β-Glucosidase Involvement in the Bioactivation of Glycosyl Conjugates

Oct 29, 2014 - Mobile glucose–pesticide conjugates in the phloem are often restricted by decreases in biological activity. However, plants can bioac...
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β‑Glucosidase Involvement in the Bioactivation of Glycosyl Conjugates in Plants: Synthesis and Metabolism of Four Glycosidic Bond Conjugates in Vitro and in Vivo Qing Xia,∥ Ying-Jie Wen,∥ Hao Wang, Yu-Feng Li, and Han-Hong 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, Guangdong 510642, People’s Republic of China S Supporting Information *

ABSTRACT: Mobile glucose−pesticide conjugates in the phloem are often restricted by decreases in biological activity. However, plants can bioactivate endogenous glucosides, which are assumed as able to bioactivate exogenous conjugates. In this study, four glycosidic bonds (O-, S-, N-, and C-glycosidic bonds) of glucose−pesticide conjugates were designed and synthesized, and then metabolism assays were carried out in vitro and in vivo. Results showed that β-glucosidases played a role in the hydrolysis of O-glycosidic bond conjugates in Ricinus communis L. The liberated aglycons possessed insecticidal activities against Plutella xylostella L. and Spodoptera litura F. These results could help establish methods of circumventing the mutual exclusivity of phloem mobility and biological activity by hydrolyzing endogenous β-glucosidases. KEYWORDS: glycosylation, glycosidic bond, β-glucosidase, hydrolysis, insecticidal activity

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INTRODUCTION Phloem mobility enhances the efficacy of pesticides and has potential economic benefits for crop protection.1,2 The addition of a glycosyl group can confer phloem mobility to immobile or weakly mobile pesticides.3 Hydroxymethyloxamyl glucuronide has improved phloem mobility since the establishment of Kleier’s mathematical model, which indicates that glucuronidation can enable aglycon conversion into phloem-mobile conjugates.2,4 The desired phloem mobility of monosaccharide−pesticide conjugates can be achieved using endogenous plant monosaccharide transporters. Our group has previously synthesized fipronil−glucose (GTF) conjugate and a series of monosaccharide−fipronil conjugates to induce satisfactory phloem mobility.5,6 However, the insecticidal activities of these compounds significantly decrease because the series of monosaccharide−fipronil conjugates is negligibly effective against Spodoptera litura F. and Plutella xylostella L. Glycosyl groups have been added to pesticides to enhance phloem mobility, but they decrease biological activity. The inherent incompatibility between mobility and activity partially explains the scarcity of synthetic phloem-mobile fungicides and insecticides.2 The conflicting requirement for activity and mobility during the conferment of phloem mobility to immobile pesticides can be solved by two methods. In the first method, the target site delivery and interaction of the active compound do not encounter interferences when the mobility-conferring moiety is tethered to the compound.7 Chollet found that fenpiclonil could become a phloem-mobile compound by introducing a carboxyl group and that the derivatives of this pesticide exhibit favorable biological activities to inhibit Eutypa lata growth.8 The other approach is the use of pro-drugs that can be converted to active compounds in the bodies of treated plants © XXXX American Chemical Society

or pests through spontaneous or enzyme-catalyzed reactions. Phloem-mobile pro-drugs (hydroxymethyloxamyl glucuronide) could be activated within the root of transgenic tobaccos, which have been modified by genes of Escherichia coli βglucuronidase.2 Several endogenous β-glycosides have been detected in plants and found to have important functions in various aspects of plant physiology, such as growth, signaling, and chemical plant defense.9−12 Studies have focused on the hydrolytic mechanisms of endogenous conjugates. Brzobohaty analyzed the bioactivation of cytokinin glucoside, which is correlated with β-glucosidase in the maize root meristem.13 Free abscisic acid (ABA) is liberated from the inactive ABA glucose ester (ABA-GE) by AtBG1, an Arabidopsis β-glucosidase homologue localized to the endoplasmic reticulum. 14 Plants have established an excellent chemical defense system against the disruption of herbivores and microorganisms throughout their evolution.11,15 Several plant defense compounds are stored in inactive glycosylated forms; these forms exhibit a wide range of effects, such as increased water solubility, improved chemical stability, and protection of plants from the toxic effects of their defense systems.16 When plant cells are disrupted because of chewing insects, defense compounds are bioactivated by the hydrolysis of the glucosidic linkage catalyzed by β-glucosidases, and the plants are immediately protected against this disruption.11 The mechanism of pro-drugs should be used to design exogenous glucose−pesticide conjugates. These conjugates can Received: July 20, 2014 Revised: October 29, 2014 Accepted: October 29, 2014

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dx.doi.org/10.1021/jf5034575 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Figure 1. Chemical structures of O-, S-, N-, and C-glycosidic bonds in glucose−fipronil conjugates.

Figure 2. Metabolism of O-glycosidic bond in glucose−fipronil conjugates as catalyzed by β-glucosidase.

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be recognized and hydrolyzed by endogenous β-glucosidases to bioactivate aglycons and control pests, which are also performed by plant defense systems. Secondary plant metabolites are glycosylated within plants to O (OH− and COOH−), N, S, and C atoms by glycosyltransferases.16−18 In this study, we synthesize O-, S-, N-, and C-glycosidic bonds of glucose−fipronil conjugates (Figure 1) to investigate their metabolisms in vitro and in vivo. To verify metabolism and insecticidal activity, we introduce another synthesized Oglycosidic bond conjugate N-{3-cyano-1-[2,6-dichloro-4(trifluoromethyl)phenyl]-4-[(trifluoromethyl)-sulfinyl]-1H-pyrazol-5-yl}-2-aminoethyl-β-D-glucopyranoside (GOF; Figure 2), which possesses moderate phloem mobility.6 This study aimed to determine metabolic differences between four glycosidic bond conjugates and the activity of endogenous β-glucosidase, which is involved in the hydrolysis of exogenous glucose− pesticide conjugates in the plant. The results can help establish methods of circumventing the mutual exclusivity of phloem mobility and biological activity through the hydrolysis of endogenous β-glucosidases.

