Synthesis of a series of monosaccharide-fipronil conjugates and their phloem mobility

Article abstract of DOI:10.1021/jf400888c

To test the effect of adding different monosaccharide groups to a non-phloem-mobile insecticide on the phloem mobility of the insecticide, a series of conjugates of different monosaccharides and fipronil were synthesized using the trichloroacetimidate method. Phloem mobility tests in castor bean (Ricinus communis L.) seedlings indicated that the phloem mobility of these conjugates varied markedly. l-Rhamnose-fipronil and d-fucose-fipronil displayed the highest phloem mobility among all of the tested conjugates. Conjugating hexose, pentose, or deoxysugar to fipronil through an O-glycosidic linkage can confer phloem mobility to fipronil in R. communis L. effectively, while the -OH orientation of the monosaccharide substantially affected the phloem mobility of the conjugates.

Full text of DOI:10.1021/jf400888c

Article
pubs.acs.org/JAFC
Synthesis of a Series of Monosaccharide−Fipronil Conjugates and
Their Phloem Mobility
Jian-Guo Yuan,†,‡,§ Han-Xiang Wu,†,‡,§ Meng-Ling Lu,†,‡ Gao-Peng Song,†,‡ 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: To test the effect of adding different monosaccharide groups to a non-phloem-mobile insecticide on the phloem
mobility of the insecticide, a series of conjugates of different monosaccharides and fipronil were synthesized using the
trichloroacetimidate method. Phloem mobility tests in castor bean (Ricinus communis L.) seedlings indicated that the phloem
mobility of these conjugates varied markedly. L-Rhamnose−fipronil and D-fucose−fipronil displayed the highest phloem mobility
among all of the tested conjugates. Conjugating hexose, pentose, or deoxysugar to fipronil through an O-glycosidic linkage can
confer phloem mobility to fipronil in R. communis L. effectively, while the −OH orientation of the monosaccharide substantially
affected the phloem mobility of the conjugates.
KEYWORDS: Fipronil, monosaccharide, phloem transport
that link with an O-glycosidic bond and describe their phloem
mobility in Ricinus communis L. seedlings.
INTRODUCTION
■
To apply pesticides to control pest efficiently, phloem-mobile
pesticides need to be developed.1−3 A strategy for developing
phloem-mobile pesticides is to add an acidic group to a non-
systemic pesticide. For instance, a conjugate of oxamyl with
glucuronic acid exhibits good phloem mobility.4 Another
efficient strategy is to conjugate an endogenous phloem mobile
substrate, such as an amino acid or carbohydrate, to the active
non-phloem-mobile pesticide molecules to make them phloem-
mobile using the plant endogenous transporters. Several
derivatives of pesticides with an α-amino acid have been
reported. ε-(2,4-Dichlorophenoxyacetic acid)-L-lysine (2,4-D-
Lys), which is one of these derivatives, exhibits good phloem
mobility, and its uptake is mediated by an active carrier
system.5−7 Glucosylation and glutathionation have been proven
important for vacuolar uptake of sulfonylurea herbicides.8
In recent studies,9,10 we synthesized a novel conjugate of the
insecticide fipronil, N-[3-cyano-1-[2,6-dichloro-4-(trifluoro-
methyl)phenyl]-4-[(trifluoromethyl)-sulfinyl]-1H-pyrazol-5-
yl]-1-(β-D-glucopyranosyl)-1H-1,2,3-triazole-4-methanamine
(GTF) (Figure 1), which contains a glucose moiety. GTF
exhibits moderate phloem mobility, and the phloem transport
property of fipronil has not yet been reported.11,12 We showed
that conjugating glucose to fipronil could confer phloem mobility
to fipronil using plant monosaccharide transporters.
The monosaccharide transporter gene superfamily consists of
many members, and many of these proteins are capable of
transporting more than one monosaccharide. For example,
AtSTP1, which is one of the Arabidopsis sugar transporters,
transports glucose but is also capable of transporting galactose,
mannose, and xylose.13,14 Therefore, we have been inspired to
research phloem mobility of the conjugates of fipronil with
different monosaccharides. Furthermore, although the linkage of
GTF is a N-glycosidic bond, O-glycosidic bonds are the most
common linkages found in natural glycosides. Herein, we report
the synthesis of a series of monosaccharide−fipronil conjugates
MATERIALS AND METHODS
■
General Information for Synthesis. Solvents were used as
purchased without further purification. Nuclear magnetic resonance
(NMR) spectra were obtained on a Bruker AV-600 instrument.
Chemical shifts were expressed in parts per million, with tetramethylsi-
lane (TMS) as the internal standard. The mass spectra (MS) of new
compounds were obtained by MAT 95 (Thermo) with electron impact
(EI) ionization or Bruker maXis 4G ESI−Q-TOF mass spectrometer.
Analytical thin-layer chromatography (TLC) was performed on silica gel
GF254. Silica gel was used for column chromatography.
Plant Materials. Castor bean seeds (R. communis L.) were obtained
from the Agricultural Science Academy of Zibo Shandong China and
grown as previously described.15 Seedlings were grown in moist
vermiculite for 7−8 days, and average-sized seedlings were selected for
the experiments.
