl acetate = 2:1) to give 3. Needle solid: yield 75%; 1H NMR (600 MHz, CDCl3) δ 7.17 (d, J = 8.8 Hz, 1H), 5.39−5.27 (m, 1H), 5.10 (t-like, J = 9.7 Hz, 1H), 4.81 (d, J = 9.2 Hz, 1H), 4.28 (dd, J = 12.5, 5.0 Hz, 1H), 4.20 (dd, J = 12.5, 2.2 Hz, 1H), 4.03 (dd, J = 19.7, 9.4 Hz, 1H), 3.88 (ddd, J = 10.0, 4.9, 2.3 Hz, 1H), 2.10 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 171.41, 170.95, 169.49, 157.78, 115.61, 87.82, 74.14, 71.72, 68.30, 61.96, 54.35, 20.81, 20.67, 20.46. N-[3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl]-1-(2-trifluoroacetamido2-deoxy-β-D-glucopyranosyl)-1H-1,2,3-triazole-4-methanamine (5). Fipronil-alkyne 4 (474 mg, 1 mmol) was added to a vigorously stirred suspension of compound 2 (426 mg, 1 mmol) in 3 mL of tert-butyl alcohol. The reaction was initiated by the addition of a solution of CuSO4·5H2O (100 mg, 0.4 mmol) and sodium ascorbate (173 mg, 0.8 mmol) in distilled water (3 mL). The deep yellow suspension was stirred vigorously at 50 °C for 3 h. Distilled water (10 mL) was added, and the aqueous layer was extracted with chloroform (10 mL × 3). The combined organic extracts were washed with aqueous sodium hydrogen carbonate and brine, dried with sodium sulfate, filtered, and evaporated in vacuo. The crude compound was added to a solution of sodium methoxide in dry methanol (0.05 M, 15 mL). The resultant solution was stirred for 30 min at room temperature. The mixture was neutralized with Amberlite IR 120 (H+) resin and filtered, and the filtrate was then evaporated. The residues were purified by column chromatography to obtain compound 5. White solid: yield 78%; 1H NMR (600 MHz, MeOD) δ 8.12 (s, 1H), 8.09* (s, 1H), 8.06 (dd, J = 7.6, 6.3 Hz, 2H), 5.88 (d, J = 5.7 Hz, 1H), 5.86* (d, J = 5.7 Hz, 1H), 4.64 (d, J = 4.1 Hz, 1H), 4.62* (d, J = 4.1 Hz, 1H), 4.45 (d, J = 2.3 Hz, 1H), 4.43* (d, J = 2.3 Hz, 1H), 4.30 (t, J = 7.3 Hz, 1H), 4.27* (t, J = 7.3 Hz, 1H), 3.92 (t-like, 1H), 3.90 (t-like, 1H), 3.84−3.73 (m, 2H), 3.63−3.55 (m, 2H); 13C NMR (150 MHz, MeOD) δ 157.93, 150.54, 144.27, 136.57, 136.45, 134.68, 126.63, 126.54, 126.44, 124.42, 123.12, 121.47, 116.66, 114.76, 110.79, 96.26, 85.99, 79.92, 79.90*, 73.76, 73.66*, 69.88, 69.88*, 60.77, 60.74*, 55.75, 55.65*, 39.66, 39.59* (*enantiomer); ESI-MS, m/z 807.4 [M − H]−.

MATERIALS AND METHODS

General Information. Reagents and anhydrous solvents were used directly. Melting points were determined on an FP62 digital micro melting point apparatus; data were left uncorrected. NMR spectra were obtained from a Bruker AV-600 instrument. Deuterated solvents were obtained from Cambridge Isotope Laboratories (Andover, MA). CD3OD and CDCl3 solvent peaks (3.31 and 7.26 ppm for 1H; 49.0 and 77.0 ppm for 13C, respectively) were used as internal chemical shift references. The mass spectra (MS) of new compounds were obtained by Bruker maXis with an electrospray ionization (ESI) spectrometer. Silica gel was used for column chromatography. GTF was prepared according to our previously described procedure.6 The compounds to be added to the incubation medium were purchased from Sigma [carbonyl cyanidem-chlorophenylhy-drazone (CCCP)] and Aladdin (phloridzin, MES, and mannitol). Chemicals (Scheme 1). 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-trifluoroacetamido-α-D-glucopyranoside (2). 2-Deoxy-2-trifluoroacetamido-D-glucopyranose (2 g, 7.24 mmol) was dissolved in pyridine (15 mL) and acetic anhydride (5 mL). The reaction mixture was kept at room temperature overnight and poured into ice water and stirred for 1 h. The mixture was extracted with dichloromethane, and the organic layer was washed with 1 M HCl and saturated aqueous NaHCO3 and dried on Na2SO4. The solution was concentrated and purified by silica gel column chromatography to obtain a light yellow solid. Slightly yellow solid: yield 78%; mp 123.5 °C; 1H NMR (600 MHz, CDCl3) δ 6.27 (d, J = 3.9 Hz, 1H), 5.32 (br,1H), 5.22 (dd, J = 10.0 Hz, 10.0 Hz, 1H), 4.44 (m, 1H), 4.28 (dd, J = 12.0 Hz, 4.2 Hz, 1H), 4.07 (dd, J = 12.0, 2.2 Hz, 1H), 4.04 (m, 1H), 2.19 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 171.8, 170.6, 169.0, 168.2, 157.7, 116.7, 89.4, 69.8, 69.8, 67.0,61.3, 52.2, 20.7, 20.6, 20.5, 20.4; ESI-MS, m/z 442.2 [M − H]−. 3,4,6-Tetra-O-acetyl-2-trifluoroacetamido-2-deoxy-β-D-glucopyranosyl Azide (3). Compound 2 (170 mg, 0.384 mmol) and azidotrimethylsilane (TMS-N3, 56 μL, 0.423 mmol) were dissolved in anhydrous CH2Cl2 (5 mL), and SnCl4 (45 μL, 0.384 mmol) was 8792