MATERIALS AND METHODS

General Information on Synthesis. All reagents and solvents were purchased from a commercial company. 1H NMR and 13C NMR spectra were obtained with a Bruker AV-600 instrument. Chemical shifts were expressed in parts per million with TMS as internal standard. High-resolution electrospray ionization mass spectra (HRESI-MS) were recorded on a Bruker maXis impact ESI-Q-TOF instrument. Analytical thin-layer chromatography (TLC) was carried out on precoated plates (silica gel GF254), and spots were visualized with a ZF-20D ultraviolet (UV) analyzer. Silica gel (200−300 mesh) was used for column chromatography. Synthesis of 2,3,4,6-Tetra-O-acetyl-(2-azidoethyl)-β-D-glucopyranoside (3a; Scheme 1). β-D-Glucose pentaacetate (5 g, 12.81 mmol) and 2-bromoethanol (3.2 g, 25.62 mmol, 2 equiv) were dissolved in 25 mL of anhydrous CH2Cl2. The solution was stirred under nitrogen and cooled to 0 °C, and then BF3·Et2O (2.1 mL, 16.65 mmol, 1.3 equiv) was added dropwise over a period of 30 min. The reaction was stirred for 1 h at 0 °C and overnight at room temperature. After dilution with CH2Cl2 and washing with cold water and saturated aqueous NaHCO3, the organic phase was dried over anhydrous Na2SO4 and evaporated under reduced pressure to yield 2a (4.55 g, 78%) as colorless oil. The product was subjected to the next step without further purification. B

dx.doi.org/10.1021/jf5034575 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Scheme 1a

Reagents and conditions: (1) 2-bromoethanol, BF3·Et2O, and CH2Cl2 overnight; (2) NaN3, 2:1 acetone/H2O, and reflux for 20 h; (3) CH2Br2, K2CO3, 6 h; (4) NaN3 and DMF at 90 °C for 12 h; (5) (ClCH2CO)2O, TEA, CH2Cl2, 0 °C and room temperature for 2 h and overnight, respectively; (6) NaN3 and DMF at 90 °C for 12 h; (7) DPPA, DBU, and DMF at 100 °C for 4 h; NaN3 at 100 °C for 12 h.

a

5.10 (t, J = 9.8 Hz, 1H), 5.06 (t, J = 9.7 Hz, 1H), 4.70 (d, J = 10.1 Hz, 1H), 4.41 (d, J = 13.5 Hz, 1H), 4.32 (d, J = 13.5 Hz, 1H), 4.23 (dd, J = 12.4, 4.9 Hz, 1H), 4.14 (dd, J = 12.4, 2.2 Hz, 1H), 3.74 (ddd, J = 10.1, 4.9, 2.3 Hz, 1H), 2.06 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H); 13 C NMR (CDCl3) δ 170.71, 170.21, 169.53, 169.45, 81.81, 76.31, 73.79, 70.12, 68.18, 62.05, 51.12, 20.81, 20.74, 20.69, 20.68. HRMS (ESI) calcd for C15H21N3NaO9S (M + Na)+ 442.0896, found 442.0891. Synthesis of 2,3,4,6-Tetra-O-acetyl-N-chloroacetyl-β-D-glucopyranosylamine (2c; Scheme 1). 2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosylamine20 (1c) was synthesized as previously described. The solution of 1c (1.74 g, 5.0 mmol) in anhydrous CH2Cl2 (30 mL) was cooled to 0 °C, anhydrous TEA (1.4 mL, 10.0 mmol, 2 equiv) was added dropwise, and then chloroacetic anhydride (1.28 g, 7.5 mmol, 1.5 equiv) was added to the solution in portions. The reaction mixture was stirred for 2 h at 0 °C, followed by stirring at room temperature overnight. After dilution with CH2Cl2 and washing with cold water and saturated aqueous NaHCO3, the organic phase was dried over anhydrous Na2SO4 and evaporated in vacuo. The residues were purified by column chromatography (hexane/EtOAc 1:1) to afford the product 2c (1.17 g, 55%) as a white solid: 1H NMR (CDCl3) δ 7.31 (d, J = 8.9 Hz, 1H), 5.32 (t, J = 9.5 Hz, 1H), 5.20 (t, J = 9.2 Hz, 1H), 5.07 (t, J = 9.7 Hz, 1H), 5.00 (t, J = 9.6 Hz, 1H), 4.29 (dd, J = 12.5, 4.4 Hz, 1H), 4.13−3.95 (m, 3H), 3.83 (ddd, J = 10.1, 4.3, 2.1 Hz, 1H), 2.08 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H); 13C NMR (CDCl3) δ 170.88, 170.69, 169.97, 169.60, 166.95, 78.63, 73.92, 72.68, 70.38, 68.21, 61.69, 42.36, 20.83−20.68 (4C). HRMS (ESI) calcd for C16H22ClNNaO10 (M + Na)+ 446.0830, found 446.0824. Synthesis of 2,3,4,6-Tetra-O-acetyl-N-azidoacetyl-β-D-glucopyranosylamine (3c; Scheme 1). NaN3 (650.0 mg, 10.0 mmol, 5 equiv) was added to a solution of 2c (847.6 mg, 2 mmol) in 20 mL of DMF. The reaction mixture was stirred at 90 °C for 12 h, diluted with CH2Cl2, and washed with saturated aqueous NaHCO3, water, and brine. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by column chromatography (hexane/EtOAc 2:1) to afford product 3c (780 mg, 91%) as a white amorphous solid: 1H NMR (CDCl3) δ 7.11 (d, J = 9.1 Hz, 1H), 5.31 (t, J = 9.5 Hz, 1H), 5.22 (t, J = 9.3 Hz, 1H), 5.07 (t, J = 9.8 Hz, 1H), 4.97 (t, J = 9.6 Hz, 1H), 4.29 (dd, J = 12.5, 4.4 Hz, 1H), 4.09 (dd, J = 12.5, 2.1 Hz, 1H), 4.03−3.90 (m, 2H), 3.83 (ddd, J = 10.1, 4.3, 2.1 Hz, 1H), 2.08 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H); 13C NMR (CDCl3) δ 170.96, 170.68, 169.96, 169.62, 167.57, 78.25, 73.90, 72.71, 70.58, 68.22, 61.71, 52.69, 20.81,

