Synthesis of Rotenone-O-monosaccharide Derivatives and Their

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Synthesis of Rotenone‑O‑monosaccharide Derivatives and Their Phloem Mobility Pei-Wen Qin,†,‡,§ Jie Wang,†,‡,§ Hao Wang,†,‡ Ying-Jie Wen,†,‡ Meng-Ling Lu,†,‡ Yu-Feng Li,†,‡ Yue-Shuo Xu,†,‡ 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 ABSTRACT: Six monosaccharide derivatives of rotenone were designed and synthesized to assess whether rotenone could become phloem mobile by the addition of a monosaccharide group. Phloem mobility experiments showed that only D-glucose conjugates exhibit phloem transport properties in castor bean (Ricinus communis L.) seedlings. Two D-glucose conjugates, 2-O-βD-glucopyranosyldemethylrotenone and 6′-O-β-D-glucopyranosyldalpanol, had significantly obtained systemicity compared with that of rotenone, and 6′-O-β-D-glucopyranosyldalpanol was more mobile than 2-O-β-D-glucopyranosyldemethylrotenone. Coupling with a monosaccharide core is a reasonable method for conferring phloem mobility on insecticides, but phloem mobility is also affected by the parent molecule and the position of the monosaccharide. KEYWORDS: rotenone, monosaccharide, glycosylation reaction, phloem-mobility

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INTRODUCTION Phloem-mobile insecticides are both economical and efficient,1−3 but there are only a few phloem systemic insecticides obtained because the development of new pesticides with that systemicity is often expensive and complicated. Two strategies have been adopted for conferring phloem mobility to nonsystemic insecticides. One strategy consists of introducing a carboxyl group to a nonsystemic pesticide. For example, after conjugating oxamyl with glucuronic acid, the oxamyl derivative exhibits an improved phloem mobility higher than that of the parent compound in transgenic tobacco.4 The other strategy is using the endogenous plant transporters to confer phloem mobility on nonphloem-mobile insecticides or to confer a phloem mobility different from that of the parent compound.5−7 In plants, sugar-transport proteins play a key role not only in long-distance transportation but also in cell-to-cell distribution of sugars throughout the plant.8 Recently, researchers found that a plant hexose-transporter bound in plasmalemma was involved in the transportation of the 2-deoxyglucose-NBD conjugate in E. coli.9 Moreover, we have reported the synthesis of a novel fipronil-glucose conjugate (GTF) and demonstrated that linking glucose can change fipronil into a phloem systemicity type, which was mediated by monosaccharide transporters.2,10 In addition, 10 kinds of fipronil-monosaccharide derivatives were synthesized in our previous work (Figure 1).11 Most of these derivatives have been verified as having phloem systemicity. These studies revealed that conjugating glucose or another monosaccharide can confer phloem mobility on a nonphloem-mobile insecticide. It remains to investigate whether any nonmobile insecticide can be converted into a phloem-mobile one by the coupling of a glucose group or other monosaccharide in the same manner as that done to fipronil. Therefore, this study focuses on rotenone, a natural product of the isoflavone family and a © 2014 American Chemical Society

Figure 1. Chemical structure of 2-O-demethylrotenone, dalpanol, GTF, and monosaccharide-fipronil conjugates.

botanical insecticide obtained from the root extracts of some leguminous plants such as Derris and Lonchocarpus.12−15 Rotenone was linked with five kinds of monosaccharides (glucose, galactose, mannose, xylose, and arabinose) to obtain Received: Revised: Accepted: Published: 4521

January 12, 2014 April 28, 2014 April 29, 2014 April 29, 2014 dx.doi.org/10.1021/jf500197k | J. Agric. Food Chem. 2014, 62, 4521−4527

Journal of Agricultural and Food Chemistry

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Figure 2. Structures of monosaccharide trichloroacetimidate donors.

Scheme 1a

a

Reagents and conditions: (1) BF3·OEt2, CH2Cl2, and 4 Å molecular sieves, −30 °C and (2) CH3OH and CH3COCl, and 25 °C.

Scheme 2a

a

Reagents and conditions: (1) TMSOTf, CH2Cl2, and 4 Å molecular sieves, −30 °C and (2) CH3OH and CH3COCl, and 25 °C. Then, six day-old seedlings (the hypocotyl was about 20 mm length) were selected for further experiments. General procedure for the Preparation of Compounds (2a− 2f). Trichloroacetimidate donors18 (Figure 2), 2-de-O-methylrotenone19,20 and dalpanol21 (Figure 1) were synthesized as previously described. The trichloroacetimidate donor (1.2 mmol), activated 4 Å molecular sieve, and 2-de-O-methylrotenone (1 mmol), or dalpanol (1 mmol) were added in anhydrous CH2Cl2 (10 mL) under nitrogen atmosphere. The mixtures were carried out at 25 °C for 0.5 h, then cooled to −30 °C. Next, boron trifluoride etherate (BF3·OEt2, 0.05 mL) was injected into the solution containing 2-de-O-methylrotenone to produce compounds 2a−2e (Scheme 1). Trimethylsilyl trifluoromethanesulfonate (TMSOTf, 0.05 mL) was injected to the solution containing dalpanol to produce compound 2f (Scheme 2). The mixtures were kept in this condition for 4 h and stirred another 1 h at 25 °C. After filtering the suspension, the filtrate was collected and

six rotenone-glucosides linked with an O-glycosidic bond, including a natural product, dalpanol-O-glucoside.16 Their phloem mobilities were tested in a Ricinus system.