Insects. Larvae of Spodoptera litura F. were obtained from the
Guangzhou Biological Control Station and raised with artificial feed at
26 2 °C and 60% relative humidity under a photoperiod of 16:8 h
(light/dark).
Membrane Potential Measurements. Plasma membrane potential
was measured according to our previously described measurements.9
The fluorescent membrane potential indicator dye, bis-(1,3-dibutylbar-
bituric acid)-trimethine oxonol [DiBAC4(3)], was selected as the
fluorescent membrane potential indicator dye. The method was
established using flow cytometric analysis of the protoplasts of R.
communis cotyledons. The protoplasts were suspended in buffer solution
without (control) or with title compounds at 100 μM concentration for
2 h, and then the protoplast suspension was incubated with 2 μM
DiBAC4(3) for 30 min at 28 °C prior to flow cytometric analysis.
Phloem Sap Collection and Analysis. The phloem sap collection
method was similar to that recently described.9 The cotyledons were
Received: February 25, 2013
Revised: April 14, 2013
Accepted: April 15, 2013
Published: April 15, 2013
© 2013 American Chemical Society
4236
dx.doi.org/10.1021/jf400888c | J. Agric. Food Chem. 2013, 61, 4236−4241

Journal of Agricultural and Food Chemistry
Article
Figure 1. Chemical structure of fipronil, GTF, and monosaccharide−fipronil conjugates.
incubated in buffered solution containing 20 mM 2-(N-morpholino)-
ethanesulfonic acid (MES), 0.25 mM MgCl2, and 0.5 mM CaCl2 at pH
5.5, and each chemical was used at 100 μM in these experiments. The
phloem sap was analyzed by high-performance liquid chromatography
(HPLC) after dilution with water (phloem sap/water, 1:5, v/v). Agilent
Technologies 1100 HPLC system with a vacuum degasser, a quaternary
pump, an autosampler, and an ultraviolet−visible (UV−vis) detector
were employed for analysis. An Agilent C18 reversed-phase column (5
μm, 250 × 4.6 mm inner diameter) was used and maintained at 30 °C.
The mobile phase consisted of acetonitrile and water (50:50, v/v), at a
flow rate of 1 mL min−1, and the injection volume was 10 μL. The
absorbance wavelength was 210 nm. An external calibration method was
used to quantify the title compounds. A series of standard solutions of
title compounds (0.5, 1, 5, 10, 25, 50, and 100 μM) for linearities were
prepared in methanol. The linear equations and limits of detection
(LODs) of the title compounds were shown in Table 1. The LODs were
calculated as a signal/noise ratio = 3.
The reaction mixture was quenched by adding ice water, and the
resultant mixture was extracted with ethyl acetate (15 mL × 3). The
combined organic layers were washed with aqueous sodium hydrogen
carbonate and brine, dried with sodium sulfate, filtered, and evaporated
in vacuo. The residues were purified by column chromatography to
obtain the desired product 1 as a yellow solid in 91% yield.
1H NMR (CDCl3) δ: 7.85−7.79 (m, 2H), 6.05 (t, J = 5.2 Hz, 1H),
4.21−4.16 (m, 2H), 3.81−3.76 (m, 1H), 3.74−3.68 (m, 1H), 1.28−1.23
(m, 3H). 13C NMR (CDCl3) δ: 168.2, 151.1, 136.8, 136.7, 135.6, 135.5,
135.4, 128.7, 127.3, 126.6, 126.5 (two), 124.6, 124.3, 122.8, 122.1, 121.0,
119.2, 110.3, 96.0, 62.7, 45.9, 14.1. EI−MS, m/z 522 M+.
Synthesis of N-[3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)-
phenyl]-4-[(trifluoromethyl)-sulfinyl]-1H-pyrazol-5-yl]-aminoetha-
nol (2) (Scheme 1). To a stirred solution of compound 1 (0.523 g, 1
mmol) in THF (10 mL) was added NaBH4 (0.072 g, 2 mmol) and
ZnCl2(0.136g, 1mmol). The reaction mixture was heated at 50 °C using
a preheated oil bath and stirred for 1 h. After completion, the pH value of
the reaction mixture was adjusted to 7−8 by 1.0 M HCl, and the
resultant mixture was extracted with ethyl acetate (15 mL × 3). The
combined organic layers were washed with aqueous sodium hydrogen
carbonate and brine, dried with sodium sulfate, filtered, and evaporated
in vacuo. The residues were purified by column chromatography to
obtain the desired product 2 as a yellow solid in 56% yield.
Table 1. Linear Equations, Correlation Coefficients, and
LODs of the HPLC Method for Quantification of Compounds
4a−4j
compound
linear equation
correlation coefficient LOD (mg/L)
1H NMR (DMSO-d6) δ: 8.36 (s, 1H), 8.34 (d, J = 1.2 Hz, 1H), 7.59
(t, J = 6.0 Hz, 1H), 4.97 (s, 1H), 3.56−3.47 (m, 2H), 3.27−3.18 (m,
2H). 13C NMR (DMSO-d6) δ: 150.7, 136.0, 135.9, 134.6, 133.8, 133.6,
133.4, 128.9, 127.0, 126.9, 126.7, 126.6, 125.6, 124.4, 123.2, 121.4, 111.6,
99.6, 94.7, 60.1, 46.6. EI−MS, m/z 480 M+.
