Glucose positions affect the phloem mobility of glucose-fipronil conjugates

Article abstract of DOI:10.1021/jf5010429

In our previous work, a glucose-fipronil (GTF) conjugate at the C-1 position was synthesized via click chemistry and a glucose moiety converted a non-phloem-mobile insecticide fipronil into a moderately phloem-mobile insecticide. In the present paper, fipronil was introduced into the C-2, C-3, C-4, and C-6 positions of glucose via click chemistry to obtain four new conjugates and to evaluate the effects of the different glucose isomers on phloem mobility. The phloem mobility of the four new synthetic conjugates and GTF was tested using the Ricinus seedling system. The results confirmed that conjugation of glucose at different positions has a significant influence on the phloem mobility of GTF conjugates.

Full text of DOI:10.1021/jf5010429

Article
pubs.acs.org/JAFC
Glucose Positions Affect the Phloem Mobility of Glucose−Fipronil
Conjugates
Zhiwei Lei,† Jie Wang,† Genlin Mao, Yingjie Wen, Yuxin Tian, Huawei Wu, Yufeng Li, and Hanhong Xu*
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, and Key Laboratory of Natural Pesticide and
Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, Guangdong 510642, People’s Republic of China
S
* Supporting Information
ABSTRACT: In our previous work, a glucose−fipronil (GTF) conjugate at the C-1 position was synthesized via click chemistry
and a glucose moiety converted a non-phloem-mobile insecticide fipronil into a moderately phloem-mobile insecticide. In the
present paper, fipronil was introduced into the C-2, C-3, C-4, and C-6 positions of glucose via click chemistry to obtain four new
conjugates and to evaluate the effects of the different glucose isomers on phloem mobility. The phloem mobility of the four new
synthetic conjugates and GTF was tested using the Ricinus seedling system. The results confirmed that conjugation of glucose at
different positions has a significant influence on the phloem mobility of GTF conjugates.
KEYWORDS: phloem mobility, glucosyl conjugates, click chemistry, glycosyl azides, glucose positions
C-1- and C-3-substituted derivatives.11 (7-Nitrobenz-2-oxa-1,3-
diazol-4-yl)-amine (NBD) replaced at C-6 of the sugar cannot be
INTRODUCTION
■
The phloem is one of the two long-distance transport pathways
phosphorylated by hexokinase, but NBD substituted at C-2 can
in plants.1 The phloem mobility of pesticides is an essential
be metabolized to 2-NBDG-6-phosphate.12 In addition, several
requirement that contributes positively to its efficacy.2 Pesticides
different (C-2, C-3, C-4, and C-6) natural glucosides are found in
with good phloem mobility can have an efficacious function in
nature.13 Therefore, biological properties of the glucosides have a
controlling hidden and soil-dwelling pests3 because these
pesticides can be distributed along with the nutrients via the
correlation with different positions of glucose.
All of the monosaccharide−fipronil conjugates were linked
at C-1 of the monosaccharide,8,9 which gives rise to consider if
parent compounds linking different positions of the glucose may
have different phloem mobilities. In the present work, four novel
glucosyl conjugates were designed and synthesized by linking the
fipronil to C-2, C-3, C-4, and C-6 of glucose via click chemistry to
study the effects of the different positions of glucose on phloem
mobility.