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MATERIALS AND METHODS

General Methods. TLC was taken on a silica gel plate (GF254, Qingdao, China). Silica gel (200−300 mesh) was used for flash column chromatography. The 1H and 13C NMR spectra were provided by a Bruker AVANCE III 600 (Brunker Corporation, Switzerland) spectrometer with tetramethylsilane (TMS) as the internal standard, and chemical shifts were recorded. The mass spectrographic analysis was recorded on a Waters ZQ4000 (Waters, MA, USA) with electronspray ionization. Materials. The seeds of castor bean No. 9, provided by the Agricultural Science Academy of Zibo (Zibo, China), and the cultivation of seedlings were followed by a previous method.17 4522

dx.doi.org/10.1021/jf500197k | J. Agric. Food Chem. 2014, 62, 4521−4527

Journal of Agricultural and Food Chemistry

Article

112.69, 105.72, 105.06, 101.57, 101.07, 87.94, 72.05, 69.89, 69.27, 67.42, 66.55, 62.80, 55.99, 44.53, 31.40, 21.08, 20.94, 20.87, 17.27. EIMS, m/z: 639.8 [M + 1]+, 661.7 [M + Na]+. 6′-O-(2,3,4,6-Tetra-O-acetyl)-β-D-glucopyranosyldalpanol (2f). White solid; yield 87%. 1H NMR (CDCl3) δ: 7.77 (d, J = 8.4 Hz, 1H), 6.72 (s, 1H), 6.42 (s, 1H), 6.41 (d, J = 9.0 Hz, 1H), 5.16 (t, J = 9.6 Hz, 1H), 4.98 (t, J = 9.6 Hz, 1H), 4.92−4.88 (m, 2H), 4.76 (d, J = 7.8 Hz, 1H), 4.66 (t, J = 9.0 Hz, 1H), 4.58 (dd, J = 12.0, 2.4 Hz, 1H), 4.17 (dd, J = 12.0, 10.0 Hz, 1H), 4.15 (d, J = 12.0 Hz, 1H), 4.09 (dd, J = 12.0, 2.4 Hz, 1H), 3.81 (d, J = 3.6 Hz, 1H), 3.77 (s, 3H), 3.72 (s, 3H), 3.67 (ddd, J = 9.6, 6.0, 2.4 Hz, 1H), 3.07 (d, J = 9.0 Hz, 2H), 2.00, 1.99, 1.95, and 1.80 (both s, 3H, Ac), 1.28 (s, 3H), 1.27(s, 3H). 13 C NMR (CDCl3) δ: 189.20, 170.75, 170.44, 169.67, 169.22, 167.42, 158.08, 149.69, 147.63, 144.03, 129.97, 113.61, 113.54, 110.55, 104.95, 104.82, 101.14, 95.83, 90.62, 78.62, 73.08, 72.38, 71.83, 71.55, 68.97, 66.46, 62.53, 56.53, 56.05, 44.80, 27.52, 24.09, 21.43, 20.83, 20.79(2C) 20.56. EI-MS, m/z: 743.7 [M + 1]+, 765.7 [M + Na]+. General Procedure for the Preparation of Title Compounds (3a−3f). The solid (2a−2f, 1 mmol) in a mixture of acetyl chloride (0.1 mL) in anhydrous methanol (10 mL) was left to stand for 4 h at 25 °C, then concentrated to dryness at 25 °C (Schemes 1 and 2). The solid was chromatographed on a silica gel column (chloroform− methanol, 15:1), and after removing solvents and washing with cooled methanol, the desired products 3a−3f were obtained. 2-O-β-D-Glucopyranosyldemethylrotenone (3a). White solid; yield 37%. 