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microscopy (Zeiss LSM780, Germany) directly. Photos of at least five sections from each treatment were taken with a 10× or 20× PlanApochromat (0.8 M27) objective using an argon laser with 488 nm excitation and 500−535 nm emission filter. Determination of 2-NBDGTF. Disk extracts and phloem sap were analyzed using an HPLC system (Shimadzu, Japan) equipped with a LC-20A fluorescence detector. The fluorescence detection conditions of 2-NBDGTF were excitation at 470 nm and emission at 530 nm. The determination of GTF was performed with a HP 1100 (Agilent) HPLC system. Separations were made with a C8 reversed-phase column. The solvent system consisted of acetonitrile and water (60:40, v/v). The injection volume was 10 μL, and the flow rate was 1 mL/ min. The calibration curve of 2-NBDGTF (ranging from 1 μM to 100 μM, r = 0.999) and GTF (ranging from 2.5 μM to 100 μM, r = 0.998) was linear. The presence of 2-NBDGTF in disk extracts and phloem sap was further identified by HPLC−HRMS (high resolution mass spectrometry) (Bruker maXis, Germany) using electrospray ionization. The mass spectrometer was operated in the positive electrospray ionization mode (ESI+). The HPLC system was an Agilent 1290 Infinity LC system which was equipped with a diode array detector. The operating conditions of the HPLC were the same as described above. Physicochemical Properties. Physicochemical properties [molecular mass (MW), ionization constant in aqueous solution (pKa), octanol/water partitioning coefficient (log Kow)] of GTF and 2NBDGTF were predicted using ACD LogD suite version 14.0 software. The chemical structures of conjugates 2-NBDGTF and GTF are shown in Figure 1, and the physicochemical properties of 2NBDGTF are summarized in Table 1. The excitation and emission fluorescence spectra are shown in Figure 2.