NaN3 (3.25 g, 50 mmol, 5 equiv) was added to a solution of 2a (4.55 g, 10.0 mmol) in acetone (20 mL) and water (10 mL). The reaction mixture was heated under reflux for 20 h, diluted with CH2Cl2, and sequentially washed with saturated aqueous NaHCO3, water, and brine. The organic layer was dried over anhydrous Na2SO4 and evaporated to a residue, which was purified by column chromatography (hexane/EtOAc 3:2) to afford 2-azidoethyl glycoside (3a) (3.75 g, 85%) as a clear glass: 1H NMR (CDCl3) δ 5.18 (t, J = 9.5 Hz, 1H), 5.06 (t, J = 9.7 Hz, 1H), 4.99 (t, J = 8.8 Hz, 1H), 4.57 (d, J = 8.0 Hz, 1H), 4.22 (dd, J = 12.3, 4.7 Hz, 1H), 4.13 (dd, J = 12.3, 2.0 Hz, 1H), 4.00 (dt, J = 10.5, 4.1 Hz, 1H), 3.72−3.63 (m, 2H), 3.46 (ddd, J = 11.7, 7.6, 3.3 Hz, 1H), 3.26 (dt, J = 13.4, 4.0 Hz, 1H), 2.06 (s, 3H), 2.02 (s, 3H), 1.99 (s, 3H), 1.97 (s, 3H); 13C NMR (CDCl3) δ 170.65, 170.26, 169.41(2C), 100.69, 72.81, 71.96, 71.10, 68.58, 68.36, 61.87, 50.55, 20.75, 20.69, 20.61 (2C). HRMS (ESI) calcd for C16H23N3NaO10 (M + Na)+ 440.1281, found 440.1276. Synthesis of Bromomethyl 2,3,4,6-Tetra-O-acetyl-1-thio-β-Dglucopyranoside (2b; Scheme 1). 2,3,4,6-Tetra-O-acetyl-β-D-1-thioglucopyranosyl19 (1b) was synthesized as previously described. K2CO3 (3.03 g, 21.9 mmol, 2 equiv) was added to a stirred solution of 1b (4 g, 10.95 mmol) in CH2Br2 (20 mL), and the mixture was stirred at room temperature. TLC was used to determine the disappearance of the starting material (about 6 h), and then the solid was removed by filtration and the filtrate was concentrated. The residue was purified by column chromatography (hexane/EtOAc 3:2) to afford product 2b (3.51 g, 70%) as a clear glass: 1H NMR (CDCl3) δ 5.28 (t, J = 9.4 Hz, 1H), 5.13−5.06 (m, 2H), 4.81 (d, J = 10.2 Hz, 1H), 4.76 (d, J = 11.0 Hz, 1H), 4.55 (d, J = 11.0 Hz, 1H), 4.25 (dd, J = 12.5, 5.0 Hz, 1H), 4.17 (dd, J = 12.4, 2.3 Hz, 1H), 3.78 (ddd, J = 10.1, 5.0, 2.3 Hz, 1H), 2.08 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H); 13 C NMR (CDCl3) δ 170.71, 170.22, 169.54, 169.50, 82.05, 76.39, 73.86, 69.91, 68.34, 62.01, 32.11, 20.86, 20.75, 20.71, 20.70. HRMS (ESI) calcd for C15H21BrNaO9S (M + Na)+ 478.9988, found 478.9982. Synthesis of Azidomethyl 2,3,4,6-Tetra-O-acetyl-1-thio-β-D-glucopyranoside (3b; Scheme 1). NaN3 (2.13 g, 32.8 mmol, 5 equiv) was added to a solution of 2b (3.0 g, 6.56 mmol) in 30 mL of DMF. The reaction mixture was stirred at 90 °C for 12 h, diluted with CH2Cl2, and washed with saturated aqueous NaHCO3, water, and brine. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residues was purified by column chromatography (hexane/EtOAc 2:1) to afford product 3b (2.48 g, 90%) as a white amorphous solid: 1H NMR (CDCl3) δ 5.23 (t, J = 9.4 Hz, 1H), C

dx.doi.org/10.1021/jf5034575 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Scheme 2a

Reagents and conditions: (1) CuSO4·5H2O, sodium ascorbate, and 1:1 t-BuOH/H2O at 60 °C for 3 h; (2) 1 M NaOMe/MeOH at room temperature for 30 min. a

Scheme 3a

Reagents and conditions: (1) CuSO4·5H2O, sodium ascorbate, and 1:1 t-BuOH/H2O at 60 °C for 3 h; (2) anhydrous FeCl3 and CH2Cl2 at room temperature. a

20.70, 20.66 (2C). HRMS (ESI) calcd for C16H22N4NaO10 (M + Na)+ 453.1234, found 453.1228. 3-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-1-propanol21 (2d; Scheme 1). 2d was synthesized as previously described to afford the compound as a white amorphous solid: 1H NMR (CDCl3) δ 7.35− 7.26 (m, 18H), 7.15−7.10 (m, 2H), 4.93 (d, J = 10.9 Hz, 1H), 4.80 (dd, J = 14.1, 10.9 Hz, 2H), 4.70 (d, J = 11.7 Hz, 1H), 4.61 (dd, J = 11.8, 10.1 Hz, 2H), 4.48 (dd, J = 18.7, 11.5 Hz, 2H), 4.07−4.00 (m, 1H), 3.80−3.53 (m, 9H), 1.82−1.77 (m, 2H), 1.72−1.58 (m, 2H); 13C NMR (CDCl3) δ 138.86, 138.42, 138.30, 138.07, 128.58−127.74 (20C), 82.54, 80.36, 78.39, 75.60, 75.17, 74.52, 73.66, 73.30, 71.32, 69.33, 62.54, 29.39, 21.10. HRMS (ESI) calcd for C37H42NaO6 (M + Na)+ 605.2879, found 605.2874. Synthesis of 3-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-1-azidopropyl (3d; Scheme 1). Compound 2d (582.73 mg, 1 mmol) was dissolved in anhydrous DMF (15 mL), DPPA (650 μL, 3 mmol, 3 equiv), and DBU (150 μL, 1 mmol, 1 equiv) added dropwise. The solution was stirred at 100 °C for 4 h and then cooled to room temperature. Sodium azide (195 mg, 3 mmol, 3 equiv) was added to

the solution in one portion, and the reaction mixture was stirred at 100 °C for an additional 12 h. After cooling to room temperature, the solution was diluted with diethyl ether (100 mL). The organic phase was then washed with H2O (100 mL) and saturated aqueous NaCl (100 mL), dried over anhydrous Na2SO4, and then evaporated in vacuo. The residue was purified by column chromatography (hexane/ EtOAc 8:1) to afford 3d (2.48 g, 90%) as a colorless oil: 1H NMR (CDCl3) δ 7.43−7.27 (m, 18H), 7.16−7.07 (m, 2H), 4.92 (d, J = 10.9 Hz, 1H), 4.80 (dd, J = 10.8, 4.7 Hz, 2H), 4.71 (d, J = 11.7 Hz, 1H), 4.66−4.55 (m, 2H), 4.47 (dd, J = 11.4, 7.1 Hz, 2H), 4.05−3.91 (m, 1H), 3.82−3.21 (m, 9H), 1.85−1.67 (m, 3H); 13C NMR (CDCl3) δ 138.88, 138.38, 138.32, 138.16, 128.61−127.75 (20C), 82.59, 80.25, 78.29, 75.63, 75.21, 73.91, 73.68, 73.43, 71.35, 69.22, 51.31, 25.16, 22.01. HRMS (ESI): calcd for C37H41N3NaO5 (M + Na)+ 630.2944, found 630.2938. General Procedure for the Synthesis of Title Compounds (4a−4d; Schemes 2 and 3). The propyn-fipronil donor5 was synthesized as previously described. Compounds 3a−3d (1 mmol) were added to a solution of propyn-fipronil donor (1 mmol) in 3 mL D

dx.doi.org/10.1021/jf5034575 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Scheme 4a

a

Reagents and conditions: (1) NaN3 and water at 85 °C for 16 h; (2) CuSO4·5H2O, sodium ascorbate, and 1:1 t-BuOH/H2O at 60 °C for 3 h.