1H NMR (DMSO) δ: 7.71 (d, J = 8.4 Hz, 1H), 6.82 (s, 1H), 6.57 (d, J = 8.4 Hz, 1H), 6.51 (s, 1H), 5.33 (t, J = 9.0 Hz, 1H), 5.15−5.14 (m, 1H), 5.12 (d, J = 5.4 Hz, 1H), 5.05−5.02 (m, 2H), 4.94 (d, J = 5.4 Hz, 1H), 4.92 (s, 1H), 4.51 (dd, J = 12.0, 3.0 Hz, 1H), 4.51 (d, J = 7.8 Hz, 1H, G1H), 4.41 (t, J = 5.4 Hz, 1H), 4.24 (d, J = 12.0 Hz, 1H), 3.92 (d, J = 3.6 Hz, 1H), 3.70 (s, 3H), 3.66−3.62 (m, 1H), 3.53 (m, 1H), 3.27 (dd, J = 15.6, 9.6 Hz, 1H), 3.23−3.13 (m, 3H), 3.08− 3.05 (m, 1H), 2.88 (dd, J = 15.6, 7.8 Hz, 1H), 1.71 (s, 3H). 13C NMR (DMSO) δ: 188.60, 166.51, 157.58, 149.78, 148.51, 143.12, 140.71, 129.25, 115.01, 113.18, 112.78, 112.36, 105.49, 104.34, 101.59, 101.54, 87.18, 76.93, 76.85, 73.15, 71.69, 69.31, 65.76, 60.32, 55.78, 43.54, 30.52, 16.91. EI-MS, m/z: 565.8 [M + Na]+. 2-O-β-D-Galactopyranosyldemethylrotenone (3b). White solid; yield 33%. 1H NMR (DMSO) δ: 7.71 (d, J = 8.4 Hz, 1H), 6.82 (s, 1H), 6.57 (d, J = 8.4 Hz, 1H), 6.51 (s, 1H), 5.33 (t, J = 9.0 Hz, 1H), 5.15−5.13 (m, 1H), 5.04 (s, 1H), 4.96−4.93 (m, 1H), 4.93−4.91 (m, 1H), 4.78−4.76 (m, 1H), 4.55−4.49 (m, 2H), 4.47 (d, J = 7.8 Hz, 1H, G1H), 4.45 (d, J = 4.2 Hz, 1H), 4.24 (d, J = 12.0 Hz, 1H), 3.92 (d, J = 3.6 Hz, 1H), 3.70 (brs, 4H), 3.56−3.52 (m, 1H), 3.49−3.45 (m, 1H), 3.37−3.30 (m, 3H), 3.28 (dd, J = 15.6, 10.2 Hz, 1H), 2.88 (dd, J = 15.6, 7.8 Hz, 1H), 1.71 (s, 3H). 13C NMR (DMSO) δ: 188.54, 166.48, 157.55, 150.10, 148.66, 143.11, 140.50, 129.21, 115.95, 113.14, 112.75, 112.31, 105.51, 104.29, 101.59, 87.15, 75.02, 73.35, 71.68, 70.40, 67.55, 65.73, 59.59, 55.78, 43.51, 40.05, 30.51, 16.89. EI-MS, m/z: 565.8 [M + Na]+. 2-O-α-D-Mannopyranosyldemethylrotenone (3c). White solid; yield 30%. 1H NMR (MeOD) δ: 7.78 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 1.0 Hz, 1H), 6.51 (s, 1H), 6.50 (d, J = 8.4 Hz, 1H), 5.31 (t, J = 9.0 Hz, 1H), 5.18 (d, J = 1.8 Hz, 1H, G1H), 5.06 (s, 1H), 5.02−5.00 (m, 1H), 4.93−4.91 (m, 1H), 4.58 (dd, J = 12.0, 3.0 Hz, 1H), 4.23 (d, J = 12.0 Hz, 1H), 4.01 (dd, J = 3.0, 1.8 Hz, 1H), 3.85−3.83 (m, 2H), 3.77 (s, 3H), 3.75−3.68 (m, 4H), 3.31 (dd, J = 15.6, 9.6 Hz, 1H), 2.95 (dd, J = 15.6, 7.8 Hz, 1H), 1.75 (s, 3H). 13C NMR δ: 191.40, 169.11, 159.66, 153.06, 151.58, 145.17, 141.33, 130.96, 120.12, 114.53, 114.48, 112.79, 107.11, 105.78, 102.83, 102.71, 89.38, 75.43, 73.72, 72.54, 72.16, 68.28, 67.58, 62.60, 56.52, 45.71, 32.22, 17.23. EI-MS, m/z: 543.8 [M + 1]+, 565.8 [M + Na]+. 2-O-β-D-Xylopyranosyldemethylrotenone (3d). Brown solid; yield 23%. 1H NMR (MeOD) δ: 7.79 (d, J = 8.6 Hz, 1H), 6.91 (s, 1H), 6.51 (s, 1H), 6.50 (d, J = 8.4 Hz, 1 H), 5.30 (d, J = 9.0 Hz, 1H), 5.05 (s, 1H), 5.00 (t, J = 3.0 Hz, 1H), 4.92−4.91 (m, 1H), 4.58(dd, J = 12.0, 3.0 Hz, 1H), 4.57 (d, J = 7.2 Hz, 1H, G1H), 4.22 (d, J = 12.0 Hz, 1H), 3.87 (dd, J = 11.4, 5.4 Hz, 1H), 3.84 (d, J = 3.6 Hz, 1H), 3.78 (s, 3H), 3.55−3.50 (m, 1H), 3.41−3.38 (m, 1H), 3.37−3.33 (m, 1H), 3.31(dd, J = 15.6, 9.6 Hz, 1H), 3.19 (dd, J = 11.4, 10.2 Hz, 1H), 2.95 (dd, J =