N-[3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl]-1-(2-[N-(7-nitrobenz-2oxa-1,3-diazol-4-yl)amino]-2-deoxy-β-D-glucopyranosyl)-1H-1,2,3triazole-4-methanamine (6). Compound 5 (774 mg, 1 mmol) was dissolved in 1 M NaOH (5 mL) and was stirred for 5 h. The mixture was extracted with ethyl acetate, dried on Na2SO4, and filtered, and the filtrate was evaporated. The residue and K2CO3 (140 mg, 2.5 mmol) were suspended in methanol (3 mL), and the reaction mixture was cooled to 0 °C on an ice bath. To the suspension was added in small portions NBD-Cl (300 mg, 1.5 mmol), and the reaction mixture was kept at room temperature for another 12 h. Solvent was evaporated under reduced pressure, and the crude product was purified by TLC. Red brown solid: yield 68%; 1H NMR (600 MHz, MeOD) δ 8.40 (d, J = 8.8 Hz, 1H), δ 8.40* (d, J = 8.8 Hz, 1H), 8.20 (s, 1H), 8.17* (s, 1H), 7.96 (m, 2H), 6.46 (d, J = 8.8 Hz, 1H), 6.43* (d, J = 8.8 Hz, 1H), 6.01* (d, J = 9.5 Hz, 1H), 5.99* (d, J = 9.5 Hz, 1H), 4.60 (s-like, 1H), 4.51 (dd, J = 16.5, 4.86 Hz, 1H), 4.30 (dd, J = 16.5, 9 Hz, 1H), 3.95 (dd, J = 12.1, 3.8 Hz, 1H), 3.96* (dd, J = 12.1, 3.8 Hz, 1H), 3.91−3.85 (m, 1H), 3.81 (dd, J = 12.1, 7.7 Hz, 1H), 3.81* (dd, J = 12.1, 7.4 Hz, 1H), 3.70−3.65 (m, 2H); 13C NMR (150 MHz, MeOD) δ 151.7, 146.2, 145.72, 145.5, 145.2, 137.8, 137.7, 137.6*, 136.2, 136.0, 135.9, 127.9, 127.8, 127.8, 124.5, 123.5, 123.3, 122.6, 112.1, 112.0, 97.4, 81.4, 77.5, 77.2*, 71.1, 62.2, 56.0, 40.7, 40.5* (*enantiomer); ESI-MS, m/z 842.0499 [M + H]+, 840.0331 [M − H]−. Plant Materials. Castor bean seeds (No. 9) were obtained from the Agricultural Science Academy of Zibo Shandong China. The seeds were treated and grown as previously described.8 Six day old seedlings were selected for the further experiments. Uptake by Ricinus communis Foliar Disks. Disks were obtained and treated with incubation medium containing 20 mM MES (pH 5.6), 250 mM mannitol, 0.25 mM MgCl2, and 0.5 mM CaCl2 as described formerly.8,15 Then the disks were transferred to the incubation medium supplemented with 2-NBDGTF to perform the time-course uptake experiment and to investigate the concentration dependence of 2-NBDGTF uptake. Disks were incubated with incubation medium, pH ranging from 4.0 to 8.0, to investigate the pH dependence, and treated at 4 or 28 °C to examine the temperature effect on the 2-NBDGTF uptake. For the inhibition experiments, the disks were preincubated with 50 μM CCCP, 1 mM phloridzin, 1 mM GTF, and 2 mM monosaccharide (glucose, xylose, arabinose, mannose, and galactose) in the incubation medium for 30 min, respectively. Then the disks were coincubated with 50 μM 2NBDGTF and these substrates for 1 h. Incubation was run under mild agitation on a reciprocal shaker at 28 °C. After the total incubation period, the disks were rinsed using the incubation solution three times to remove the residuals. Then the disks were freeze-dried using liquid nitrogen and then ground with 10 mL of methanol and ultrasonically treated for 30 min. The extract solutions were centrifuged at 12000 rpm/min for 10 min and then filtered with 0.22 μm filter. Then the extract solution was analyzed by HPLC system (Shimadzu, Japan). Phloem Sap Collection. As described previously,6 the cotyledons, from which the endosperm had been removed, were incubated in the buffer solution containing test compound 2-NBDGTF at a 100 μM concentration. GTF synthesized in our previous work was set as the positive control in this experiment. The roots of seedlings were immersed in 0.5 mM CaCl2 solution. After 1 h of incubation, the hypocotyl was severed in the hook region for phloem exudation. The interval of phloem sap collection was 1 h, and the duration was 5 h. The collected phloem sap was quantified by an HPLC system after dilution with pure water (phloem sap/pure water, 1/4, v/v). Visualization of Cotyledon Uptake and Translocation of 2NBDGTF in R. communis Seedlings. Visualization of uptake and translocation of 2-NBDGTF was investigated using 6 day old castor bean seedlings as described in our previous work.16 After treatment with 100 μM 2-NBDGTF incubation solution, cotyledons and stems were excised at 1 h, 2 h, and 3 h and washed thoroughly with incubation solution to remove the residual conjugate. Freehand cross sections of midveins and petioles were taken from the treated cotyledons, and sections of hypocotyl were immediately taken above the hook. Then sections were observed with confocal laser scanning

Table 1. Properties of the Tested Conjugatesa properties

GTF

2-NBDGTF

mol wt LogP pKa

680.4 2.06 12.8 ± 0.7 (acid)

842.5 3.31 12.8 ± 0.7 (acid)

a

The value of log Kow and pKa of GTF and 2-NBDGTF were predicted by ACD Laboratories Percepta program, version 14.0. Log Kow and pKa value was classic value.

Figure 2. Excitation and emission fluorescence spectra of fluorescent conjugate 2-NBDGTF. The maximum fluorescence excitation wavelength was at 470 nm, and the maximum emission wavelength was at 530 nm.

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RESULTS AND DISCUSSION Synthesis of 2-NBDGTF. As outlined in Scheme 1, glucosamine hydrochloride was chosen as starting material, and the amino group was protected by the trifluoroacetyl group to obtain compound 2. Reaction of compound 2 and TMS-N3 in the presence of SnCl4 afforded compound 3.17 Compound 5 was synthesized via click chemistry by introducing an azide group or an alkyne moiety to the corresponding compounds 3 8793