of tert-butyl alcohol. Then, a solution of CuSO4·5H2O (100 mg, 0.4 mmol) and sodium ascorbate (173 mg, 0.8 mmol) in distilled water (3 mL) was further added. The yellow solution was vigorously stirred at 60 °C for 3 h and quenched by adding water (10 mL). The aqueous layer was extracted with CH2Cl2 (10 mL × 3). The combined organic extracts were washed with aqueous sodium hydrogen carbonate and brine, dried with anhydrous Na2SO4, filtered, and evaporated in vacuo. The residues were purified by column chromatography (hexane/ EtOAc 1:1) to afford the desired products as solids. N-{3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)-phenyl]-4[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl}-1-[2,3,4,6-tetra-O-acetyl(2-triazolethyl)-β-D-glucopyranoside]-1H-1,2,3-triazole-4-methanamine (4a): white solid; yield 85%; 1H NMR (CDCl3) δ 7.75 (d, J = 10.2 Hz, 2H), 7.53 (s, 1H), 6.24 (t, J = 5.6 Hz, 1H), 5.16 (t, J = 9.5 Hz, 1H), 5.03 (t, J = 9.7 Hz, 1H), 4.86 (dd, J = 9.5, 7.9 Hz, 1H), 4.50 (t, J = 4.9 Hz, 2H), 4.46 (d, J = 7.8 Hz, 1H), 4.37 (ddd, J = 30.0, 14.7, 5.6 Hz, 2H), 4.21 (dd, J = 12.4, 5.0 Hz, 1H), 4.17−4.10 (m, 1H), 4.08 (dd, J = 12.4, 2.3 Hz, 1H), 3.91 (dt, J = 10.8, 5.4 Hz, 1H), 3.68 (ddd, J = 9.9, 4.9, 2.3 Hz, 1H), 2.03 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.89 (s, 3H); 13C NMR (CDCl3) δ 170.64, 170.11, 169.60, 169.47, 151.24, 142.78, 136.66, 136.53, 135.48, 135.06 (q, J = 34.4 Hz), 127.32, 126.49, 126.32, 125.46 (q, J = 336.3 Hz), 123.77, 121.93 (q, J = 274.1 Hz), 110.53, 100.70, 96.18, 72.32, 72.14, 71.10, 68.26, 67.84, 61.79, 50.37, 41.21, 20.71, 20.62 (2C), 20.59. HRMS (ESI): calcd for C31H30Cl2F6N7O11S (M+H)+ 892.1005, found 892.1000. N-{3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)-phenyl]-4[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl}-1-(2,3,4,6-Tetra-O-acetyl-β- D-glucopyranosylthiomethyl)-1H-1,2,3-triazole-4-methanamine (4b). White solid; yield 85%. 1H NMR (CDCl3) δ 7.77 (s, 2H), 7.70 (d, J = 2.7 Hz, 1H), 6.19 (d, J = 6.3 Hz, 1H), 5.61 (dd, J = 14.7, 10.2 Hz, 1H), 5.31 (dd, J = 14.7, 10.5 Hz, 1H), 5.19 (td, J = 9.4, 3.4 Hz, 1H), 5.09−4.99 (m, 2H), 4.59 (dd, J = 10.1, 5.6 Hz, 1H), 4.45 (dd, J = 15.0, 6.0 Hz, 1H), 4.32 (dd, J = 14.8, 5.0 Hz, 1H), 4.09−4.00 (m, 2H), 3.67−3.63 (m, 1H), 2.02 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.99 (s, 3H); 13C NMR (CDCl3) δ 170.78, 170.09, 169.53, 169.43, 151.24, 143.94, 136.62 (2C), 135.37, 135.26 (q, J = 34.7 Hz), 127.44, 126.65, 126.44, 125.49 (q, J = 336.1 Hz), 122.60, 121.96 (q, J = 274.0 Hz), 110.46, 96.06, 82.30, 76.52, 73.49, 69.55, 68.02, 61.44, 49.04, 40.84, 20.86, 20.66 (3C). HRMS (ESI): calcd for C30H28Cl2F6N7O10S2 (M + H)+ 894.0620, found 894.0615. N-{3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)-phenyl]-4[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl}-1-(2,3,4,6-tetra-O-acetylN-triazolylacetyl-β-D-glucopyranosylamine)-1H-1,2,3-triazole-4methanamine (4c): white solid; yield 85%; 1H NMR (CDCl3) δ 7.75 (s, 2H), 7.67 (d, J = 9.7 Hz, 1H), 7.00 (dd, J = 26.1, 8.9 Hz, 1H), 6.29 (dd, J = 10.4, 6.1 Hz, 1H), 5.28 (t, J = 9.5 Hz, 1H), 5.19 (t, J = 9.2 Hz, 1H), 5.09−4.94 (m, 3H), 4.84 (dt, J = 11.9, 9.6 Hz, 1H), 4.52 (dd, J = 15.0, 6.7 Hz, 1H), 4.37−4.18 (m, 2H), 4.07 (d, J = 11.2 Hz, 1H), 3.84−3.78 (m, 1H), 2.04 (s, 3H), 2.02 (s, 3H), 2.00 (s, 6H); 13C NMR (CDCl3) δ 171.18, 170.73, 169.94, 169.68, 165.58, 151.19, 143.78, 136.55 (2C), 135.29, 135.12 (q, J = 34.5 Hz), 127.36, 126.46 (2C), 125.45 (q, J = 336.7 Hz), 124.08, 121.91 (q, J = 274.1 Hz), 110.51, 96.29, 78.37, 73.89, 72.59, 70.62, 68.13, 61.74, 52.58, 40.83, 20.77, 20.70, 20.65, 20.60. HRMS (ESI): calcd for C31H28Cl2F6N8NaO11S (M + Na)+ 927.0777, found 927.0772.