dried in vacuo, followed by chromatography (chloroform−methanol, 99:1) to produce compounds 2a−2f as solids. 2-O-(2,3,4,6-Tetra-O-acetyl)-β-D-glucopyranosyldemethylrotenone (2a). White solid; yield 88%. 1H NMR (CDCl3) δ: 7.81(d, J = 8.4 Hz, 1H), 7.07 (s, 1H), 6.52 (d, J = 8.4 Hz, 1H), 6.43 (s, 1H), 5.26−5.20 (m, 3H), 5.16 (m, 1H), 5.07 (brs, 1H), 4.93−4.92 (m, 2H), 4.76 (dd, J = 6.0, 2.4 Hz, 1H), 4.60 (dd, J = 12.0, 3.6 Hz, 1H), 4.30 (dd, J = 12.0, 4.8 Hz, 1H), 4.19−4.14 (m, 2H), 3.80(d, J = 4.2 Hz, 1H), 3.74 (s, 3H), 3.70(ddd, J = 10.2, 4.8, 2.4 Hz, 1H), 3.31 (dd, J = 15.6, 9.6 Hz, 1H), 2.95 (dd, J = 15.6, 8.4 Hz, 1H), 2.14, 2.04, 2.03, and 2.01 (s, both 3H, Ac), 1.76 (s, 3H). 13C NMR (CDCl3) δ: 188.50, 171.09, 170.46, 169.62, 169.57, 167.51, 157.94, 151.17, 150.25, 143.19, 141.11, 130.04, 118.89, 113.49, 113.19, 112.80, 105.74, 105.18, 101.57, 101.44, 88.03, 72.91, 72.17, 72.13, 71.27, 68.39, 66.54, 61.95, 56.18, 44.57, 31.47, 20.98, 20.83, 20.80 (2C), 17.31. EI-MS, m/z: 711.7 [M + 1]+, 733.7 [M + Na]+. 2-O-(2,3,4,6-Tetra-O-acetyl)-β-D-galactopyranosyldemethylrotenone (2b). White solid; yield 85%. 1H NMR (CDCl3) δ: 7.81 (d, J = 8.4 Hz, 1H), 7.05 (s,1H), 6.50 (d, J = 8.4 Hz, 1H), 6.42 (s, 1H), 5.41 (dd, J = 10.2, 7.8 Hz 1H), 5.39 (d, J = 3.0 Hz, 1H), 5.23 (t, J = 8.4 Hz, 1H), 5.06 (s, 1H), 5.03 (dd, J = 10.2, 3.0 Hz, 1H), 4.92 (m, 2H), 4.71 (d, J = 8.4 Hz 1H), 4.59 (dd, J = 12.0, 3.0 Hz, 1H), 4.22 (dd, J = 10.8, 7.2 Hz, 1H), 4.16 (d, J = 12.0 Hz, 1H), 4.11 (dd, J = 10.8, 6 Hz, 1H), 3.88 (t, J = 6.6 Hz, 1H), 3.79 (d, J = 4.2 Hz, 1H), 3.74 (s, 3H), 3.29 (dd, J = 15.6, 9.6 Hz, 1H), 2.94 (dd, J = 15.6, 7.8 Hz, 1H), 2.16, 2.07, 2.04, and 1.98 (both s, 3H, Ac), 1.75 (s, 3H). 13C NMR (CDCl3) δ: 188.46, 170.62, 170.50, 170.30, 169.65, 167.46, 157.89, 151.23, 150.24, 143.18, 141.07, 130.05, 119.26, 113.45, 113.13, 112.74, 105.70, 105.13, 101.98, 101.49, 87.97, 72.13, 70.96 (2C), 68.85, 67.03, 66.50, 61.18, 56.12, 44.55, 31.43, 20.87, 20.85 (2C), 20.76, 17.27. EI-MS, m/z: 711.7 [M + 1]+, 733.7 [M + Na]+. 2-O-(2,3,4,6-Tetra-O-acetyl))-α-D-mannopyranosyldemethylrotenone (2c). White solid; yield 85%. 1H NMR (CDCl3) δ: 7.81 (d, J = 8.4 Hz, 1H), 6.94 (s,1H), 6.49 (d, J = 8.4 Hz, 1H), 6.43 (s, 1H), 5.51− 5.45 (m, 2H), 5.32 (t, J = 9.6 Hz, 1H), 5.24 (s, 1H), 5.23 (t, J = 9.0 Hz, 1H), 5.05 (s, 1H), 4.92−4.89 (m, 2H), 4.60 (dd, J = 12.0, 3.0 Hz, 1H), 4.31 (ddd, J = 10.2, 4.8, 2.4 Hz, 1H), 4.24 (dd, J = 12.0, 4.8 Hz, 1H), 4.17 (d, J = 12.0 Hz, 1H), 4.06 (dd, J = 12.0, 2.4 Hz, 1H), 3.77 (d, J = 3.6 Hz, 1H), 3.75 (s, 3H), 3.29 (dd, J = 15.6, 9.6 Hz, 1H), 2.93 (dd, J = 15.6, 7.8 Hz, 1H), 2.16, 2.09, 2.05, and 1.99 (both s, 3H, Ac), 1.75 (s, 3H). 13C NMR (CDCl3) δ: 188.60, 170.95, 170.10, 170.01, 169.97, 167.54, 157.92, 151.78, 150.55, 143.16, 139.32, 130.21, 120.04, 113.36, 113.08, 112.71, 105.76, 105.15, 101.64, 98.67, 87.97, 72.04, 69.55(2C), 69.17, 66.51, 66.15, 62.45, 55.92, 44.49, 31.40, 21.08, 20.89 (2C), 20.83, 17.28. EI-MS, m/z: 733.7 [M + Na]+. 2-O-(2,3,4,-Tri-O-acetyl)-β- D-xylopyranosyldemethylrotenone (2d). White solid; yield 82%. 1H NMR (CDCl3) δ: 7.78 (d, J = 8.4 Hz, 1H), 6.95 (s, 1H), 6.48 (d, J = 8.4 Hz, 1H), 6.41 (s, 1H), 5.22 (t, J = 9.0 Hz, 1H), 5.16 (t, J = 7.8 Hz, 1H), 5.10 (dd, J = 8.4, 6.6 Hz, 1H), 5.04 (s, 1H), 4.96 (td, J = 7.8, 4.8 Hz, 1H), 4.92−4.88 (m, 3H), 4.57 (dd, J = 12.0, 3.0 Hz, 1H), 4.18 (dd, J = 12.0, 6.4 Hz, 1H), 4.16 (d, J = 12.6 Hz, 1H), 3.78 (d, J = 3.6 Hz, 1H), 3.72 (s, 3H), 3.39 (dd, J = 12.0, 7.8 Hz, 1H), 3.28 (dd, J = 15.6, 9.6 Hz, 1H), 2.93 (dd, J = 15.6, 7.8 Hz, 1H), 2.05−2.02 (m, 9H), 1.74 (s, 3H). 13C NMR (CDCl3) δ: 188.68, 170.22, 170.05, 169.60, 167.52, 157.96, 151.31, 150.07, 143.15, 140.55, 130.10, 118.69, 113.43, 113.14, 112.76, 105.74, 105.11, 101.58, 100.78, 88.00, 72.15, 70.96, 70.33, 68.79, 66.47, 62.06, 56.10, 44.51, 31.42, 20.94, 20.91, 20.86 17.28. EI-MS, m/z: 639.8 [M + 1]+, 661.8 [M + Na]+. 2-O-(2,3,4,-Tri-O-acetyl)-α-D-arabopyranosyldemethylrotenone (2e). White solid; yield 81%. 1H NMR (CDCl3) δ: 7.79 (d, J = 8.4 Hz, 1H), 7.00 (s, 1H), 6.48 (d, J = 8.4 Hz, 1H), 6.43 (s, 1H), 5.35 (dd, J = 8.4, 6 Hz, 1H), 5.25 (m, 1H), 5.22 (t, J = 9 Hz, 1H), 5.09 (dd, J = 8.4, 3.6 Hz, 1H), 5.05 (s, 1H), 4.91 (m, 1H), 4.90 (m, 1H), 4.82 (d, J = 6.6 Hz, 1H), 4.60 (dd, J = 12, 3.0 Hz, 1H), 4.17 (d, J = 12 Hz, 1H), 4.08 (dd, J = 12.6, 3.6 Hz, 1H), 3.78 (d, J = 3.6 Hz, 1H), 3.74 (s, 3H), 3.60 (dd, J = 12.6, 1.8 Hz, 1H), 3.28 (dd, J = 15.6, 10.2 Hz, 1H), 2.92 (dd, J = 15.6, 7.8 Hz, 1H), 2.13, 2.07, and 2.04 (both s, 3H, Ac), 1.75 (s, 3H). 13C NMR (CDCl3) δ: 188.60, 170.61, 170.40, 169.67, 167.47, 157.88, 151.61, 150.44, 143.17, 140.09, 130.18, 120.75, 113.39, 113.03, 4523