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Figure 3. Uptake of 2-NBDGTF by cotyledon disks obtained from castor bean seedlings. (A) Time course of 2-NBDGTF uptake by disks. Disks were incubated in a buffered solution containing 50 μM 2-NBDGTF at pH 5.6. Shown are the averages of 12 disks ± SE (n = 4). (B) Concentration dependence of 2-NBDGTF uptake. Disks were incubated in a buffered solution at pH 5.6 containing 2-NBDGTF concentration ranged from 0.005 mM to 1 mM. Disks coincubated with 50 μM CCCP were set as the complementary set (b). Each point was the mean of 12 disks ± SE (n = 6). (C) pH dependence of 2-NBDGTF uptake by disks. Each bar represents the mean of 12 disks ± SE (n = 4). (D) Effect of low temperature (4 °C) and inhibitor (50 μM CCCP and 1 mM phloridzin) on the 2-NBDGTF uptake. Each bar represents the mean of 12 disks ± SE (n = 5). (E) Effect of 2 mM monosaccharides on the uptake of 50 μM 2-NBDGTF at pH 5.6. Data are mean of 12 disks ± SE (n = 4). (F) Effect of 1 mM GTF on the uptake of 2-NBDGTF. Shown are the averages of 12 disks ± SE (n = 4). Statistical analysis was performed using the ANOVA test, and Dunnett’s test was used to analyze differences in the mean of each group with control group. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

identified by HRMS-ESI analysis (calculated for 2-NBDGTF [M + H]+, 842.0420; found, 842.0499;). From these results, the synthesized product was identified as 2-NBDGTF. The Foliar Disk Uptake of 2-NBDGTF Is CarrierMediated. When castor bean foliar disks were incubated with buffer solution supplemented with 50 μM 2-NBDGTF at pH 5.6 for up to 360 min, the rate of 2-NBDGTF uptake increased rapidly and almost linearly during the 120 min incubation period and then decreased until 360 min (Figure 3A). The results suggested that the uptake of 2-NBDGTF will reach equilibrium rapidly with increasing incubation times. To investigate the concentration dependence of 2-NBDGTF uptake, castor bean foliar disks were incubated with buffer solution supplemented with 2-NBDGTF at concentrations ranging from 0.005 to 1 mM for 1 h. Concentration dependence data clearly show that there were two components involved in 2-NBDGTF uptake. The first component is a saturable component that occurs between 0.005 and 0.2 mM,

and 4. NaOMe/MeOH was used to remove the acetyl and trifluoroacetyl groups. However, the trifluoroacetyl group remained intact after reaction to get compound 5. The trifluoroacetyl group was finally deprotected with 1 M NaOH solution and then reacted with NBD-Cl to obtain target product 2-NBDGTF. All products were identified by 1 H and 13 C NMR spectroscopy and mass spectrometry. The 1H NMR spectra of these compounds are complicated because fipronil has a symmetrical sulfoxide and forms two enantiomers.18,19 Their configurations were confirmed on the basis of a chemical shift, coupling constants, and band shape of H-1. For example, the NMR signals of product 2-NBDGTF were immediately measured after the dissolution of the glucosyl conjugates in denatured methanol. The doublet, observed at 6.01 and J1,2 = 9.5 Hz, was assigned to H-1 of the β-D-pyranose anomer. The synthesized compound C27H19Cl2F6N11O8S (2-NBDGTF) was further 8794



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solutes readily. Therefore, the castor bean seedling system is employed to evaluate phloem mobility of 2-NBDGTF in the present work. When the cotyledons were incubated in buffer solution containing 100 μM 2-NBDGTF, the conjugate was clearly detected in the phloem sap. Time-course experimental results indicated that the concentrations of 2-NBDGTF in the phloem sap increased almost linearly during the experimental period, which is consistent with GTF (Figure 4). The measured 2-