General Procedure for Synthesis Title Compounds (4a−4j).
Trichloroacetimidate donors (a−j) (Scheme 2) were synthesized as
previously described.16,17
4a
4b
4c
4d
4e
4f
y = 20.08x + 7.53
y = 20.17x + 6.05
y = 20.92x − 0.69
y = 20.11x − 7.23
y = 20.99x + 4.08
y = 20.82x − 2.95
y = 20.15x − 1.88
y = 20.98x − 0.39
y = 20.28x − 0.72
y = 20.77x − 2.55
0.9994
0.9995
0.9997
0.9997
0.9991
0.9998
0.9997
0.9996
0.9995
0.9997
0.04
0.06
0.02
0.09
0.07
0.06
0.07
0.04
0.05
0.05
4g
4h
4i
Step 1 (Scheme 3): A solution of trichloroacetimidate donor (a−j) (1
mmol) and compound 2 (1 mmol) in anhydrous CH2Cl2 (10 mL) was
stirred at room temperature with activated 4 Å molecular sieves for 30
min. The reaction mixture was then cooled to −30 °C, and BF3·OEt2
(0.5 mmol) was added dropwise. The reaction was carried out under an
atmosphere of nitrogen. After stirring at −30 °C for 1 h and then at room
temperature for 0.5 h, the reaction mixture was quenched with Et3N and
filtered through celite. The filtrate was concentrated and purified by flash
chromatography to afford compounds 3a−3j as solids.
4j
Synthesis of Ethyl N-[3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)-
phenyl]-4-[(trifluoromethyl)-sulfinyl]-1H-pyrazol-5-yl]-aminoace-
tate (1) (Scheme 1). In an ice bath, sodium hydride (0.96 g, 40 mmol)
was added in a portion to the solution of fipronil (4.37 g, 10 mmol) in
dry tetrahydrofuran (THF, 40 mL), and then ethyl bromoacetate (1.11
mL, 10 mmol) was added dropwise. The mixture was then stirred at
room temperature for 3 h.
Step 2 (Scheme 3): The solid above (0.5 mmol) was added to a
solution of sodium methoxide in dry methanol (0.05 M, 5 mL). The
resultant solution was stirred for 30 min at room temperature. The
Scheme 1. Reagents and Conditions: (1) NaH, THF, and 2 h and (2) NaBH4, ZnCl2, THF, 50 °C, and 1 h
4237
dx.doi.org/10.1021/jf400888c | J. Agric. Food Chem. 2013, 61, 4236−4241

Journal of Agricultural and Food Chemistry
Article
Scheme 2. Monosaccharide Trichloroacetimidate Donors
mixture was neutralized with Amberlite IR120 (H+) resin and filtered,
and the filtrate was then evaporated. The residues were purified by
column chromatography to obtain the desired products 4a−4j (Figure
2).
Figure 2. Structures of designed monosaccharide−fipronil conjugates.
The monosaccharides of these conjugates fall within the following three
categories: hexose (4a−4c), pentose (4d and 4e), and deoxysugar (4f−
4j).
N-[3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(tri-
fluoromethyl)-sulfinyl]-1H-pyrazol-5-yl]-2-aminoethyl β-D-Gluco-
pyranoside (4a). Yield of 62% for two steps, white solid, and the
product was isolated as a pair of diastereoisomers in a ratio of 1:1. 1H
NMR (CD3OD) δ: 8.11−8.08 (m, 2H), 4.26 (d, J = 7.7 Hz, 1H), 4.25*
(d, J = 7.7 Hz, 1H), 4.06−4.01 (m, 1H), 3.86 (t, J = 11.6 Hz, 1H), 3.85*
(t, J = 11.6 Hz, 1H), 3.75−3.71 (m, 1H), 3.70−3.66* (m, 1H), 3.63−
3.56 (m, 2H), 3.52−3.48 (m, 1H), 3.45−3.41 (m, 1H), 3.37−3.22 (m,
2H), 3.19 (t, J = 9.2 Hz, 1H), 3.19* (t, J = 9.2 Hz, 1H) (∗ represents
another diastereoisomer). 13C NMR (CD3OD) δ: 152.5, 152.4, 138.0
(two), 137.9 (two), 136.3, 136.2, 136.1, 135.9, 130.3, 128.1, 128.0,
127.9, 127.8, 127.7, 125.9, 124.6, 122.8, 120.9, 112.3, 104.3, 104.1, 96.8,
78.1, 78.0, 77.9, 75.1, 75.0, 71.7, 71.6, 69.4, 69.3, 62.9, 62.8, 46.3, 45.7.
HRMS (EI) calcd for C20H18Cl2F6N4O7S, 642.0172; found, 642.0169.