phloem to pest-damaged sites.4 In addition, the agricultural
sector needs novel pesticides that have sufficient phloem
mobility in plants because of their evolved resistance, environ-
mental regulations, and economic considerations.5 For modern
synthetic insecticides, the most effective mode of transport in
plants is via the xylem, and only a few phloem-mobile insecticides
are available. An efficient and economical strategy to develop
novel systemic pesticides has been mentioned, in which the
structure of the parent compounds is modified by adding
endogenous carboxylic acid, amino acid, and hexose derivatives.2,3,6,7
For example, ε-(2,4-dichlorophenoxyacetyl)-L-Lys, a conjugate of an
α-amino acid and an auxenic herbicide, exhibits high phloem
mobility in broad bean (Vicia faba).7 IPGN, a novel fluorescent
conjugate containing glucose, could enter Ricinus communis phloem.8
Our research team focuses on exploring the possibility of
glycosylation to develop new phloem-mobile pesticides. N-[3-
Cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluor-
omethyl)-sulfinyl]-1H-pyrazol-5-yl]-1-(β-D-glucopyranosyl)-
1H-1,2,3-triazole-4-methanamine (GTF) has been synthesized,
and a systemicity test demonstrated that the glucose core confers
phloem mobility to fipronil.9 To fully understand the function
of the sugar core in the phloem-mobile molecule, a series of
monosaccharide−fipronil conjugates were synthesized in our
further study. Phloem mobility tests in castor bean seedlings
indicated that the hydroxyl of the monosaccharide has a
significant effect on the phloem mobility of the conjugates.10
Glucosyl derivatives, which are conjugated at different carbons
of glucose, have different properties. For example, the new
glucosyl dopamine conjugates substituted at C-6 of the sugar
were more potent inhibitors of glucose transport compared to
MATERIALS AND METHODS
■
General Information for Synthesis. Melting points were
determined on a FP62 digital micro melting point apparatus. Nuclear
magnetic resonance (NMR) spectra were obtained on a Bruker AV-600
instrument. Deuterated solvents were obtained from Cambridge
Isotope Laboratories (Andover, MA). CD3OD and CDCl3 solvent
1
peaks (3.31 and 7.26 ppm for H and 49.0 and 77.0 ppm for 13C,
respectively) were used as internal chemical shift references. The mass
spectra (MS) of new compounds were obtained by a Waters ZQ 4000
with electronspray ionization (ESI) or a Bruker maXis 4G ESI-Q-TOF
mass spectrometer. Data were reported as m/z. Silica gel was used for
column chromatography. Reagents and anhydrous solvents were used as
purchased without further purification.
Plant Materials. Castor bean seeds (R. communis) No. 9, obtained
from the Agricultural Science Academy of Zibo, Shandong, China, were sown
and grown as previously described.6 Six-day-old seedlings (the hypocotyl at
about 20 mm in length) were relatively selected for further experiments.
Received: March 4, 2014
Revised: May 20, 2014
Accepted: June 11, 2014
Published: June 11, 2014
© 2014 American Chemical Society
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a
Table 1. Properties of the Tested Compounds
properties
GTF
2-GTF
680.37
3-GTF
680.37
4-GTF
680.37
5-GTF
680.37
1.58
MW
680.37
log Kow
pKa
2.06
1.11
1.42
1.41
12.8 0.7
11.9 0.7
12.0 0.7
12.0 0.7
12.1 0.7
HBD
HBA
5
5
5
5
5
13
13
13
13
13
a
The physicochemical properties of tested compounds were predicted by ACD Laboratories Percepta program, version 14.0. Log Kow and pKa values
were classic values. 2-GTF, 3-GTF, 4-GTF, and 6-GTF mean that fipronil was conjugated at the C-2, C-3, C-4, and C-6 positions of glucose,
respectively. All values (MW, HBD, and HBA) are the same for the different compounds.
Membrane Potential Measurements. Plasma membrane poten-
tial of the protoplasts of R. communis cotyledons was performed with
flow cytometric analysis using a fluorescent membrane potential
indicator dye bis(1,3-dibutylbarbituric acid)-trimethine oxonol
[DiBAC4(3)]. The protoplasts were incubated with buffer solution
without (control) or with the five glucosyl fipronil conjugates at 100 μM
concentration for 5 h, and then the protoplast suspension was co-
incubated with 3 μM DiBAC4(3) for 30 min at 28 °C prior to flow
cytometric analysis.
Phloem Sap Collection and Analysis. The seedlings with 6 days
of development were prepared for phloem sap collection. The phloem
sap was collected from the upper part of the hypocotyl, which was similar
to that recently described.6,14 The cotyledons were removed from
endosperm carefully without bending or crushing the cotyledons. The
cotyledons were incubated in buffered solution supplemented with
100 μM glucosyl fipronil conjugtes. After 1 h of preincubation, the
hypocotyl was severed in the hook region for phloem exudation. Phloem
sap was collected from the upper part of the hypocotyl at a 1 h interval
for 5 h and was stored at 4 °C until analysis.15
The collected phloem sap, diluted with pure water [1:4 (v/v) phloem
sap/H2O], was quantified by an Agilent 1100 HPLC system.