suggesting that part of 2-NBDGTF uptake is carrier-mediated at low concentrations. The second component is a roughly linear component at higher concentrations, indicating the existence of passive diffusion (Figure 3B). Upon cotreatment with 50 μM CCCP, which is widely utilized in studies of transmembrane transport to dissipate the proton motive force,8,15,20 the saturable component of 2-NBDGTF uptake appeared to be apparently inhibited (Figure 3b). To study the pH effect of incubation medium on 2-NBDGTF uptake, the disks were incubated with different pH buffer solution (pH ranging from 4.0 to 8.0). The results showed that uptake of 2NBDGTF was sensitive to pH changes in the incubation medium. The uptake of 2-NBDGTF apparently increased with decreasing incubation pH, and the reduction of pH from 8.0 to 4.0 increased the uptake of 2-NBDGTF approximately 2-fold (Figure 3C). The results above confirmed that 2-NBDGTF uptake in lower concentrations is proton motive force dependent. To further understand the uptake mechanism, the effect of temperature on 2-NBDGTF uptake was investigated. The uptake of 2-NBDGTF maintained at low temperature (4 °C) was reduced about 47% compared with normal incubation conditions (28 °C), suggesting that the 2-NBDGTF uptake machinery is energy dependent (Figure 3D). Phloridzin is an inhibitor of the sodium-linked glucose transporters,21 and it is also used as an inhibitor of plant hexose transporter.22,23 When treated with 1 mM phloridzin or 50 μM CCCP alone, the 2NBDGTF uptake was remarkably decreased about 29% and 31%, respectively (Figure 3D). The results suggested that the 2NBDGTF uptake machinery is also sensitive to phloridzin. To clarify the competitive inhibition of substrates, the effect of monosaccharides on the uptake of 2-NBDGTF was investigated. At 2 mM concentration, both monosaccharides significantly inhibited 2-NBDGTF uptake by 10−15% (Figure 3E). In the case of the CCCP-sensitive component of the conjugate, the influx was only about 31% of the total uptake when the disks were incubated with 50 μM 2-NBDGTF. At 2 mM concentrations, these monosaccharides led to a 0.05 mM 2-NBDGTF uptake of the CCCP sensitive component, totaling 30−50%. In addition, the effect of GTF on 2-NBDGTF uptake was also evaluated. When treated with 1 mM GTF, the uptake of 2-NBDGTF was dramatically reduced about 42% at 10 μM and 24% at 25 μM, respectively (Figure 3F). The results above suggested that both monosaccharides and GTF exhibited strong competition with the uptake of 2-NBDGTF. The characteristics of carrier-mediated transport mainly include that it is concentration dependent (saturable), subject to inhibition, more structure specific compared to passive transport, and cell type specific, which requires expression of the transporter.24 In the present research, the disk results showed that the uptake of 2-NBDGTF was concentration dependent, energy dependent, and subject to CCCP and phloridzin inhibition and involved competitive inhibition of monosaccharides. Besides, GTF exhibited strong competition with the uptake of 2-NBDGTF. Altogether, these disk uptake results demonstrated the involvement of a carrier-mediated system in the 2-NBDGTF uptake process. Phloem Sap Determination of 2-NBDGTF. Castor bean seedling is an ideal system to identify endogenous molecules moving in sieve tubes25,26 and to evaluate phloem systemicity of xenobiotics.27 Thanks to the cotyledon epidermis being thin and highly permeable and making contact with the external space directly, the system could respond to incubation with

Figure 4. Time course of the concentration of conjugates 2-NBDGTF and GTF in R. communis phloem sap. The cotyledons were incubated in a buffered solution containing 100 μM 2-NBDGTF or GTF at pH 5.0. The hook of the hypocotyl was severed after 1 h incubation, and then the sap was collected per hour during 5 h. Each bar represents the mean of 12 plants ± SE, n = 4.

NBDGTF concentrations in the phloem sap were 5.68 ± 0.69 μM, 10.61 ± 1.11 μM, and 22.45 ± 0.83 μM (mean ± SE) at 1 h, 3 h, and 5 h, respectively. These values represent GTF concentrations in the phloem sap of 0.68-fold, 0.64-fold, and 0.70-fold, respectively. The results showed that 2-NBDGTF exhibited comparable phloem mobility with GTF in castor bean seedlings. Moreover, the fluorescent glucose−fipronil conjugate present in the foliar disk extracts and phloem sap at 5 h was further identified by HPLC−HRMS (high resolution mass spectrometry) analysis. Calculated for 2-NBDGTF (C27H19Cl2F6N11O8S) was [M + H]+ 842.0420; and the found mass [M + H]+ of 2-NBDGTF in disk extracts was 842.0482 (Supporting Information, Figure 1B) and in phloem sap was 842.0480 (Supporting Information, Figure 1D), respectively. These results established that it was the intact molecule that was detected in the disk extracts and phloem sap, indicating that the fluorescent molecule 2-NBDGTF was relatively stable during the experimental period. The “Kleier model” was widely employed to predict the mobility of xenobiotics based on the physicochemical properties (log P and pKa).3,28 Taking the given physicochemical properties into consideration, 2-NBDGTF is a compound ranging into the non-phloem-mobile area which is similar to GTF (Figure 5). However, 2-NBDGTF presented comparable phloem mobility with GTF in castor bean seedlings. Most xenobiotics fitted well into this model except for some xenobiotics whose transport was carrier-mediated. This inconsistency between the predicting results and phloem mobility test confirmed that 2-NBDGTF was an exception to the “Kleier model” and the involvement of a carrier-mediated system in the uptake process. Introduction of fluorophores will inevitably bring some changes to physicochemical properties of the parent com8795



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Figure 5. Prediction of phloem mobility of GTF and 2-NBDGTF using Kleier map;1,32 log Kow and pKa values were calculated by ACD Laboratories Percepta program version 14.0.