N-[3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(tri-
fluoromethyl)-sulfinyl]-1H-pyrazol-5-yl]-2-aminoethyl β-D-Galacto-
pyranoside (4b). Yield of 57% for two steps, white solid, and the
product was isolated as a pair of diastereoisomers in a ratio of 1:1. 1H
NMR (CD3OD) δ: 8.10−8.06 (m, 2H), 4.21 (d, J = 7.7 Hz, 1H), 4.20*
(d, J = 7.7 Hz, 1H), 4.06−4.01 (m, 1H), 3.81−3.79 (m, 1H), 3.73−3.66
(m, 3H), 3.60−3.55 (m, 1H), 3.50−3.40 (m, 4H). 13C NMR (CD3OD)
δ: 152.5, 152.4, 138.1, 138.0, 137.9, 136.3, 136.2, 136.1 (two), 135.9
(two), 130.3, 128.1, 128.0, 127.9, 127.8, 126.4, 125.9, 124.6, 122.8,
112.3, 104.9, 104.7, 96.8, 76.8, 76.7, 75.0, 74.8, 72.4, 70.3, 70.2, 69.4,
69.2, 62.5 (two), 46.4, 45.8. HRMS (ESI) calcd for
C20H18Cl2F6N4NaO7S [M + Na]+, 665.0075; found, 665.0073.
N-[3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(tri-
fluoromethyl)-sulfinyl]-1H-pyrazol-5-yl]-2-aminoethyl α-D-Manno-
pyranoside (4c). Yield of 52% for two steps, white solid, and the
product was isolated as a pair of diastereoisomers in a ratio of 1.3:1. 1H
NMR (CD3OD) δ: 8.12−8.07 (m, 2H), 4.77 (d, J = 1.3 Hz, 1H), 4.71*
(d, J = 1.2 Hz, 1H), 3.95−3.85 (m, 1H), 3.84−3.80 (m, 1H), 3.79−3.74
Scheme 3. Reagents and Conditions: (1) BF3·OEt2, CH2Cl2, and 4 Å molecular sieves and (2) CH3OH and CH3ONa
4238
dx.doi.org/10.1021/jf400888c | J. Agric. Food Chem. 2013, 61, 4236−4241

Journal of Agricultural and Food Chemistry
Article
(m, 1H), 3.63 (dd, J = 6.4, 11.6 Hz, 1H), 3.60−3.55 (m, 1H), 3.56−3.47
(m, 4H), 3.46−3.41 (m, 1H). 13C NMR (CD3OD) δ: 152.4, 152.3,
138.1, 138.0, 137.9, 137.8, 136.2 (two), 136.0 (two), 128.2, 128.0, 127.8
(two), 126.0, 124.6, 122.8, 112.4, 102.7, 102.1, 96.4, 74.9, 74.7, 72.5,
72.4, 71.9, 71.8, 69.1, 68.6, 68.5, 68.2, 62.9, 45.5. HRMS (ESI) calcd for
C20H18Cl2F6N4NaO7S [M + Na]+, 665.0075; found, 665.0071.
N-[3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(tri-
fluoromethyl)-sulfinyl]-1H-pyrazol-5-yl]-2-aminoethyl α-D-Arabo-
pyranoside (4d). Yield of 56% for two steps, white solid, and the
product was isolated as a pair of diastereoisomers in a ratio of 1:1. 1H
NMR (CD3OD) δ: 8.10−8.07 (m, 2H), 4.18 (d, J = 6.6 Hz, 1H), 4.17*
(d, J = 6.6 Hz, 1H), 3.94−3.88 (m, 1H), 3.81−3.77 (m, 2H), 3.72−3.68
(m, 1H), 3.66−3.62* (m, 1H), 3.55−3.40 (m, 5H). 13C NMR
(CD3OD) δ: 152.4, 138.1, 137.9 (two), 136.3, 136.1 (two), 128.1,
128.0, 127.9, 127.8 (two), 126.4, 125.9, 124.6, 123.7, 122.7, 120.9, 112.3,
104.9, 104.8, 97.7, 74.2, 74.1, 72.3, 69.5, 69.2 (two), 66.9 (two), 46.3,
45.9. HRMS (EI) calcd for C19H16Cl2F6N4O6S, 612.0066; found,
612.0066.
anoside (4i). Yield of 41% for two steps, white solid, and the product was
isolated as a pair of diastereoisomers in a ratio of 1.1:1. H NMR
1
(CD3OD) δ: 8.09−8.05 (m, 2H), 4.18 (d, J = 7.2 Hz, 1H), 3.99−3.93
(m, 1H), 3.72−3.63 (m, 1H), 3.62−3.59 (m, 1H), 3.58−3.54 (m, 2H),
3.52−3.46 (m, 1H), 3.43−3.38 (m, 2H), 1.25−1.22 (m, 3H). 13C NMR
(CD3OD) δ: 152.5 (two), 138.1, 138.0, 137.9 (two), 136.2, 136.1 (two),
135.9 (two), 128.0 (two), 127.9, 127.8, 125.9, 124.5, 122.8, 112.3, 104.8,
104.6, 102.9, 73.0, 72.9, 72.2 (two), 72.0 (two), 69.3, 69.2, 46.3, 45.9,
16.7 (two). HRMS (ESI) calcd for C20H19Cl2F6N4O6S [M + H]+,
627.0307; found, 627.0303.