Separations were made with a C8 reversed-phase column (5 μm,
250 × 4.6 mm inner diameter, Agilent Co.) at 30 °C. The injection was
10 μL, and the flow rate was 1 mL/min. The elution system consisted of
acetonitrile and water (50:50, v/v). The conjugates detected in the
phloem sap were further identified using ultra-performance liquid
chromatography−mass spectrometry (UPLC−MS, Waters, Milford,
MA). Samples were separated using an ACQUITY UPLC BEH C18
column (1.7 μm, 2.1 × 100 mm, Waters). The parameters were similar
to our previously reported method.16
Physicochemical Properties. The physicochemical properties
[molecular weight (MW), ionization constant in aqueous solution
(pKa), octanol/water partitioning coefficient (log Kow), number of
hydrogen bond donors (HBDs), and number of hydrogen bond
acceptors (HBAs)] of different positions of glucose conjugates were
predicted using ACD LogD suite version 14.0 software (Table 1).
Synthesis of 1,3,4,6-Tetra-O-acetyl-2-azido-2-deoxy-β-D-gluco-
side (2a) (Scheme 1). 2-Azido-2-deoxy-D-glucose (205 mg, 1 mmol)
was dissolved in pyridine (5 mL), and then acetic anhydride (Ac2O, 3
mL) was added with catalytic dimethylaminopyridine (DMAP, 6.1 mg,
0.05 mmol). After the solvents were stirred for 12 h at room
temperature, they were removed under reduced pressure and the
residue was diluted with CH2Cl2. The solution was washed with 1 M
HCl and saturated NaHCO3 solution, dried with MgSO4, concentrated
under reduced pressure, and purified by column chromatography (1:1
ethyl acetate/hexane) to obtain compound 2a. White power; yield, 78%;
melting point (mp), 80.4 °C; 1H NMR (600 MHz, CDCl3) δ, 5.54 (d, J
= 8.6 Hz, 1H, H-1), 5.08 (t, J = 9.6 Hz, 1H, H-4), 5.03 (t, J = 9.6 Hz, 1H,
H-3), 4.29 (dd, J = 12.5 and 4.5 Hz, 1H, H-6-a), 4.07 (dd, J = 12.5 and
2.1 Hz, 1H, H-6-b), 3.80 (ddd, J = 9.8, 4.5, and 2.2 Hz, 1H, H-5), 3.65
(dd, J = 9.9 and 8.6 Hz, 1H, H-2); 13C NMR (150 MHz, CDCl3) δ,
170.6, 69.8, 169.7, 168.6, 92.6, 72.8, 72.8, 67.9, 62.7, 61.5, 21.0, 20.8,
20.7, 20.6; MS−ESI [M + Na]+, 396.1.
solvent afforded 3-azido-3-deoxy-D-glucose without further purification.
The crude 3-azido-3-deoxy-D-glucose was per-acetylated according to
the procedure of compound 2a to obtain compound 3a. α anomer:
syrup; yield, 35%; 1H NMR (CDCl3, 600 MHz) δ, 6.25 (d, J = 3.6 Hz,
1H, H-1), 4.98 (t, J = 10.1 Hz, 1H, H-4), 4.90 (dd, J = 10.6 and 3.5 Hz,
1H, H-2), 4.17 (dd, J = 12.0 and 4.6 Hz, 1H, H-6-a), 4.05−3.99 (m, 1H,
H-6-b), 3.93 (t, J = 10.3 Hz, 1H, H-3), 3.75 (m, H-5), 2.15−2.03 (4 s,
12H, COCH3); 13C NMR (CDCl3, 150 MHz) δ, 171.0, 169.7, 168.6,
87.8, 73.2, 69.9, 69.8, 61.5, 60.5, 21.1, 20.9, 20.8, 20.8. β anomer: syrup;
yield, 35%; 1H (CDCl3, 600 MHz) δ, 5.63 (d, J = 8.2 Hz, 1H, H-1), 4.99
(dd, J = 10.1 and 8.2 Hz, 1H, H-2), 4.18 (dd, J = 12.0 and 4.6 Hz, 1H, H-6-
a), 4.04−3.96 (m, 3H), 3.66 (t, J = 10.1 Hz, 1H, H-3), 2.15−2.03 (4s, 12H,
COCH3); 13C (CDCl3, 150 MHz) δ, 171.0, 169.7, 168.6, 91.3, 69.6, 67.4,
66.0, 64.4, 61.5, 21.1, 21.0, 20.9, 20.8; MS−ESI [M + Na]+, 396.1.