pounds, including enlarging the molecular weight, increasing the octanol/water partitioning coefficient value, etc., which is consequently to have some effects on the parent compound characteristics. Compared with fluorescein (Fl) and Alexa Fluor 647 (AF), which have been employed successfully to label the gibberellins and castasterone,13,14 NBD is a fluorophore with lower molecular weight and good cell permeability and thus has been widely utilized to label the glucose. The fluorescent conjugate of glucose 2-NBDG, fluorescently modified with a small fluorescent moiety NBD of glucose at the C-2 position, has been employed as a fluorescent glucose replacement and widely used to monitor the cellular glucose uptake activity.29,30 It seems that the effect of the fluorescent tag NBD on the parent conjugate is tolerable. In comparison with GTF, 2NBDGTF is indeed a compound with larger MW, and higher log P and HBA value (Table 1). Our results showed that the fluorescent conjugate 2-NBDGTF retained the carrier-mediated uptake mechanism and moderate phloem mobile property of GTF. Hence, 2-NBDGTF is regarded as a reasonable fluorescent surrogate of GTF and is believed to be capable of revealing the movement of glucose−fipronil analogues within plants. Visualization of Uptake and Transport of 2-NBDGTF in Castor Bean Seedlings. The visualization of uptake and transport of 2-NBDGTF within castor bean seedlings was performed using confocal laser scanning microscopy (CLSM). The CLSM observation results showed the presence of remarkable fluorescence in epidermal cells and mesophyll cells (Figure 6B) of the midveins of the cotyledons treated with 2-NBDGTF for 1 h. Moreover, enormous fluorescence was observed present in the phloem. Ricinus is a symplastic− apoplastic loader, meaning that endogenous molecules from the seedling endosperm or incubation solution found in the phloem sap via the symplastic pathway may originate from the transfer cells of the lower epidermis. These molecules may have also been taken up directly from the phloem apoplast.26,27 The observed results of the presence of remarkable

Figure 6. Representative fluorescence photographs of six day old caster bean seedlings: The cotyledons were incubated in a buffered solution containing 100 μM 2-NBDGTF. (A, B) Cross sections of midveins of cotyledons, (C, D) cross sections of petioles of cotyledons, and (E, F) cross sections of hypocotyl of caster bean seedlings. All photos were observed by confocal laser scanning microscope (Zeiss780, Germany). No significant fluorescence was observed in the mesophyll cells or phloem of control set (A, C, E). The presence of fluorescent xenobiotics was indicated by arrowheads. EC: epidermal cells. CS: cell wall space. MC: mesophyll cells. PH: phloem. X: xylem.

fluorescence in epidermal cells and mesophyll cells obtained using 2-NBDGTF suggest that cotyledons could take up 2NBDGTF from the medium readily via lower epidermal cells. The cotyledons could then load the 2-NBDGTF into sieve tubes from the apoplast after transission through mesophyll cells in an indirect apoplasmic loading process or via plasmodesmata after uptake by mesophyll cells in a symplasmic loading process. The obvious fluorescence observed in the cell wall and intercellular space (Figure 6B) suggests that cotyledons may load 2-NBDGTF into sieve tubes directly via an apoplasmic loading pathway. The observed results show that the loading pathway of 2-NBDGTF is consistent with the phloem loading pathway of sucrose in Ricinus cotyledons.26 After a 2 h incubation, intense fluorescence was also detected in the phloem of cotyledon petiole (Figure 6D), signifying that the 2-NBDGTF efflux can rapidly export via the phloem of the 8796



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cotyledon petiole. Intense fluorescence was also observed in the phloem of the hypocotyls after receiving 3 h of treatment (Figure 6F). These results indicate that 2-NBDGTF is phloem mobile and can translocate at a distance (from cotyledon to hypocotyl) via phloem. The cells of 6 day old Ricinus seedling cotyledons appear as meristematic cells with well-developed organelles lacking large central vacuoles.31 Fluorescence present in epidermal and mesophyll cells was homogeneously distributed in the cytoplasm, but was not caused by the compartmentation trapping by the vacuole. Alternatively, the intense fluorescence present in the xylem of the treated cotyledons was likely caused by the direct loading into the vessels. The fluorescence observed in the xylem of petioles and hypocotyls may be the result of lateral diffusion during phloem translocation and the adsorption of the lipophilic xenobiotic on lignins. Since 2-NBDGTF was stable in the phloem during the experimental period, the fluorescence observed in the epidermal cells, mesophyll cells, and phloem could reflect the distribution of 2-NBDGTF, which would in turn reveal the distribution of GTF within plants. Accumulation spots of 2NBDGTF likely represent the tissue sites of GTF, meaning that GTF was also loaded into the sieve tubes after transiting through epidermal cells and mesophyll cells along a symplastic−apoplastic loading route and was able to translocate at a distance (from cotyledon to hypocotyl) via phloem within seedlings. In conclusion, a fluorescent glucose−fipronil conjugate was obtained via click chemistry. The uptake of disks of 2NBDGTF was demonstrated to involve a carrier-mediated uptake process, and the phloem mobility in castor bean seedlings was comparable with GTF. Visualization of 2NBDGTF movement showed that this fluorescent glucose− fipronil conjugate could be loaded into sieve tubes after transiting through epidermal cells and mesophyll cells and translocate at a distance via phloem within seedlings. From this information, 2-NBDGTF was determined to be a reasonably fluorescent surrogate of GTF and capable of revealing the movement of GTF analogues. The wide utilization of 2-NBDG provides us a substantial belief that 2-NBDGTF is a promising fluorescent conjugate. Moreover, this fluorescently labeled approach presents a great potential for revealing the activities of other glucose insecticides in plants.