dx.doi.org/10.1021/jf500197k | J. Agric. Food Chem. 2014, 62, 4521−4527

Journal of Agricultural and Food Chemistry

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Figure 3. MRM chromatograms of conjugates glucopyranosyldemethylrotenone (3a) and glucopyranosyldalpanol (3f). (A) Standard solution of conjugates 3f (RT, 2.84 min) and (B) standard solution of conjugates 3a (RT, 3.14 min); (a) conjugates 3f in phloem sap (RT, 2.84 min) and (b) conjugates 3a in phloem sap (RT, 3.14 min) whose cotyledons were incubated in a buffered solution for 6 h including 100 μM conjugates 3a and 3f, respectively.

Figure 4. Structures of designed rotenone-O-monosaccharide conjugates: (3a) 2-O-β-D-glucopyranosyldemethylrotenone; (3b) 2-O-β-Dgalactopyranosyldemethylrotenone; (3c) 2-O-β-D-mannopyranosyldemethylrotenone; (3d) 2-O-β-D-xylopyranosyldemethylrotenone; (3e) 2-O-βD-arabopyranosyldemethylrotenone; and (3f) 6′-O-β-D-glucopyranosyldalpanol. 15.6, 7.8 Hz, 1H), 1.75 (s, 3H). 13C NMR δ: 191.34, 169.08, 159.68, 152.18, 151.26, 145.15, 142.03, 130.92, 118.51, 114.59, 114.49, 112.79, 107.15, 105.76, 104.50, 102.76, 89.38, 77.56, 74.74, 73.77, 71.17, 67.59, 66.98, 56.71, 45.75, 32.21, 17.24. EI-MS, m/z: 535.8 [M + Na]+. 2-O-α-D-Arabopyranosyldemethylrotenone (3e). White solid; yield 28%. 1H NMR (MeOD) δ: 7.77 (d, J = 8.4 Hz, 1H), 6.91 (s, 1H), 6.51 (s, 1H), 6.50 (d, J = 8.4 Hz, 1H), 5.29 (t, J = 9.0 Hz, 1H), 5.05 (s, 1H), 5.01−4.98 (m, 1H), 4.91 (br s, 1H), 4.66 (d, J = 6.0 Hz, 1H, G1H), 4.58 (dd, J = 12.6, 3.0 Hz, 1H), 4.22 (d, J = 12.6 Hz, 1H), 3.88−3.83 (m, 3H), 3.80−3.77 (m, 4H), 3.62 (dd, J = 8.4, 3.0 Hz, 1H), 3.55 (dd, J = 12.0, 1.8 Hz, 1H), 3.30 (dd, J = 15.6, 9.6 Hz, 1H), 2.94 (dd, J = 15.6, 7.8 Hz, 1H), 1.75 (s, 3H). 13C NMR (DMSO) δ: 188.55, 166.51, 157.58, 150.65, 149.18, 143.09, 139.68, 129.19, 117.91, 113.16, 112.79, 112.32, 105.56, 104.33, 101.92, 101.59, 87.17, 71.97, 71.59, 70.38, 66.54, 65.79, 64.06, 55.78, 43.43, 30.54, 16.90. EI-MS, m/ z: 535.8 [M + Na]+. 6′-O-β-D-Glucopyranosyldalpanol (3f). White solid; yield 35%.1H NMR (CDCl3) δ: 7.72 (d, J = 8.4 Hz, 1H), 6.72 (s, 1H), 6.40 (s, 1H), 6.39 (d, J = 8.4 Hz, 1H), 4.94 (s, 1H), 4.74 (t, J = 9.0 Hz, 1H), 4.60−

4.55 (m, 1H), 4.53 (d, J = 7.2 Hz, 1H, G1H), 4.12 (d, J = 11.4 Hz, 1H), 3.83−3.76 (m, 3H), 3.76 (s, 3H), 3.73 (s, 3H), 3.58−3.48 (m, 2H), 3.31 (t, J = 7.8 Hz, 1H), 3.28 (s, 1H), 3.11 (dd, J = 15.6, 9.6 Hz, 1H), 3.00 (dd, J = 15.6, 8.4 Hz, 1H), 1.22 (s, 3H), 1.21 (s, 3H). 13C NMR (CDCl3) δ: 189.29, 167.16, 158.02, 149.73, 147.61, 144.09, 130.07, 113.61, 113.35, 110.63, 104.99, 104.86, 101.16, 97.38, 90.31, 79.10, 76.53, 75.66, 73.56, 72.44, 69.69, 66.42, 61.75, 56.56, 56.05, 44.73, 28.01, 22.59, 21.77.EI-MS, m/z: 597.8 [M + Na]+. Assessment of Membrane Potential. The membrane potential of protoplast was estimated by the previously reported method.2 Duncan’s multiple range test was employed to determine statistical differences between the control and treatments at a 5% probability level. Collection and Analysis of Phloem Sap. The phloem sap collection method was as previously reported with some modifications.2,5 The buffered solution contained 100 μM monosacchariderotenone conjugates or rotenone (1% Tween-80 V/V). The phloem sap was analyzed by the previously described method with some modifications.2 A C8 reversed-phase column (Agilent, 5 μm, 4524