Synthesis of 1,2,3,6-Tetra-O-acetyl-4-azido-4-deoxy-α-D-gluco-
side (4a). To a solution of methyl 4-azido-4-deoxy-α-D-glucopyranoside
(2 g, 9.13 mmol) in glacial acetic acid (10 mL) and acetic anhydride
(10 mL) was added concentrated sulfuric acid (2 mL) dropwise with
stirring. The solution was left at room temperature for 48 h. Then, 6.13 g
of sodium acetate was added to neutralize the sulfuric acid, and
chloroform (100 mL) was added. The mixture was pour into ice−water.
The chloroform layer was washed with aqueous NaHCO3 and aqueous
NaCl, dried, and evaporated to dryness in vacuo. The brown oil was
purified by column chromatography (1:1 ethyl acetate/hexane) to
obtain compound 4a. Syrup; yield, 68%; 1H NMR (600 MHz, CDCl3) δ,
6.12 (d, J = 3.6 Hz, 1H, H-1), 5.31 (t, J = 10.0 Hz, 1H, H-3), 4.88 (dd, J =
10.2 and 3.7 Hz, 1H, H-2), 4.18 (dd, J = 12.4 and 1.8 Hz, 1H, H-6-a),
4.12 (dd, J = 12.4 and 3.9 Hz, 1H, H-6-b), 3.80−3.72 (m, 1H, H-5), 3.58
(t, J = 10.2 Hz, 1H, H-4), 2.01 (s, 3H, COCH3), 1.97 (s, 3H, COCH3),
1.96 (s, 3H, COCH3), 1.85 (s, 3H, COCH3); 13C (150 MHz, CDCl3) δ,
170.1, 169.5, 169.4, 168.5, 88.8, 70.1, 69.9, 69.0, 62.1, 59.6, 20.5, 20.4,
20.3, 20.1; MS−ESI [M + Na]+, 396.1.
Synthesis of 1,2,3,4-Tetra-O-acetyl-6-azido-6-deoxy-D-glucopyr-
anose (6a). 1,2,3,4-Tetra-O-acetyl-6-O-p-tolylsulfonyl-D-glucopyra-
nose17 (7 g, 13.95 mmol) and sodium azide (5.0 g, 154 mmol)
dissolved in dry N,N-dimethylformamide (DMF) (50 mL) were stirred
at 50 °C for 4 h and then at room temperature for 16 h. The mixture was
poured onto icy water and extracted with ether (4 × 50 mL). The
organic extracts were washed twice with water and then dried over
calcium chloride. After evaporation to dryness, 1,2,3,4-tetra-O-acetyl-6-
azido-6-deoxy-α-D-glucopyranose (6a) was obtained (2.03 g). Needle
solid; yield, 39%; mp, 146.2 °C; 1H NMR (600 MHz, CDCl3) δ, 6.33 (d,
J = 3.7 Hz, 1H, H-1), 5.44 (t, J = 9.9 Hz, 1H, H-3), 5.09 (t-like, J = 9.6 Hz,
1H, H-4), 5.07 (dd, J = 10.3 and 3.7 Hz, H-2), 4.06 (m, 1H, H-5), 3.38
(dd, J = 13.5 and 2.7 Hz, 1H, H-6-a), 3.28 (dd, J = 13.5 and 5.5 Hz, 1H,
H-6-b), 2.17 (s, 3H, COCH3), 2.03 (s, 3H, COCH3), 2.01 (s, 3H,
COCH3), 2.00 (s, 3H, COCH3); 13C NMR (150 MHz, CDCl3) δ, 170.3,
169.7, 169.5, 168.8, 88.9, 70.8, 69.7, 69.3, 69.1, 50.8, 20.9, 20.7, 20.7,
20.5; MS−ESI [M + Na]+, 396.1.