N-{3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl}-1-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)propyl-1H-1,2,3-triazole-4-methanamine (4d): white solid; yield 85%; 1H NMR (CDCl3) δ 7.73 (d, J = 5.7 Hz, 2H), 7.41 (s, 1H), 7.35−7.23 (m, 18H), 7.17−7.10 (m, 2H), 6.20 (d, J = 16.0 Hz, 1H), 4.89 (d, J = 10.9 Hz, 1H), 4.79 (t, J = 11.1 Hz, 2H), 4.70 (d, J = 11.7 Hz, 1H), 4.57 (dd, J = 11.6, 9.3 Hz, 2H), 4.52−4.21 (m, 6H), 3.98 (d, J = 9.8 Hz, 1H), 3.71 (d, J = 2.4 Hz, 2H), 3.64 (s, 2H), 3.57−3.48 (m, 2H), 2.04−1.85 (m, 2H), 1.79−1.63 (m, 2H); 13C NMR (CDCl3) δ 151.43, 143.08, 138.65, 138.18, 138.08, 137.99, 136.52 (2C), 135.50, 135.24 (q, J = 34.7 Hz), 128.65−127.76 (20C), 127.34, 126.54, 126.38, 125.50 (q, J = 336.5 Hz), 121.89 (q, J = 274.1 Hz), 121.73, 110.38, 95.61, 82.26, 79.86, 78.03, 75.54, 75.19, 73.68, 73.66, 73.47, 71.55, 69.22, 50.25, 40.95, 26.71, 21.89. HRMS (ESI): calcd for C52H48Cl2F6N7O6S (M + H)+ 1082.2668, found 1082.2663. General Procedure for the Synthesis of Title Compounds (5a−5c; Scheme 2). The peracetylated compounds 4a−4c (1 mmol) were dissolved in 10 mL of dry MeOH. To this solution was added freshly prepared NaOMe (1 M, 0.5 mL) with stirring at room temperature for 30 min. The reaction mixture was neutralized with Amberlite IR 120 (H+) resin and filtered, and then the filtrate was evaporated. The residues were purified by column chromatography (chloroform/methanol 15:1) to afford the desired products as solids. N-{3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl}-1-(2-triazolethyl-β-D-glucopyranoside)-1H-1,2,3-triazole-4-methanamine (5a): white solid; yield 60%; 1H NMR (MeOD) δ 8.08 (d, J = 3.9 Hz, 2H), 8.00 (s, 1H), 4.66−4.57 (m, 3H), 4.45 (d, J = 16.1 Hz, 1H), 4.31 (dd, J = 7.8, 5.5 Hz, 1H), 4.20 (ddd, J = 10.1, 6.9, 4.0 Hz, 1H), 3.99 (dq, J = 6.6, 3.7 Hz, 1H), 3.86 (d, J = 11.9 Hz, 1H), 3.66−3.60 (m, 1H), 3.39−3.32 (m, 1H), 3.29−3.22 (m, 2H), 3.17 (ddd, J = 9.1, 7.9, 4.9 Hz, 1H); 13C NMR (MeOD) δ 151.87, 144.90, 137.90 (2C), 136.13, 136.12 (q, J = 34.3 Hz), 127.95, 127.86, 126.95 (q, J = 335.8 Hz), 125.13 (2C), 123.63 (q, J = 273.2 Hz), 112.18, 104.75, 97.59, 78.13, 78.03, 74.96, 71.62, 69.28, 62.78, 51.72, 41.20. HRMS (ESI): calcd for C23H22Cl2F6N7O7S (M + H)+ 724.0582, found 724.0577. N-{3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl}-1-(β-D-glucopyranosylthiomethyl)-1H-1,2,3-triazole-4-methanamine (5b): white solid; yield 60%; 1H NMR (MeOD) δ 8.08 (d, J = 3.1 Hz, 1H), 8.06 (s, 2H), 5.82 (dd, J = 14.6, 2.7 Hz, 1H), 5.53 (dd, J = 14.6, 2.4 Hz, 1H), 4.61 (dd, J = 16.1, 3.3 Hz, 1H), 4.50−4.40 (m, 2H), 3.92−3.82 (m, 1H), 3.62 (dd, J = 11.9, 5.7 Hz, 1H), 3.35−3.17 (m,4H); 13C NMR (DMSO) δ 150.18, 143.54, 136.02 (2C), 134.50, 133.61 (q, J = 33.7 Hz), 126.87 (2C), 125.73, 125.46 (q, J = 337.7 Hz), 123.51, 122.23 (q, J = 274.2 Hz), 111.53, 95.59, 83.32, 81.29, 79.16, 78.07, 73.30, 70.16, 61.40, 47.44. HRMS (ESI): calcd for C22H20Cl2F6N7O6S2 (M + H)+ 726.0197, found 726.0192. N-{3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl}-1-(N-triazolylacetyl-β-Dglucopyranosylamine)-1H-1,2,3-triazole-4-methanamine (5c): white solid; yield 60%; 1H NMR (MeOD) δ 8.00 (d, J = 2.3 Hz, 2H), 7.81 (s, 1H), 5.20−5.08 (m, 2H), 4.92 (d, J = 9.1 Hz, 1H), 4.43 (s, 2H), 3.82 (dd, J = 11.9, 2.1 Hz, 1H), 3.65 (dd, J = 11.9, 5.2 Hz, 1H), 3.41 (t, J = 8.8 Hz, 1H), 3.36−3.33 (m, 1H), 3.32−3.26 (m, 2H); 13 C NMR (MeOD) δ 175.20, 161.33, 152.19, 145.12 (2C), 144.21, E

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After incubation at 37 °C for 1 h, the reaction was stopped by boiling for 3 min. The reaction mixture was analyzed with an Agilent 1100 HPLC system on a C18 reversed-phase column (5 μm; 250 × 4.6 mm i.d.; Agilent Co.) with a flow rate of 1.0 mL min−1 at 30 °C, and the eluent was made of water and acetonitrile (50:50, v/v). Glucoside and aglycon were detected by UV absorption at 210 nm. For the identification of reaction products, UHPLC-HRMS analyses were performed. The UHPLC system consisting of an autosampler, a binary pump, and a diode array detector (Agilent Technologies 1290 infinity) was interfaced to a time-of-flight (TOF) mass spectrometer (Bruker Maxis, Germany). SB-C18 RRHD (1.8 μm, 50 × 2.1 mm, Agilent Technologies) was used for the separation. Column temperature was set at 30 °C, and injection volume was 5 μL. The binary mobile phase consisted of (A) 100% water and (B) 100% acetonitrile, and the 8 min gradient was described as follows: 0−1 min, 10% B isocratic; 1−4 min, 90% B; 4−8 min, 10% B, at a flow rate of 0.2 mL min−1. The detection wavelength was 210 nm. Electrospray ionization in positive mode was performed under the following conditions: nebulizer, 0.3 bar; drying gas, 4.0 L min−1; drying temperature, 180 °C; and capillary, 3500 V. The full scan mass spectra of the compounds were recorded within m/z 50−1000. Glucoside Metabolism Assays in Vivo. These assays were performed following a previous method.25 In a typical procedure, an aqueous solution (10 mM, equal volumes of 10 μL in each petiole) was injected into the petioles of the two mature leaves using a 10 μL microsyringe. After the application of glucosides, the plants were grown for 4 h, and then the blades of the mature leaves were separated from the plants. The compounds were extracted from the leaves (2.0 g) by grinding in 10 mL of methanol with a glass mortar and pestle. After ultrasonic treatment for 20 min, a portion (2.0 mL) of the extract was centrifuged at 15000 rpm for 5 min and the supernatant passed through a 0.22 μm filter. Then, the treated solutions were analyzed by HPLC. Recoveries of parent compound were >85% conducted by adding known amounts of glucosides with three replicates. Insects. Larvae of S. litura F. were obtained from the Guangzhou Biological Control Station and raised with artificial feed under cage conditions for three generations. Healthy larvae of P. xylostella L. were collected from the experimental farm of Dongguan Agricultural Science Research Center, Guangdong, China, and reared on Chinese cabbage (Brassica rapa) under cage conditions for three generations. The feeding conditions were as follows: temperature, 26 ± 2 °C; relative humidity, 70%; and photoperiod, 16:8 h (light/dark). Insecticidal Activity. To investigate the insecticidal activities of FTO (Scheme 4) and FOH,6 we synthesized the aglycons beforehand. The insecticidal activities of the compounds GOTF, FTO, GOF, FOH, and fipronil against P. xylostella L. and S. litura F. were evaluated by leaf disk dipping assay.26 The leaf disks (1.8 cm diameter) were cut from fresh cabbage grown in a greenhouse. The compounds were dissolved in acetone and suspended in distilled water containing Tween 80 (0.1%). Leaf disks were dipped in each test solution for 30 s and allowed to dry for 2 h. After air-drying, the treated leaf disks were placed in Petri dishes (9 cm diameter) lined with filter paper, and then the second-instar larvae of P. xylostella L. and S. litura F. were transferred to a Petri dish. Three replicates (10 larvae per replicate) were carried out. Fipronil was used as a standard. Petri dishes were kept in an incubator at 26 °C and 85% relative humidity under a photoperiod of 16:8 h (light/dark). Mortalities were determined 24 h after treatment.