N-[3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(tri-
fluoromethyl)-sulfinyl]-1H-pyrazol-5-yl]-2-aminoethyl α-D-Rhamno-
pyranoside (4j). Yield of 49% for two steps, white solid, and the product
1
was isolated as a pair of diastereoisomers in a ratio of 1.4:1. H NMR
(CD3OD) δ: 8.12−8.08 (m, 2H), 4.69 (d, J = 1.2 Hz, 1H), 4.64* (d, J =
1.2 Hz, 1H), 3.90−3.86 (m, 1H), 3.80−3.75 (m, 1H), 3.73−3.68 (m,
1H), 3.58−3.50 (m, 3H), 3.44−3.49 (m, 1H), 3.35 (t, J = 9.5 Hz, 1H),
1.25 (d, J = 6.3 Hz, 3H), 1.23* (d, J = 6.1 Hz, 3H). 13C NMR (CD3OD)
δ: 152.4, 152.3, 138.0 (two), 137.9, 137.8, 136.2, 136.1, 136.0, 128.2,
128.0 (two), 127.9, 127.8, 126.0, 124.6, 122.8, 112.4, 102.8, 102.2, 96.6,
96.5, 79.5, 73.8, 73.7, 72.3, 72.2, 72.1, 71.9, 70.1, 69.9, 69.0, 68.1, 45.4,
45.3, 18.0 (two). HRMS (ESI) calcd for C20H18Cl2F6N4NaO6S [M +
Na]+, 649.0126; found, 649.0123.
Insecticidal Activity of Compounds 4a−4j against S. litura F.
Assessments of compounds 4a−4j and fipronil bioactivities on the third
instar larvae of S. litura F. were according to the previously described
procedure9,18 by the leaf disk-dipping assay.
Test compounds were dissolved in acetone and suspended in distilled
water containing Tween 80 (0.1%), and the concentration of acetone is
below 5%. Leaf disks were dipped in each test solutions for 30 s, after air-
drying, the treated leaf disks were placed into Petri dishes (9 cm in
diameter). A total of 10 third-instar larvae of S. litura were released into
each dish. Distilled water containing acetone (5%) and Tween 80
(0.1%) solutions was used as the control. Petri dishes were incubated at
26 2 °C and 60% relative humidity under a photoperiod of 16:8 (light/
dark). All treatments were repeated 3 times. Mortalities were observed
48 h later.
N-[3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(tri-
fluoromethyl)-sulfinyl]-1H-pyrazol-5-yl]-2-aminoethyl β-D-Xylopyra-
noside (4e). Yield of 45% for two steps, white solid, and the product was
1
isolated as a pair of diastereoisomers in a ratio of 1:1.2. H NMR
(CD3OD) δ: 8.10−8.07 (m, 2H), 4.19 (d, J = 7.6 Hz, 1H), 4.18* (d, J =
7.6 Hz, 1H), 3.93−3.89 (m, 1H), 3.80−3.72 (m, 2H), 3.69−3.64 (m,
1H), 3.60−3.56* (m, 1H), 3.48−3.36 (m, 2H), 3.28 (dd, J = 9.0, 1.6 Hz,
1H), 3.26* (dd, J = 9.0, 1.6 Hz, 1H), 3.17−3.09 (m, 2H). 13C NMR
(CD3OD) δ: 152.5, 152.3, 138.0, 137.9 (two), 136.2, 136.2, 136.1,
130.3, 128.1, 128.0, 127.9, 127.8, 126.4, 125.9, 124.5, 123.7, 120.9, 112.3,
105.1, 104.9, 96.9, 77.8, 77.7, 74.9, 74.7, 71.1, 71.0, 69.6, 69.3, 66.9, 46.2,
45.7. HRMS (EI) calcd for C19H16Cl2F6N4O6S, 612.0066; found,
612.0064.
N-[3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(tri-
fluoromethyl)-sulfinyl]-1H-pyrazol-5-yl]-2-aminoethyl α-L-Rhamno-
pyranoside (4f). Yield of 51% for two steps, white solid, and the product
1
was isolated as a pair of diastereoisomers in a ratio of 1:1. H NMR
(CD3OD) δ: 8.12−8.08 (m, 2H), 4.69 (d, J = 1.4 Hz, 1H), 4.64* (d, J =
1.4 Hz, 1H), 3.87 (dd, J = 3.4, 1.4 Hz, 1H), 3.82−3.74 (m, 1H), 3.73−
3.68 (m, 1H), 3.58−3.48 (m, 3H), 3.39−3.43 (m, 1H), 3.37 (t, J = 9.4
Hz, 1H), 3.36* (t, J = 9.4 Hz, 1H), 1.26 (d, J = 6.1 Hz, 3H), 1.24* (d, J =
6.1 Hz, 3H). 13C NMR (CD3OD) δ: 152.4, 152.3, 138.0 (two), 137.9,
137.8, 136.2, 136.0, 130.4, 128.2, 128.0, 127.9, 127.8, 126.4, 126.0, 124.6,
123.8, 122.7, 120.9, 112.4, 102.8, 102.2, 96.6, 73.8, 73.7, 72.3, 72.2, 72.1,
71.9, 70.1, 69.9, 69.0, 68.1, 45.5, 45.4, 18.0 (two). HRMS (EI) calcd for
C20H18Cl2F6N4O6S, 626.0223; found, 626.0220.