General Procedure for Synthesis of the Conjugates 2-GTF, 3-GTF,
4-GTF, and 6-GTF (Scheme 2). Fipronil−alkyne (474 mg, 1 mmol) was
added to a vigorously stirred suspension of azide-containing glucoses
(373 mg, 1 mmol) in 3 mL of tert-butyl alcohol. The reaction was
initiated by the addition of a solution of CuSO4·5H2O (100 mg,
0.4 mmol) and sodium ascorbate (173 mg, 0.8 mmol) in distilled water
(3 mL). The deep yellow suspension was stirred vigorously at 50 °C for
3 h. Distilled water (10 mL) was added, and the aqueous layer was
Synthesis of 1,2,4,6-Tetra-O-acetyl-3-azido-3-deoxy-D-glucoside
(Anomers α/β = 1:1) (3a). A solution of 1,2:5,6-di-O-isopropylidene-3-
azido-3-deoxy-α-D-glucofuranose (2 g, 7 mmol) in 80% CF3COOH
(10 mL) was stirred at room temperature for 3 h. Evaporation of the
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Scheme 1. Synthesis of Azido-Containing Acetylglucoses: (A) 1, TMSN3, SnCl4, CH2Cl2; (B) 1, TfN3, CuSO4, H2O, MeOH,
CH2Cl2; 2, Ac2O, DMAP, Pyridine; (C) 1, Acetone, ZnCl2, H3PO4; 2, Pyridinium Dichromate, Ac2O, CH2Cl2; 3, NaBH4, EtOH; 4,
Tf2O, CH2Cl2, Pyridine; 5, NaN3, MeCN; 6, TFA, Ac2O, DMAP, Pyridine; (D) 1, Benzoyl Chloride, Pyridine; 2, Tf2O, CH2Cl2,
Pyridine; 3, NaN3, MeCN; 4, NaOMe, MeOH; 5, H2SO4, Ac2O; and (E) 1, TsCl, Ac2O, Pyridine; 2, NaN3, DMF
extracted with chloroform (10 mL × 3). The combined organic extracts
were washed with aqueous sodium hydrogen carbonate and brine, dried
with sodium sulfate, filtered, and evaporated in vacuo. The crude
compound was added to a solution of sodium methoxide in dry
methanol (0.05 M, 15 mL). The resultant solution was stirred for 30 min
at room temperature. The mixture was neutralized with Amberlite IR
120 (H+) resin and filtered, and the filtrate was then evaporated. The
residues were purified by column chromatography (9:1 CH2Cl2/
MeOH) to obtain the glucosyl fipronil conjugates.
41.0*; HRMS (ESI) calcd for C21H17Cl2F6N7O6SNa [M + Na]+,
702.0140; found, 702.0135.
D-Glucopyranose, 3-Deoxy-3-(4-(((1-(2,6-dichloro-4-
(trifluoromethyl)phenyl)-4-((trifluoromethyl)sulfinyl)-3-cyano-1H-
pyrazol-5-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)- (3-GTF). White
1
solid; yield, 80%; mp, 156.8 °C; H NMR (CD3OD, 600 MHz) δ,
8.07 (s, 2H), 7.86 (s, 1H), 7.85* (s, 1H), 5.23 (d, J = 3.1 Hz, 1H), 4.50
(d, J = 3.0 Hz, 2H), 4.47* (d, J = 3.0 Hz, 2H), 4.29 (t, J = 10.1 Hz, 1H),
4.28* (t, J = 10.1 Hz, 1H), 4.04−4.08 (m, 1H), 4.02 (d, J = 10.0 Hz, 1H),
3.96* (d, J = 10.0 Hz, 1H), 3.93−3.96 (m, 1H), 3.89 (d, J = 11.9 Hz,
1H), 3.82* (d, J = 11.9 Hz, 1H), 3.72−3.80 (m, 2H); 13C NMR
(CD3OD, 150 MHz) δ, 151.8, 144.7, 144.4*, 137.9, 136.3*, 136.0,
135.8, 127.9, 127.8, 125.9, 125.7, 124.5, 123.5, 122.5 112.1, 93.4, 79.2,
73.4, 71.7, 69.4, 67.8, 62.3, 41.0; HRMS (ESI) calcd for
C21H17Cl2F6N7O6SNa [M + Na]+, 702.0140; found, 702.0135.