143.30 (q, J = 34.2 Hz), 137.76, 136.39 (2C), 133.91, 131.86 (q, J = 274.2 Hz), 129.39 (q, J = 325.2 Hz), 119.61, 100.91, 89.14, 88.65, 88.19, 86.82, 82.12, 79.38, 70.35, 49.67. HRMS (ESI): calcd for C23H21Cl2F6N8O7S (M + H)+ 737.0535, found 737.0530. General Procedure for the Synthesis of Title Compound 5d (Scheme 3). Anhydrous FeCl3 (12 mmol) was added to a stirred solution of compound 4d (1 mmol) in dry CH2Cl2 (10 mL) at room temperature under nitrogen. The reaction was left undisturbed until the color of the reaction mixture became brown. The solution was then evaporated under reduced pressure, and the residue was purified by column chromatography (chloroform/methanol 15:1) to afford the desired product 5d as a white solid. N-{3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl}-1-(β-D-glucopyranosyl)propyl-1H-1,2,3-triazole-4-methanamine (5d): white solid; yield 55%; 1H NMR (MeOD) δ 8.08 (s, 2H), 7.87 (d, J = 2.3 Hz, 1H), 4.61 (dd, J = 16.1, 3.8 Hz, 1H), 4.50−4.36 (m, 3H), 3.93−3.87 (m, 1H), 3.78 (dd, J = 11.8, 2.1 Hz, 1H), 3.59 (ddd, J = 15.3, 10.6, 6.0 Hz, 2H), 3.47 (t, J = 9.0 Hz, 1H), 3.36−3.32 (m, 1H), 3.24−3.17 (m, 1H), 2.13−2.02 (m, 1H), 1.96−1.85 (m, 1H), 1.74−1.58 (m, 2H); 13C NMR (MeOD) δ 151.87, 145.11, 137.88 (2C), 136.14, 136.13 (q, J = 34.6 Hz), 127.97, 127.85, 126.94 (q, J = 335.8 Hz), 123.98 (2C), 123.62 (q, J = 272.9 Hz), 112.17, 97.60, 76.66, 75.18, 74.67, 72.94, 72.35, 63.24, 51.23, 41.18, 27.78, 22.65. HRMS (ESI): calcd for C24H24Cl2F6N7O6S (M + H)+ 722.0790, found 722.0785. Synthesis of N-{3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4- [(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl}-1-ethanol1H-1,2,3-triazole-4-methanamine (FTO; Scheme 4). 2-Bromoethanol (3.2 g, 25.62 mmol) was dissolved in water (12 mL), and sodium azide (2.5 g, 38.5 mmol, 1.5 equiv) was added to the solution. The reaction mixture was stirred at 85 °C for 16 h and quenched by adding cold water (10 mL). Diethyl ether (50 mL) was added, and the organic layer was washed with saturated aqueous NaHCO3, water, and brine. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure to yield 2-azidoethanol (1.56 g, 70%) as a colorless oil. The product was subjected to the next step without further purification. 2-Azidoethanol (0.5 g, 5.74 mmol) was added to a solution of propyn-fipronil (2.72 g, 5.74 mmol) in 15 mL of tert-butyl alcohol. Then, a solution of CuSO4·5H2O (575 mg, 2.3 mmol, 0.4 equiv) and sodium ascorbate (911.3 mg, 4.6 mmol, 0.8 equiv) in distilled water (15 mL) was added. The yellow solution was vigorously stirred at 60 °C for 3 h and then quenched by adding water (10 mL). The aqueous layer was extracted with CH2Cl2 (15 mL × 3). The combined organic extracts were washed with aqueous sodium hydrogen carbonate and brine, dried with anhydrous Na2SO4, filtered, and evaporated in vacuo. The residues were purified by column chromatography (hexane/ EtOAc 2:3) to afford FTO (2.64 g, 82%) as a white solid: 1H NMR (CDCl3) δ 7.76 (d, J = 2.6 Hz, 1H), 7.60 (s, 1H), 6.04 (t, J = 5.6 Hz, 1H), 4.47−4.44 (m, 1H), 4.40 (dd, J = 15.1, 6.2 Hz, 1H), 4.28 (dd, J = 15.1, 5.8 Hz, 1H), 4.08−4.01 (m, 1H), 2.23 (s, 1H); 13C NMR (CDCl3) δ 153.75, 146.32, 137.81 (2C), 136.59, 136.03 (q, J = 34.6 Hz), 129.81, 127.53, 126.78 (q, J = 334.6 Hz), 125.79 (2C), 122.13 (q, J = 272.8 Hz), 110.90, 96.81, 60.58, 48.74, 43.17. HRMS (ESI): calcd for C17H12Cl2F6N7O2S (M + H)+ 562.0055, found 562.0049. β-Glucosidase Enzymes. β-Glucosidase from almonds was purchased from Sigma-Aldrich. A 10 U/mL solution was prepared. Growth of Plants. This procedure is reported in detail elsewhere.22,23 Six-day-old castor bean seedlings were selected for cotyledon absorption experiments. Adult plants of castor bean were grown in half-strength Hoagland’s solution for 3−4 weeks until they possessed four true leaves. The cotyledons and primary leaves were cut, and the plants were tested 2−3 days later. Glucoside Metabolism Assays in Vitro. A modification of the phosphate-based method of Masayuki24 was used for glucoside metabolism assays. Glucosides were hydrolyzed in 0.1 M citrate−0.2 M phosphate buffer (pH 5.5) containing 1 unit of β-glucosidase, and the volume was brought up to 1 mL. Then, 5 μL of 20 mM concentrated standard solution was added to these centrifugal tubes.