RESULTS AND DISCUSSION
■
Synthesis and Characterization. To create a glycosidic
linkage between fipronil and the monosaccharide, we used the
trichloroacetimidate method.19−21 In glycosylation reactions,
glycosyl donors possess an acyloxyl group with a participating
function at C2, which exclusively yields the corresponding 1,2-
trans glycoside with quite high stereoselectivity.21 Finally, a series
of conjugates of different monosaccharides and fipronil with 1,2-
trans O-glycosidic linkage (4a−4j) were synthesized.
N-[3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(tri-
fluoromethyl)-sulfinyl]-1H-pyrazol-5-yl]-2-aminoethyl β-L-Fucopyr-
anoside (4g). Yield of 39% for two steps, white solid, and the product
1
was isolated as a pair of diastereoisomers in a ratio of 1.1:1. H NMR
(CD3OD) δ: 8.09−8.06 (m, 2H), 4.18 (d, J = 7.2 Hz, 1H), 3.99−3.93
(m, 1H), 3.72−3.63 (m, 1H), 3.62−3.55 (m, 2H), 3.53−3.46 (m, 1H),
3.45−3.38 (m, 3H), 1.24 (d, J = 5.3 Hz, 3H), 1.23* (d, J = 5.3 Hz, 3H).
13C NMR (CD3OD) δ: 149.6 (two), 135.2, 135.1, 135.0, 133.4, 133.3,
133.0, 127.5, 127.4, 125.2, 125.0, 123.0, 121.7, 119.9, 109.4 (two), 101.8,
101.7, 94.0 (two), 76.6, 72.3, 72.1, 70.1, 70.0, 69.3, 69.2, 69.1, 43.4, 43.0,
13.8 (two). HRMS (ESI) calcd for C20H19Cl2F6N4O6S [M + H]+,
627.0307; found, 627.0301.
N-[3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(tri-
fluoromethyl)-sulfinyl]-1H-pyrazol-5-yl]-2-aminoethyl β-D-6-Deoxy-
glucopyranoside (4h). Yield of 53% for two steps, white solid, and the
product was isolated as a pair of diastereoisomers in a ratio of 1.1:1. 1H
NMR (CD3OD) δ: 8.10−8.07 (m, 2H), 4.22 (d, J = 9.6 Hz, 1H), 4.21*
(d, J = 9.6 Hz, 1H), 4.00−3.93 (m, 1H), 3.73−3.69 (m, 1H), 3.60−3.65
(m, 1H), 3.50−3.45 (m, 1H), 3.29−3.24 (m, 2H), 3.18−3.09 (m, 1H),
2.99−2.93 (m, 1H), 1.24 (d, J = 6.2 Hz, 3H), 1.23* (d, J = 6.2 Hz, 3H).
13C NMR (CD3OD) δ: 152.6, 152.4, 137.9 (two), 136.2 (two), 135.9,
128.1, 127.9, 127.8, 127.7, 125.9, 124.6, 122.8, 112.3 (two), 104.2, 104.0,
102.9, 96.9, 79.5, 77.8, 77.6, 76.9, 75.3, 75.2, 73.4, 73.3, 66.7, 69.2, 46.2,
45.4, 18.1, 18.0. HRMS (ESI) calcd for C20H19Cl2F6N4O6S [M + H]+,
627.0307; found, 627.0303.
1
The structures of compounds 4a−4j were confirmed via H
NMR, 13C NMR, and mass spectrometry. The 1H NMR spectra
of compounds 4a−4j showed the presence of a pair of
diastereoisomers,22 and from these spectra, we could determine
1
the ratio of these diastereoisomers. For example, the H NMR
spectrum of compound 4a showed two doublets at 4.26 and 4.25
ppm with J = 7.7 Hz, indicating both the presence of
diastereoisomers and the β configuration of the glucosidic
linkage.
Phloem Mobility. To evaluate the phloem mobility of
compounds 4a−4j, we used the castor bean system.15,23,24 As
previously reported,9 the plasma membrane potential with
compounds 4a−4j treatments was measured by flow cytometric
analysis of the protoplasts of R. communis cotyledons. In
comparison to the control, the relative fluorescence data were
not significantly different during the treatment period (Figure 3).
The results indicate that compounds 4a−4j have no depolarizing
effects on the transmembrane potential difference (PD) and the
xenobiotics tested are not phytotoxic in these short-term
N-[3-Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(tri-
fluoromethyl)-sulfinyl]-1H-pyrazol-5-yl]-2-aminoethyl β-D-Fucopyr-
4239
dx.doi.org/10.1021/jf400888c | J. Agric. Food Chem. 2013, 61, 4236−4241

Journal of Agricultural and Food Chemistry
Article
These results indicate that the phloem mobility of these
conjugates varies significantly. Several conjugates exhibit
moderate systemicity, and it can be inferred that some plant
sugar carriers may translocate xenobiotics much larger than their
natural substrate and with a lipophilic moiety. The mono-
saccharides of these conjugates fall within the following three
categories: hexose, pentose, and deoxysugar. Among the hexose
conjugates of fipronil, the analysis of phloem sap showed that the
concentrations of compounds 4a and 4c were 23.0 1.9 and 7.6
1.6 μM, respectively. Furthermore, compound 4b was not
detected, suggesting that the equatorial −OH group of hexose
improved phloem mobility. Among the conjugates of pentose
and fipronil, compound 4d (21.8 1.9 μM) exhibited better
phloem mobility than compound 4e (15.3 2.2 μM), suggesting
that the 4-axial −OH group of pentose improved phloem
mobility. Among the deoxysugar−fipronil conjugates, com-
pounds 4f (27.3
2.1 μM) and 4i (27.3
1.9 μM) both
exhibited good phloem mobility, and compound 4h exhibited no
phloem mobility, suggesting that the 4-axial −OH or 2-axial
−OH groups of deoxysugar improved phloem mobility. In our
previous work, fipronil was not detected in phloem sap under the
same experimental conditions.9 These results demonstrate that
conjugating hexose, pentose, or deoxysugar to fipronil through
O-glycosidic linkage can confer phloem mobility to fipronil in R.