D-Glucopyranose, 2-Deoxy-2-(4-(((1-(2,6-dichloro-4-
(trifluoromethyl)phenyl)-4-((trifluoromethyl)sulfinyl)-3-cyano-1H-
pyrazol-5-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)- (2-GTF). White
1
solid; yield, 88%, mp, 151.3 °C; H NMR (CD3OD, 600 MHz) δ,
8.08 (s, 2H), 8.00 (s, 1H), 7.89* (s, 1H), 5.28 (d, J = 3.2 Hz, 1H), 5.11*
(d, J = 7.3 Hz, 1H), 4.09−4.21 (m, 2H), 3.93 (d, J = 11.3 Hz, 1H), 3.85
(d, J = 11.5 Hz, 1H), 3.74−3.81 (m, 1H), 3.44−3.55 (m, 2H), 3.32 (brs,
1H); 13C NMR (CD3OD, 150 MHz) δ, 151.7, 144.7, 144.4*, 137.9,
137.9, 137.8*, 136.0, 127.9, 127.9, 125.8, 125.7, 124.4, 123.5, 123.4*,
122.7, 112.2, 97.5, 96.1*, 78.1, 75.4, 73.2, 69.5, 66.7, 62.5, 62.4*, 41.1,
D-Glucopyranose, 4-Deoxy-4-(4-(((1-(2,6-dichloro-4-
(trifluoromethyl)phenyl)-4-((trifluoromethyl)sulfinyl)-3-cyano-1H-
pyrazol-5-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)- (4-GTF). White
1
solid; yield, 78%, mp, 177.9 °C; H NMR (CD3OD, 600 MHz) δ,
8.08 (s, 2H), 7.88 (d, J = 3.1 Hz, 1H), 7.86* (d, J = 3.1 Hz, 1H), 5.27 (d,
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Scheme 2. Synthesis of Fipronil Conjugates for Replacement of C-1, C-2, C-3, C-4, and C-6 of D-Glucose: 1, tert-Butyl Alcohol,
H2O, CuSO4·5H2O, Sodium Ascorbate, 50 °C, 3 h; 2, NaOMe, MeOH; Room Temperature, 30 min
J = 3.1 Hz, 1H), 4.47−4.49 (m, 2H), 4.32−4.37* (m, 2H), 4.09 (dd, J =
9.1, 6.0 Hz, 1H), 4.02−4.05* (m, 1H), 3.53 (dd, J = 9.4, 3.6 Hz, 1H),
3.47−3.50 (m, 2H), 3.29 (t, J = 9.1 Hz, 1H), 3.29* (t, J = 9.1 Hz, 1H),
3.11−3.18 (m, 2H); 13C NMR (CD3OD, 150 MHz) δ, 151.8, 144.6,
137.9, 136.2, 136.0, 127.9, 127.8, 125.5, 125.3, 125.2, 124.5, 122.7, 112.1,
98.3, 94.2*, 76.9, 75.8, 75.3*, 74.4, 72.0, 71.1, 63.8, 63.6*, 61.6, 61.5*,
41.0, 40.9*; HRMS (ESI) calcd for C21H17Cl2F6N7O6SNa [M + Na]+,
702.0140; found, 702.0135.
RESULTS AND DISCUSSION
■
Various strategies for increasing the phloem mobility of
pesticides have been considered. A feasible strategy is the use
of endogenous transporter proteins to carry pesticides. In this
work, D-glucose was chosen as a vector for several reasons. First,
it is the most abundant and important monosaccharide in nature.