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RESULTS AND DISCUSSION Synthesis and Characterization. Click chemistry27 was performed to create four glucose−fipronil conjugates, 4a−4d (Figure S1). CuSO4·5H2O/sodium ascorbate and CuI/DIPEA are commonly used as catalysts for this method. The CuSO4· 5H2O/sodium ascorbate system28 was selected to optimize the reaction yield (>85%) and ensure the stability of conjugates. Compounds 4a−4c were deprotected using 1 M NaOMe in F

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MeOH to yield products 5a−5c with 60% yields. The structures of 4a−4c and 5a−5c were confirmed by 1H NMR, 13 C NMR, and mass spectrometry. The acetyl groups of 4a exhibited characteristic signals at 1.89, 2.00, 2.01, and 2.03 ppm in the 1H NMR spectra, as well as 20.59, 20.62, and 20.71 ppm in the 13C NMR spectra. However, the characteristic 1H NMR and 13C NMR spectrum signals of 5a were not observed. The acetyl groups of 4a disappeared after deprotection. Generally, palladium-catalyzed hydrogenolysis was used to deprotect benzyl ether. However, 4d exhibited a sulfur atom in sulfinyl that could cause permanent catalytic poisoning. Accordingly, FeCl3 was introduced to synthesize 5d with 55% yield because of its high efficacy.29 The structures of 4d and 5d were confirmed by 1 H NMR, 13 C NMR, and mass spectrometry. Debenzylation was proved by 1H and 13C NMR spectra. The characteristic peaks between 7.35 and 7.1 ppm in the 1H NMR spectra, as well as the peaks at 128.65− 127.76, 137.99, 138.08, 138.18, and 138.65 ppm in the 13C NMR spectra, were not observed after deprotection. Hydrolysis of Glucosides. To examine the ability of βglucosidase to hydrolyze glucose−pesticide conjugates in vitro, 1 mL of buffer solutions containing 1 unit of almond βglucosidase was individually incubated with four glucose− fipronil conjugates for 1 h. Each solution was analyzed by HPLC upon termination of the reaction to detect potential hydrolysis products. Figure 3A shows that GOTF solution displayed a novel peak, and the original peak at the GOTF position cannot be clearly detected. This result indicated that 1 unit of β-glucosidase could hydrolyze nearly all GOTF within 1 h. However, no peak was observed from the other conjugate solutions, indicating that these analogues could not be hydrolyzed by β-glucosidases under experimental conditions (Figure 3B−D). In the absence of enzymes, hydrolysis was not observed in the control reaction, demonstrating the stability of glucosides. Considering the metabolite accumulation caused by the β-glucosidase-mediated degradation of GOTF, the single metabolite observed in the in vitro assay was analyzed by UHPLC-HRMS to determine its structure and define the enzymatic reactions (Figure 4A,B). The GOTF metabolite in the positive-ion mode resulted in pseudomolecular [M + Na]+ at m/z 583.9904, which was consistent for its reduced mass (mass loss = 162) indicating the removal of a single glucose molecule. This result determined the chemical structure (FTO) of the metabolite (Figure 2). The identity of the GOTF metabolite was verified by 1H NMR (Figure S2). These data indicated that the almond β-glucosidase hydrolyzed GOTF to FTO and exhibited high specificity for the O-glycosidic bond. The in vitro activity of β-glucosidases implied that it could hydrolyze glucose−fipronil conjugates in vivo. To confirm this result, tests were conducted in adult Ricinus communis L. A peak was detected by HPLC in the leaf extracts when 20 μL of a 10 mM GOTF solution was injected into the petioles after 4 h (Figure 3A). The retention time of the metabolic product suggested that for GOTF the observed products of castor bean in vivo were similar to those generated from the in vitro assays using β-glucosidase solutions (UHPLC-HRMS, Figure 4C−E). GOTF was also identified in the leaf extract (Figure 4C,D). By contrast, peaks were undetected in the extracted solution by HPLC when analogues were treated under the same experimental condition (Figure 3B−D). Thus, the analogues were stable within 4 h of injection, but we cannot rule out the possibility of other metabolism mechanisms occurring within the plant with prolonged time. Given the results of in vivo and

Figure 3. HPLC chromatograms of four glucosides measured at 210 nm for (A) GOTF, (B) GTTF, (C) GNTF, and (D) GCTF. Lane 1 (black line) describes the standard solutions of the corresponding glucosides. Lane 2 (red line) represents the glucosides incubated in buffer solutions containing of 1 unit of β-glucosidase for 1 h. Lane 3 (blue line) corresponds to the leaf extraction of castor bean at 4 h after injection.

in vitro assays, β-glucosidase was associated with the metabolism of glucose−fipronil conjugates in the body of adult castor beans and was specific for the hydrolysis of Oglycosidic bonds. β-Glucosidase mediated the hydrolysis of glucose−pesticide conjugates, in which glucose was tethered to pesticides by Oglycosidic bonds. To verify this result, another glucose−fipronil conjugate, GOF, was selected for additional tests under the same experimental condition; this conjugate has been synthesized, and its phloem mobility in the castor bean system has been examined.6 After 1 h of incubation with β-glucosidase, a peak in the HPLC chromatogram was observed and analyzed by UHPLC-HRMS (Figure 5A). Results showed that GOF (molecular mass = 642.0177) had [M + Na]+ at m/z 665.0077 (calculated for C20H18Cl2F6N4NaO7S; Figure 5B). The hydrolysis products (FOH) indicated a reduction in glucose molecule, and the majority of these displayed [M + H]+ at m/z 480.9733 (calculated for C14H9Cl2F6N4O2S; Figure 5C). The glucose−pesticide conjugate was metabolized within the body of an adult castor bean 4 h after injection. The reaction G

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Figure 4. UHPLC-HRMS analysis of the metabolites present in the buffer solution of β-glucosidases and adult castor bean after GOTF application: (A) UV chromatogram of the reaction products in vitro; (B) positive-ion ESI mass spectrum of the peak in panel A (red arrow) showing [M + Na]+ at m/z 583.9904; (C) UV chromatogram of the leaf extracts; (D) positive-ion ESI mass spectrum of the peak in panel C (blue arrow) showing [M + K]+ at m/z 762.0148, which corresponds to the mass of GOTF; (E) positive-ion ESI mass spectrum of the peak in panel C (red arrow) indicating [M + H]+ at m/z 562.0063, which corresponds to the metabolite in vitro.