communis L. effectively and that the −OH orientation of
monosaccharide has a significant effect on the phloem mobility
of the conjugates.
Insecticidal Activity. The insecticidal activity of test
compounds against S. litura F. was evaluated over a wide range
of concentrations. The LC50 (95% confidence limits) of fipronil
is 33.49 mg L−1 (25.43−44.12 mg L−1). The title compounds
(4a−4j) did not exhibit insecticidal activity against S. litura F. at
100 mg L−1. As noted by Hsu et al.,4 making derivative pesticides
phloem-mobile is often a challenge because of the concomitant
loss of activity. Adding the monosaccharide group into fipronil
enhanced the phloem systemicity but decreased the insecticidal
activity. In our previous study,9 GTF was reconverted into the
parent molecule in the plants; however, whether the conjugates
synthesized in the present study can be reconverted into the
parent molecule in the different plant organs requires further
investigation.
Figure 3. Plasma membrane potential in the protoplasts of R. communis
cotyledons after 2 h of treatment. (A) Fluorescent intensity histogram
marked DiBAC4(3) of the treatment of compounds 4a−4j and the
control (C). (B) Relative fluorescence of the treatment of compounds
4a−4j and the control (C). The protoplasts were suspended in a
buffered solution at pH 5.8 with or without (control) compounds 4a−4j
(200 μM) for 2 h. The protoplasts were analyzed by flow cytometry for
DiBAC4(3) fluorescence. The data (mean SE; n = 3) within a column
are not significantly different, as shown by the Duncan’s multiple range
test (p > 0.05).
experiments. Therefore, the castor bean system is suitable for
testing the phloem systemicity for these conjugates.
The results of the analysis of phloem sap are summarized in
Table 2. Among these conjugates, D-fucose−fipronil (4i; 27.3
2.1 μM) and L-rhamnose−fipronil (4f; 27.3 1.9 μM) exhibited
the best phloem mobility, whereas D-galactose−fipronil (4b) and
D-6-deoxyglucose−fipronil (4h) were not detected, thereby
indicating that these conjugates did not exhibit phloem mobility.
In summary, a series of conjugates of fipronil and
monosaccharide were synthesized. The phloem-mobility tests
in the castor bean system demonstrated that conjugating hexose,
pentose, or deoxysugar to fipronil can confer phloem mobility to
fipronil in R. communis L. effectively and that the −OH
orientation of the monosaccharide substantially affected the
phloem mobility of the conjugates. Understanding the
mechanisms of phloem systemicity of the monosaccharide−
fipronil conjugates and whether these conjugates can be
reconverted into the parent molecule require further study.
Table 2. R. communis Phloem Sap Analysis after Soaking
Cotyledons in a Solution Containing Various Xenobiotics
a
xenobiotics
incubation medium (μM)
phloem sap (μM)
4i
100
100
100
100
100
100
100
100
100
100
200
27.3 2.1 a
27.3 1.9 a
23.0 1.9 ab
21.8 1.9 b
15.3 2.2 c
10.6 1.8 cd
7.6 1.6 d
6.1 1.4 d
ND
4f
4a
4d
4e
4g
4c
AUTHOR INFORMATION
■
4j
Corresponding Author
4b
4h
fipronil
*Telephone: +86-20-85285127. Fax: +86-20-38604926. E-mail:
hhxu@scau.edu.cn.
ND
b
ND
Author Contributions
a
§Jian-Guo Yuan and Han-Xiang Wu contributed equally to this
work.
Phloem sap collection started 2 h after the beginning of soaking and
lasted for 2 h. Mean SE; n = 3. ND = not detected. Duncan’s
multiple range tests at a 5% probability level were used to determine
statistical differences among treatments. The data in the table are the
mean SE, and those followed by different letters in the same column
Funding
Financial support was from the National Natural Science
Foundation of China (Grant 31171886) and the Doctor Science
are significantly different at the 5% level. Data are from Yang et al.9
b
4240
dx.doi.org/10.1021/jf400888c | J. Agric. Food Chem. 2013, 61, 4236−4241

Journal of Agricultural and Food Chemistry
Article
(17) Hou, S. J.; Kovac, P. A convenient synthesis of furanose-free D-
fucose per-O-acetates and a precursor for anthrose. Eur. J. Org. Chem.