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Figure 5. Membrane potential of protoplasts treated with conjugates 3a−3f at 6 h. (A) Fluorescent intensity histogram. (B) Mean relative fluorescence. Protoplasts in buffer (pH 5.8) with conjugates 3a−3f and rotenone (100 μM) were suspended for 6 h. Cont = control, without any treatment; Rot = rotenone. The letters on tops the columns show that there were not significant differences between treatment and the control by Duncan’s multiple range test (P > 0.05). Mean ± SE; n = 3.

Table 1. Analysis of Phloem Sap compds

incubation medium (μM)

phloem sapa (μM)

compds

incubation medium (μM)

phloem sapb (μM)

3a 3b 3c 3d 3e 3f rotenone

100 100 100 100 100 100 100

4.90 ± 0.58 ND ND ND ND 14.71 ± 0.77 ND

4a 4b 4c 4d 4e

100 100 100 100 100

23.0 ± 1.9 ND 7.6 ± 1.6 21.8 ± 1.9 15.3 ± 2.2

The collection of phloem sap started from 2 h to the end of 6 h (mean ± SE; n = 3). ND represents not detected. bData are from Yuan et al.,11 and the phloem sap collection of monosaccharide-fipronil conjugates lasted for 2 h. a

250 mm × 4.6 mm i.d.) was used at 25 °C for separation. The eluting solvent was composed of CH3CN and H2O (40/60, v/v). The method of standard curve was employed to determine the concentrations of 2O-β-D-glucopyranosyldemethylrotenone (3a) and 6′-O-β-D-glucopyranosyldalpanol (3f). The linear equations of 3a and 3f were y = 24.153x + 4.3533 (r = 0.9997) and y = 19.078x + 2.1597 (r = 0.9996), respectively. The conjugates 3a of 3f in phloem sap were identified by a previously reported method with some modifications.10 The eluting solvent was composed of CH3CN and H2O (5/95, v/v, at 0.5 min; 80/ 20, v/v, at 3 min; and 5/95, v/v, at 4.5 min). The optimized parameters were as follows: capillary voltage, 300 kV; cone voltage, 35 V; desolvation temperatures, 350 °C; and source temperatures, 105 °C. At last, the molecule ion peaks of compounds 3a and 3f were determined as 541.7 and 573 (Figure 3), and those of the fragment ion peak were 379 and 393, respectively.

saccharide derivatives with O-glycosidic linkage (3a−3f, Figure 4) was obtained. The structures of conjugates 2a−2f and 3a−3f were confirmed by mass spectrometry, 1H and 13C NMR. The characteristic signal of the acetyl groups could be observed in the 1H and 13C NMR spectra of compounds 2a−2f. The 1H NMR spectrum of conjugate 2a showed signals of the acetyl groups at 2.14, 2.04, 2.03, and 2.01 ppm, and the 13C NMR spectrum showed them at 171.09, 170.46, 169.62, 169.57, 20.98, 20.83, and 20.80 ppm. Compared with compounds 2a− 2f, there were no peaks corresponding to acetyl groups in the 1 H NMR and 13C NMR spectra in compounds 3a−3f. This demonstrates that the acetyl groups were totally removed in the desired products. The characteristic signal of G1H was also confirmed in 1H NMR spectra. For example, in compound 3a, the signal of G1H was observed at 4.51 ppm with J = 7.8 Hz, indicating that the glucosidic linkage is in the β configuration. Phloem Mobility. Phloem mobility of conjugates 3a−3f was measured using the castor bean system. The previously reported method2 was used to measure the membrane potential of the protoplast after treatment with conjugates 3a−3f. Each treatment was repeated three times, each repetetition was taken twice, and then the data were analyzed by the Duncan’s multiple range test (Figure 5). Figure 5B indicates that there is no significant difference in relative fluorescence within the treatment period compared to that of the control. Consequently, they can be used to test their phloem mobility using the Ricinus system. The detection results in Table 1 showed that only 3a (4.90 ± 0.58 μM at 6h) and 3f (14.71 ± 0.77 μM at 6 h) exhibited phloem mobility, whereas the other conjugates were not found

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RESULTS AND DISCUSSION Synthesis and Characterization. The trichloroacetimidate method22 was adopted to couple monosaccharide with rotenone producing the conjugates 2a−2f in yields of over 80%. A widely used catalyst, BF3·OEt2, was used to prepare compounds 2a−2e, but it was not efficient enough to obtain conjugate 2f because the hydroxyl group of dalpanol is less active than that of 2-de-O-methylrotenone. Therefore, a more effective catalyst, TMSOTf, was introduced to the condensation of dalpanol with glucose, resulting in a yield of 87%. A classic reaction system of acetyl deprotection, NaOMe/MeOH, was attempted in the preparation of compounds 3a−3f; however, it was unsuccessful because rotenone is unstable under strong alkaline conditions. Therefore, an unusual system, AcCl/ MeOH, was used to prepare the desired products, with a yield of 23−37%. Finally, a series of rotenone-O-mono4525

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Table 2. Physicochemical Properties of Tested Compoundsa properties