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Notes

The authors declare no competing financial interest.

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ASSOCIATED CONTENT

S Supporting Information *

HPLC and mass spectra analysis of fluorescent molecule present in disk extracts and phloem sap from 2-NBDGTF treated caster bean seedlings. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

(1) Kleier, D. A. Phloem mobility of xenobiotics. V. Structural requirements for phloem-systemic pesticides. Pestic. Sci. 1994, 42 (1), 1−11. (2) Hsu, F. C.; Sun, K.; Kleier, D. A.; Fielding, M. J. Phloem mobility of xenobiotics VI. Preparation of a phloem-mobile pro-nematocide based on oxamyl, exhibiting root-specific activation in transgenic tobacco. Pestic. Sci. 1995, 44, 9−19. (3) Chollet, J. F.; Rocher, F.; Jousse, C.; Delétage-Grandon, C.; Bashiardes, G.; Bonnemain, J. L. Synthesis and phloem mobility of acidic derivatives of the fungicide fenpiclonil. Pest Manage. Sci. 2004, 60, 1063−1072. (4) Chollet, J.; Deletage, C.; Faucher, M.; Miginiac, L.; Bonnemain, J. Synthesis and structure-activity relationships of some pesticides with an alpha-amino acid function. Biochim. Biophys. Acta 1997, 1336 (2), 331−341. (5) Qin, P.; Wang, J.; Wang, H.; Wen, Y.; Lu, M.; Li, Y.; Xu, Y.; Xu, H. Synthesis of Rotenone-O-monosaccharide derivatives and their phloem mobility. J. Agric. Food Chem. 2014, 62 (20), 4521−4527. (6) Yang, W.; Wu, H.; Xu, H.; Hu, A.; Lu, M. Synthesis of glucosefipronil conjugate and its phloem mobility. J. Agric. Food Chem. 2011, 59, 12534−12542. (7) Yuan, J.; Wu, H.; Lu, M.; Song, G.; Xu, H. Synthesis of a series of monosaccharide-fipronil conjugates and their phloem mobility. J. Agric. Food Chem. 2013, 61, 4236−4241. (8) Wu, H. X.; Yang, W.; Zhang, Z.; Huang, T.; Yao, G.; Xu, H. Uptake and phloem transport of glucose-fipronil conjugate in Ricinus communis involve a carrier-mediated mechanism. J. Agric. Food Chem. 2012, 60, 6088−6094. (9) Aajoud, A.; Raveton, M.; Azrou-Isghi, D.; Tissut, M.; Ravanel, P. How can the fipronil insecticide access phloem? J. Agric. Food Chem. 2008, 56 (10), 3732−3737. (10) Alsayeda, H.; Pascal-Lorber, S.; Nallanthigal, C.; Debrauwer, L.; Laurent, F. Transfer of the insecticide [14C] imidacloprid from soil to tomato plants. Environ. Chem. Lett. 2008, 6 (4), 229−234. (11) Liu, Z. Q.; Gaskin, R. E. Visualisation of the uptake of two model xenobiotics into bean leaves by confocal laser scanning microscopy: diffusion pathways and implication in phloem translocation. Pest Manage. Sci. 2004, 60 (5), 434−439. (12) Wright, K. M.; Horobin, R. W.; Oparka, K. J. Phloem mobility of fluorescent xenobiotics in Arabidopsis in relation to their physicochemical properties. J. Exp. Bot. 1996, 47 (304), 1779−1787. (13) Shani, E.; Weinstain, R.; Zhang, Y.; Castillejo, C.; Kaiserli, E.; Chory, J.; Tsien, R. Y.; Estelle, M. Gibberellins accumulate in the elongating endodermal cells of Arabidopsis root. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (12), 4834−4839. (14) Irani, N. G.; Di Rubbo, S.; Mylle, E.; Van den Begin, J.; Schneider-Pizoń, J.; Hniliková, J.; Šíša, M.; Buyst, D.; Vilarrasa-Blasi, J.; Szatmári, A. M.; et al. Fluorescent castasterone reveals BRI1 signaling from the plasma membrane. Nat. Chem. Biol. 2012, 8 (6), 583−589. (15) Delétage-Grandon, C.; Chollet, J. F.; Faucher, M.; Rocher, F.; Komor, E.; Bonnemain, J. L. Carrier-mediated uptake and phloem systemy of a 350-Dalton chlorinated xenobiotic with an α-amino acid function. Plant Physiol. 2001, 125, 1620−1632. (16) Lei, Z.; Wang, J.; Mao, G.; Wen, Y.; Xu, H. Phloem mobility and translocation of fluorescent conjugate containing glucose and NBD in castor bean (Ricinus communis). J. Photochem. Photobiol., B 2014, 132, 10−16. (17) Cagnoni, A. J.; Varela, O.; Gouin, S. G.; Kovensky, J.; Uhrig, M. L. Synthesis of multivalent glycoclusters from 1-Thio-β-d-galactose and their inhibitory activity against the β-Galactosidase from E. coli. J. Org. Chem. 2011, 76 (9), 3064−3077. (18) Teicher, H. B.; Kofoed Hansen, B.; Jacobsen, N. Insecticidal activity of the enantiomers of fipronil. Pest Manage. Sci. 2003, 59 (12), 1273−1275.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-20-85285127. Fax: +8620-38604926. Author Contributions §