2008, 11, 1947−1952.
Research Foundation of the Education Ministry of China (Grant
20114404110020).
Notes
(18) Sawada, Y.; Yanai, T.; Nakagawa, H.; Tsukamoto, Y.; Tamagawa,
Y.; Yokoi, S. J.; Yanagi, M.; Toya, T.; Sugizaki, H.; Kato, Y.; Shirakura,
H.; Watanabe, T.; Yajima, Y.; Kodama, S.; Masui, A. Synthesis and
insecticidal activity of benzoheterocyclic analogues of N′-benzoyl-N-
(tert-butyl)benzohydrazide: Part 3. Modification of N-tert-butylhydra-
zine moiety. Pest Manage. Sci. 2003, 59, 49−57.
(19) Schmidt, R. R.; Kinzy, W. Anomeric-oxygen activation for
glycoside synthesis: The trichloroacetimidate method. Adv. Carbohydr.
Chem. Biochem. 1994, 50, 21−123.
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
GTF, N-[3-cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-
[(trifluoromethyl)-sulfinyl]-1H-pyrazol-5-yl]-1-(β-D-glucopyra-
nosyl)-1H-1,2,3-triazole-4-methanamine; DiBAC4(3), bis-(1,3-
dibutylbarbituric acid)-trimethine oxonol
(20) Liu, Q. C.; Wang, P.; Zhang, L.; Guo, T. T.; Lv, G. K.; Li, Y. X.
Concise synthesis of two natural triterpenoid saponins, oleanolic acid
derivatives isolated from the roots of Pulsatilla chinensis. Carbohydr. Res.
2009, 344, 1276−1281.
(21) Zeng, Y.; Ning, J.; Kong, F. Z. Pure α-linked products can be
obtained in high yields in glycosylation with glucosyl trichloroacetimi-
date donors with a C2 ester capable of neighboring group participation.
Tetrahedron Lett. 2002, 43, 3729−3733.
(22) Zhao, Q. Q.; Li, Y. Q.; Xiong, L. X.; Wang, Q. M. Design, synthesis
and insecticidal activity of novel phenylpyrazoles containing a 2,2,2-
trichloro-1-alkoxyethyl moiety. J. Agric. Food Chem. 2010, 58, 4992−
4998.
(23) Antognoni, F.; Fornale, S.; Grimmer, C.; Komor, E.; Bagni, N.
Long-distance translocation of polyamines in phloem and xylem of
Ricinus communis L. plants. Planta 1998, 204, 520−527.
(24) 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, 1684−1693.
REFERENCES
■
(1) Pimentel, D. Amounts of pesticides reaching target pests:
Environmental impacts and ethics. J. Agric. Environ. Ethics 1995, 8,
17−29.
(2) Lichtner, F. Phloem mobility of crop protection products. Funct.
Plant Biol. 2000, 27, 609−614.
(3) Brueck, E.; Elbert, A.; Fischer, R.; Krueger, S.; Kuehnhold, J.;
Klueken, A. M.; Nauen, R.; Niebes, J. F.; Reckmann, U.; Schnorbach, H.
J.; Steffens, R.; van Waetermeulen, X. Movento, an innovative
ambimobile insecticide for sucking insect pest control in agriculture:
Biological profile and field performance. Crop Prot. 2009, 28, 838−844.
(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) Dufaud, A.; Chollet, J. F.; Rudelle, J.; Miginiac, L.; Bonnemain, J. L.
Derivatives of pesticides with an α-amino acid function: Synthesis and
effect on threonine uptake. Pestic. Sci. 1994, 41, 297−304.
(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, Gen. Subj. 1997,
1336, 331−341.
(7) Deletage-Grandon, C.; Chollet, J. F.; Faucher, M.; Rocher, F.;
Komor, E.; Bonnemain, J. L. Carrier-mediated uptake and phloem
system of a 350-Dalton chlorinated xenobiotic with an α-amino acid
function. Plant Physiol. 2001, 125, 1620−1632.
(8) Bartholomew, D. M.; Van Dyk, D. E.; Lau, S. C.; O’Keefe, D. P.;
Rea, P. A.; Viitanen, P. V. Alternate energy-dependent pathways for the
vacuolar uptake of glucose and glutathione conjugates. Plant Physiol.
2002, 130, 1562−1572.
(9) Yang, W.; Wu, H. H.; 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.
(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) 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.
(12) 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.
(13) Sauer, N.; Friedlander, K.; Graml-Wicke, U. Primary structure,
genomic organization and heterologous expression of a glucose
transporter from Arabidopsis thaliana. EMBO J. 1990, 9, 3045−3050.
(14) Buettner, M. The Arabidopsis sugar transporter (AtSTP) family:
An update. Plant Biol. 2010, 12, 35−41.
(15) Chollet, J. F.; Rocher, F.; Jousse, C.; Deletage-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.
(16) Su, Y. H.; Xie, J. S.; Wang, Y. G.; Hu, X.; Lin, X. F. Synthesis and
antitumor activity of new shikonin glycosides. Eur. J. Med. Chem. 2010,
45, 2713−2718.
4241
dx.doi.org/10.1021/jf400888c | J. Agric. Food Chem. 2013, 61, 4236−4241