3a

3b

3c

3d

3e

3f

rotenone

dalpanol

molecular mass log Ko/w pKa PSA(A2) HBD HBA

542.53 1.67 12.7 ± 07 (acid) 153.37 4 11

542.53 1.67 12.7 ± 0.7 (acid) 153.37 4 11

542.53 1.67 12.7 ± 0.7 (acid) 153.37 4 11

512.51 2.64 12.9 ± 0.7 (acid) 133.14 3 10

512.51 2.64 12.9 ± 0.7 (acid) 133.14 3 10

574.57 1.19 12.9 ± 0.7 (acid) 162.60 4 12

394.42 4.24 no 63.22 0 6

412.43 3.35 14.3 ± 0.3 (acid) 83.45 1 7

a The physicochemical properties of tested compounds were predicted by ACD Laboratories Perceptaprogram, version 14.0. log Ko/w and pKa values were classic values.

in the phloem sap. The mobility of 3f was about three times that of 3a, proving that the position of glucose coupling with rotenone significantly affected the phloem mobility of conjugates. Both 3a and 3f are D-glucose-rotenone conjugates, indicating that D-glucose is better as a substrate for conferring phloem mobility on rotenone than the other monosaccharides. However, in our previous work,11 almost any kind of monosaccharide-fipronil conjugate exhibited phloem mobility. The wide discrepancy of phloem mobility between monosaccharide-fipronil and monosaccharide-rotenone suggests that the parent compound is involved in phloem mobility. Furthermore, fipronil is transported in the sunflower xylem,23 while there are no reports of rotenone xylem mobility in any plant. Therefore, the results above demonstrate that phloem mobility can be acquired by a molecule by coupling it with a monosaccharide but that the extent of phloem mobility is affected by the parent molecule. Whether the influence of the parent molecule could be weakened by conjugating another monosaccharide needs further research. “Rule of Five” was widely employed to predict whether the endogenous compounds or xenobiotics can cross membranes.24−26 Given the physicochemical properties of tested compounds 3a−3f, rotenone, and dalpanol in Table 2, the tested compounds are predicted to exhibit a very poor diffusion through the membranes. The Kleier model is widely utilized to predict the phloem mobility of a xenobiotic based on its physicochemical properties (pKa and log Ko/w).10,27 Most of the tested xenobiotics fit well, except for several xenobiotics whose transport is carrier-mediated.5,27 Taking the physicochemical properties (pKa and log Ko/w) of tested compounds into consideration (Table 2), compounds 3a−3f, rotenone and dalpanol were predicted to be nonphloem mobile in terms of the Kleier Model (Figure 6). However, both 3a and 3f were demonstrated to be phloem mobile using the Ricinus system and therefore violate both this model (Figure 6) and “Rule of Five” (Table 2). The phloem mobility of some small xenobiotics were proved to involve active carrier processes, which can recognize and load these compounds into phloem sieve tubes.28,29 Therefore, it is likely that an active carriermediated mechanism is also involved in glucose-rotenone conjugate uptake as well as in the systemicity of glucose-fipronil conjugates.10 The carrier-mediated mechanisms of these large xenobiotics deserved further research. In summary, six conjugates containing both rotenone or dalpanol and monosaccharide moieties were obtained. The tests of phloem systemicity demonstrate that conjugates with different monosaccharides have different phloem mobility and that only D-glucose-type conjugates exhibit phloem systemicity. By contrast, conjugating hexoses, pentoses, and deoxysugars to fipronil can confer phloem transport (Table 1).11 The data suggest that this mobility is affected by the parent compound.

Figure 6. Prediction of phloem mobility of the tested compounds (3a−3f, rotenone and dalpanol) using the Kleier model.30 Log Ko/w and pKa in Table 2. rot = rotenone; dal = dalpanol.

The current article offers examples of successful conversions of nonphloem-transport natural pesticide molecules into phloemtransport ones and presents useful data for developing novel insecticides with phloem mobility.

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AUTHOR INFORMATION

Corresponding Author

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

P.-W.Q. and 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 (grants 20114404110020 and 20134404130003). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED NBD, 7-nitrobenz-2-oxa-1,3-diazole; GTF, N-[3-cyano-1-[2,6dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazol-5-yl]-1-(β-D-gluco-pyranosyl)-1H-1,2,3-triazole-4-methanamine; TMSOTf, trimethylsilyl trifluoromethanesulfonate; MRM, multiple reaction monitoring 4526