Figure 5. UHPLC-HRMS analysis of metabolites present in the buffer solution of β-glucosidases and leaf extract after treatment with GOF: (A) GOF solution incubated with β-glucosidases in vitro; (B) positive-ion ESI mass spectrum of the peak in panel A (blue arrow) showing [M + Na]+ at m/z 665.0077, which corresponds to the mass of GOF; (C) positive-ion ESI mass spectrum of the peak in panel A (red arrow) showing the reduced mass of the hydrolysis products to remove the glucose molecule; [M + H]+ is located at m/z 480.9733; (D) UV chromatogram of the leaf extracts; (E) positive-ion ESI mass spectrum of the peak in panel D (blue arrow) indicating [M + Na]+ at m/z 665.0101; (F) positive-ion ESI mass spectrum of the peak in panel D (red arrow) showing [M + H]+ at m/z 480.9717, which corresponds to the metabolite in vitro.

mobility of GOF is considered to be preferred over GOTF to be loaded in the phloem and achieve the slow release of insecticidal activities. The difference in hydrolyzing degree between GOF and GOTF may be due to the distinction of βglucosidase affinity. Thus, the effects of different glucose− pesticide conjugates’ chemical construction on hydrolyzing degree is worthy of further investigation. A pro-drug was designed to solve the incompatibility between phloem mobility and activity. This material could be cleaved inside the plant or pest organism by spontaneous chemical conversion or enzyme-catalyzed metabolism. The enzyme unique to the plant crucially determines the proper approach to the preparation of pro-drugs.2,30 Related works have achieved this enzyme through engineering mechanisms to release the active molecule within the plants. For instance, a sulfonylurea pro-herbicide can be activated in transgenic

products identified by UHPLC-HRMS (Figure 5D−F) were similar to those of β-glucosidase hydrolysis in vitro. These data demonstrated that β-glucosidase was associated with the hydrolysis of GOF within the plant and the O-glycosidic bond. To determine whether the O-glycosidic bond conjugates GOTF and GOF would be hydrolyzed in epidermal or mesophyll cells before reaching the phloem, a cotyledon absorption experiment was performed using castor bean seedlings (Supporting Information, Materials and Methods). After 15 min of incubation, both GOTF and GOF were hydrolyzed. GOTF was almost entirely hydrolyzed to be FTO within the cotyledons, whereas about 75% of GOF was hydrolyzed (Figure S3). Combined with in vitro and in vivo hydrolysis, GOF can be speculated to be not as readily hydrolyzed as GOTF. Given that the phloem mobility of GOF has been confirmed,6 the slow hydrolysis and moderate phloem H

dx.doi.org/10.1021/jf5034575 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

commercialized fipronil (Tables 1 and 2). LC50 value (95% confidence limits, CL) after 24 h was found to be 31.49 mg L−1 (18.19−54.49 mg L−1) against the larvae of P. xylostella L., which was analogous to that of fipronil (25.50 mg L−1 (16.10− 40.40 mg L−1)). Meanwhile, the LC50 (95% CL) of FOH was 36.34 mg L−1 (26.15−50.52 mg L−1) against the second-instar larvae of S. litura F; the LC50 for fipronil was 33.87 mg L−1 (25.81−44.45 mg L−1). FTO (the aglycon of GOTF) exhibited unsatisfactory insecticidal activity at 500 mg L−1, with mortality rates of only 30% against P. xylostella L. and 9.7% against S. litura F. after 24 h (Table 1). Thus, we inferred that triazolyl could significantly reduce the insecticidal activity of fipronil. However, the results of this study should be further analyzed. Overall, four glucose−fipronil conjugates were designed and synthesized by click chemistry. Metabolic assays of conjugates in vitro and in vivo were performed. Results demonstrated that the O-glycosidic bond could be recognized and hydrolyzed only by β-glucosidases. Another O-glycosidic bond conjugate GOF was metabolized in R. communis L., which released the aglycon FOH with highly analogous efficacy to the commercialized fipronil against P. xylostella L. and S. litura F. However, further analyses of the location and rate of GOF hydrolysis are required. These results conformed to our initial hypothesis that endogenous β-glucosidases were involved in the hydrolysis of exogenous phloem-mobile conjugates and the release of active aglycons, which can reasonably and feasibly achieve phloem mobility and biological activity of pesticides.

tobacco using the TA29 promoter to express a bacterial P450 gene in a tissue-specific manner.31 Tobacco could hydrolyze oxamyl glucuronide into its active compound by expressing the gene of E. coli β-glucuronidase in the root meristem and central cylinder.2 However, the environmental effects of genetically modified crops could limit the use of this approach.32 Plants contain endogenous β-glucosidases that catalyze the hydrolysis of glucosidic bonds to bioactivate plant glucosides (e.g., prohormones and phytoanticipins) and design parts as special active sites.33 Although the importance of β-glucosidase in catalyzing glucoside hydrolysis is well documented, few studies have focused on the utilization of endogenous β-glucosidase to hydrolyze exogenous glucosides. This study revealed the involvement of endogenous β-glucosidase in the hydrolysis of glucose−pesticide conjugates. Furthermore, tethering of the mobility-conferring glucose to the pesticide through Oglycosidic bonds can circumvent the conflicting functions of mobility and activity. Insecticidal Activity. The insecticidal activities of GOTF, FTO, GOF, and FOH were evaluated and compared with those of fipronil over a wide range of concentrations (Tables 1 and Table 1. Insecticidal Activities of GOTF, FTO, GOF, FOH, and Fipronil against P. xylostella L. and S. litura F. toxicities against P. xylostella L. compd GOTF

FTO

concn (mg L−1)

toxicities against S. litura F.

larvicidal activity (%)

concn (mg L−1)

larvicidal activity (%)

500 250 125

16.6 0

500 250 125

4.0 3.3 0

500 250 125

30.0 10.0 0

500 250 125

9.7 0

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ASSOCIATED CONTENT

S Supporting Information *

Additional spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author GOF

500 250 125

20.0 16.6 6.70

500 250 125

17.2 10.0 3.3

50 25 12.5

63.3 50.0 20.0

100 50 25

86.7 43.3 30.0

50 25 12.5

73.3 36.7 33.3

100 50 25

90.0 50.0 33.3

*(H.-H.X.) Phone: +86-20-85285127. E-mail: [email protected]. cn. Author Contributions ∥

FOH

fipronil

Q.X. and Y.-J.W. contributed equally to this work.

Funding

Financial support was from the National Natural Science Foundation of China (Grant 31171886) and the Specialized Research Fund for the Doctoral Program of Education of China (Grant 20134404130003). Notes

The authors declare no competing financial interest.

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2). GOTF and GOF exhibited negligible insecticidal activities against the second-instar larvae of P. xylostella L. and S. litura F. (mortality rate at 500 mg L−1,