Authors contributed equally to this work.

Funding

This research was supported by the National Natural Science Foundation of China (Grant No. 31171886) and The Doctor Science Research Foundation of the Education Ministry of China (Grant No. 2011440411002) 8797



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(19) Zhao, Q.; Li, Y.; Xiong, L.; Wang, Q. Design, synthesis and insecticidal activity of novel phenylpyrazoles containing a 2, 2, 2trichloro-1-alkoxyethyl moiety. J. Agric. Food Chem. 2010, 58 (8), 4992−4998. (20) Hu, A.; Yang, W.; Xu, H. Novel fluorescent conjugate containing glucose and NBD and its carrier-mediated uptake by tobacco cells. J. Photochem. Photobiol., B 2010, 101 (3), 215−223. (21) Ehrenkranz, J. R.; Lewis, N. G.; Ronald Kahn, C.; Roth, J. Phlorizin: a review. Diabetes/Metab. Res. Rev. 2005, 21 (1), 31−38. (22) Etxeberria, E.; Gonzalez, P.; Tomlinson, P.; Pozueta-Romero, J. Existence of two parallel mechanisms for glucose uptake in heterotrophic plant cells. J. Exp. Bot. 2005, 56 (417), 1905−1912. (23) Stubbs, V.; Standing, D.; Knox, O.; Killham, K.; Bengough, A. G.; Griffiths, B. Root border cells take up and release glucose-C. Ann. Bot. 2004, 93 (2), 221−224. (24) Sugano, K.; Kansy, M.; Artursson, P.; Avdeef, A.; Bendels, S.; Di, L.; Ecker, G. F.; Faller, B.; Fischer, H.; Gerebtzoff, G.; Lennernaes, H.; Senner, F. Coexistence of passive and carrier-mediated processes in drug transport. Nat. Rev. Drug Discovery 2010, 9 (8), 597−614. (25) Kallarackal, J.; Orlich, G.; Schobert, C.; Komor, E. Sucrose transport into the phloem of Ricinus communis L. seedlings as measured by the analysis of sieve-tube sap. Planta 1989, 177 (3), 327− 335. (26) Orlich, G.; Komor, E. Phloem loading in Ricinus cotyledons: sucrose pathways via the mesophyll and the apoplasm. Planta 1992, 187 (4), 460−474. (27) Rocher, F.; Chollet, J. F.; Legros, S.; Jousse, C.; Lemoine, R.; Faucher, M.; Bush, D. R.; Bonnemain, J. L. Salicylic acid transport in Ricinus communis involves a pH-dependent carrier system in addition to diffusion. Plant Physiol. 2009, 150 (4), 2081−2091. (28) Rocher, F.; Chollet, J. F.; Jousse, C.; Bonnemain, J. L. Salicylic acid, an ambimobile molecule exhibiting a high ability to accumulate in the phloem. Plant Physiol. 2006, 141 (4), 1684−1693. (29) Yoshioka, K.; Takahashi, H.; Homma, T.; Saito, M.; Oh, K.; Nemoto, Y.; Matsuoka, H. A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli. Biochim. Biophys. Acta 1996, 1289 (1), 5−9. (30) Zou, C.; Wang, Y.; Shen, Z. 2-NBDG as a fluorescent indicator for direct glucose uptake measurement. J. Biochem. Biophys. Methods 2005, 64 (3), 207−215. (31) Komor, E.; Orlich, G.; Bauer-Ruckdeschel, H. Relationship between apoplastic nutrient concentrations and the long-distance transport of nutrients in the Ricinus communis L. seedling. In The Apoplast of Higher Plants: Compartment of Storage, Transport and Reactions; Springer: The Netherlands, 2007; pp 231−249. (32) Kleier, D. A. Phloem Mobility of Xenobiotics I. Mathematical model unifying the weak acid and intermediate permeability theories. Plant Physiol. 1988, 86 (3), 803−810.

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A Novel Fluorescent Conjugate Applicable To Visualize the

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