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Article

and stereoselective isotopic labelling of (E)- or (Z)-prenyl methyl groups in (6aS, 12aS)-rot-2′-enonic acid. J. Chem. Soc. Perkin Trans. 1982, 1, 779−788. (20) Unai, T.; Cheng, H. M.; Yamamoto, I.; Casida, J. E. Chemical and biological O-demethylation of rotenone derivatives. Agric. Biol. Chem. 1972, 8, 1937−1944. (21) Crombie, L.; Kilbee, G. W.; Moffatt, F.; Proudfoot, G.; Whiting, D. A. Identification and isotopic labelling of the diastereotopic methyl groups of the prenyl-derived 2-hydroxyisopropyl residues of natural products: the rotenoid dalpanol. J. Chem. Soc., Perkin Trans. 1991, 1, 3143−3148. (22) Ješelnik, M.; Plavec, J.; Polanc, S.; Kočevar, M. Novobiocinrelated compounds: synthesis of 3-benzoylamino-2-oxo- 2H-1benzopyran-7-yl D-glycopyranosides by the trichloroacetimidate methodology. Carbohydr. Res. 2000, 4, 591−597. (23) Aajoud, A.; Raveton, M.; Aouadi, H.; Tissut, M.; Ravanel, P. Uptake and xylem transport of fipronil in sunflower. J. Agric. Food Chem. 2006, 54, 5055−5060. (24) Bhal, S. K.; Kassam, K.; Peirson, L. G.; Pearl, G. M. The rule of five revisited: Applying log D in place of log p in drug-likeness filters. Mol. Pharmaceutics 2007, 4, 556−560. (25) Palm, K.; Stenberg, P.; Luthman, K.; Artursson, P. Polar molecular surface properties predict the intestinal absorption of drugs in humans. Pharm. Res. 1997, 5, 568−571. (26) Lei, Z. W.; Wang, J.; Mao, G. L.; Wen, Y. J.; Xu, H. H. Phloem mobility and translocation of fluorescent conjugate containing glucose and NBD in castor bean (Ricinus communis). J. Photochem. Photobiol., B 2014, 10−16. (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, 4, 2081−2091. (28) Besson, J.; Delrot, S.; El Ibaoui, H.; Bonnemain, J. L. Uptake and release of a phloem-mobile (glyphosate) and of a non-phloemmobile (iprodione) xenobiotic by broad bean leaf tissues. Physiol. Veg. 1986, 24, 431−432. (29) Hart, J. J.; DiTomaso, J. M.; Linscott, D. L.; Kochian, L. V. Transport interactions between paraquat and polyamines in roots of intact maize seedlings. Plant Physiol. 1992, 4, 1400−1405. (30) Kleier, D. A.; Hsu, F. C. Phloem mobility of xenobiotics. VII. The design of phloem systemic pesticides. Weed Sci. 1996, 44, 749− 756.

REFERENCES

(1) Nauen, R.; Reckmann, U.; Thomzik, J.; Thielert, W. Biological profile of spirotetramat (Movento®)−a new two-way systemic (ambimobile) insecticide against sucking pest species. Bayer Crop Sci. J. 2008, 2, 245−278. (2) Yang, W.; Wu, H. X.; Xu, H. H.; Hu, A. L.; Lu, M. L. Synthesis of glucose−fipronil conjugate and its phloem mobility. J. Agric. Food Chem. 2011, 59, 12534−12542. (3) Smith, K.; Evans, D. A.; El-Hiti, G. A. Role of modern chemistry in sustainable arable crop protection. Philos. Trans. R. Soc., B 2008, 1491, 623−637. (4) Hsu, F. C.; Sun, K.; Kleier, D. A.; Fielding, M. J. Phloem mobility of xenobiotics VI. A phloem-mobile pro-nematicide based on oxamyl exhibiting root-specific activation in transgenic tobacco. Pestic. Sci. 1995, 44, 9−19. (5) Deletage-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. (6) Chollet, J. F.; Deletage, C.; Faucher, M.; Miginiac, L.; Bonnemain, J. L. Synthesis and structure-activity relationships of some pesticides with an α-amino acid function. Biochim. Biophys. Acta 1997, 1336, 331−341. (7) Jiang, D. X.; Lu, X. L.; Hu, S.; Zhang, X. B.; Xu, H. H. A new derivative of fipronil: Effect of adding a glycinyl group to the 5-amine of pyrazole on phloem mobility and insecticidal activity. Pestic. Biochem. Physiol. 2009, 95, 126−130. (8) Williams, L. E.; Lemoine, R.; Sauer, N. Sugar transporters in higher plants − a diversity of roles and complex regulation. Trends Plant Sci. 2000, 5, 283−290. (9) Yoshioka, K.; Takahashi, H.; Homma, T.; Saito, M.; Oh, K. B.; 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, 5−9. (10) Wu, H. X.; Yang, W.; Zhang, Z. X.; Huang, T.; Yao, G. K.; Xu, H. 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. (11) Yuan, J. G.; Wu, H. X.; Lu, M. L.; Song, G. P.; Xu, H. H. Synthesis of a series of monosaccharide-fipronil conjugates and their phloem mobility. J. Agric. Food Chem. 2013, 61, 4236−4241. (12) Isman, M. B. Botanical insecticides, deterrents, and repellents in modern agriculture and an incresasingly regulated world. Annu. Rev. Entomol. 2006, 51, 45−66. (13) Dimetry, N. Z. Prospects of botanical pesticides for the future in integrated pest management programme (IPM) with special reference to neem uses in Egypt. Arch Phytopathol. Plant Prot. 2012, 10, 1138− 1161. (14) Sariaslani, F. S.; Rosazza, J. P. Microbial transformations of natural antitumor agents: products of rotenone and dihydrorotenone transformation by Cunninghamella blakesleeana. Appl. Environ. Microbiol. 1983, 2, 616−621. (15) CaboniLi, P.; Sherer, T. B.; Zhang, N.; Taylor, G.; Na, H. M.; Greenamyer, J. T.; Casida, J. E. Rotenone, deguelin, their metabolites, and the rat model of Parkinson’s disease. Chem. Res. Toxicol. 2004, 17, 1540−1548. (16) Li, L.; Wang, H. K.; Chang, J.-J.; Mcphail, A. T.; Mcphnl, D. R.; Estes, J. R.; Lee, K.-H. Antitumor agents, 138. Rotenoids and isoflavones as cytotoxic constituents from Amorpha f ruticosa. J. Nat. Prod. 1993, 5, 690−698. (17) 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, 3, 327−335. (18) Tian, Q.; Zhang, S.; Yu, Q.; He, M.; Yang, J. Amberlyst 15 as a mild and effective activator for the glycosylation with disarmed glycosyl trichloroacetimidate donors. Tetrahedron 2007, 10, 2142− 2147. (19) Carson, D.; Crombie, L.; Kilbee, G. W.; Moffatt, F.; Whiting, D. A. Regioselective ether cleavages of rotenoids: spiro-ether formation 4527

dx.doi.org/10.1021/jf500197k | J. Agric. Food Chem. 2014, 62, 4521−4527