Second, it could be transported by various monosaccharide
transporters and their homologues through diverse plant
species.18 Third, D-glucose confers moderate phloem mobility
to the non-phloem-mobile insecticide fipronil, as presented in
our previous paper.9,16 In the present work, to evaluate the effects
of the different positions of glucose on phloem mobility, four
novel GTF conjugates, namely, 2-GTF, 3-GTF, 4-GTF, and
6-GTF, were synthesized via click chemistry based on the
synthetic method of GTF.9 All of the novel structures were
D-Glucopyranose, 6-Deoxy-6-(4-(((1-(2,6-dichloro-4-
(trifluoromethyl)phenyl)-4-((trifluoromethyl)sulfinyl)-3-cyano-1H-
pyrazol-5-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)- (6-GTF). White
1
solid; yield, 88%, mp, 188.4 °C; H NMR (CD3OD, 600 MHz) δ,
8.08 (s, 2H), 7.89 (d, J = 3.1 Hz, 1H), 7.84* (d, J = 3.1 Hz, 1H), 5.06 (d,
J = 3.2 Hz, 1H), 4.43−4.46 (m, 2H), 4.06−4.11 (m, 1H), 3.68 (d, J = 9.2
Hz, 1H), 3.68* (d, J = 9.2 Hz, 1H), 3.28−3.39 (m, 2H), 3.10 (t, J = 9.2
Hz, 1H), 3.04* (t, J = 9.2 Hz, 1H); 13C NMR (CD3OD, 150 MHz) δ,
151.7, 144.9, 137.8, 137.7, 135.9, 127.9, 127.8, 127.8, 125.4, 125.3, 124.4,
122.6, 112.1, 98.2, 94.0*, 77.6, 76.0, 75.6, 74.5, 73.5, 72.7, 72.6, 71.0,
52.3, 52.3*, 41.0, 40.9*. HRMS (ESI) calcd for C21H17Cl2F6N7O6SNa
[M + Na]+, 702.0140; found, 702.0135.
1
confirmed via H and 13C NMR spectroscopy and ESI mass
spectrometry.
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Synthesis of Azido-Containing Acetylglucoses (Scheme 1).
2,3,4,6-Tetra-O-acetyl-1-azido-1-deoxy-D-glucoside (1a) was
prepared from glucose pentaacetate in the presence of
azidotrimethylsilane and SnCl4 (Scheme 1A).19 Glucosamine
hydrochloride was transformed into 2-azido-2-deoxy-D-glucose
by diazo transfer from triflyl azide (TfN3). 2-Azido-2-deoxy-
D-glucose was subsequently acetylated using acetic anhydride
with catalytic DMAP in pyridine to yield 1,3,4,6-tetra-O-acetyl-2-
azido-2-deoxy-D-glucose (2a) (Scheme 1B).20 The introduction
of nitrogen at C-3 of glucose required a double inversion of the
configuration. Thus, the usual oxidation/reduction sequence led
to the fomation of diacetone allose, which was then converted to
azide via a triflation/azide displacement sequence.21 Treatment
of furanose diacetonide with aqueous trifluoroacetic acid was
followed by the complete acetylation of the crude products via
treatment with acetic anhydride and pyridine, and pyranose
acetates were produced as 1,2,4,6-tetra-O-acetyl-3-azido-3-deoxy-
D-glucosise (3a)22 (Scheme 1C). Methyl α-D-galactopyranoside
(1 equiv) was benzoylated by benzoyl chloride (3.5 equiv) in
pyridine to link selectively to methyl 2,3,6-tri-O-benzoyl-α-D-
galactopyranoside because the axial C-4 hydroxyl in the favored
conformation has a lower reactivity than C-2, C-3, and C-6.23
Methyl 2,3,6-tri-O-benzoyl-α-D-galactopyranoside was then sulfo-
nated by trifluoromethanesulfonic anhydride. The displacement of
the trifluoromethanesulfonate by sodium azide in dimethylforma-
mide gave a good yield of methyl 4-azido-2,3,6-tri-O-benzoyl-
4-deoxy-α-D-glucopyranoside in syrup form.24 The debenzoylation
of methyl 2,3,6-tri-O-benzoyl-4-azido-4-deoxy-α-D-glucopy-rano-
side with sodium methoxide resulted in crystalline methyl 4-azido-
4-deoxy-α-D-glucopyranoside, which was subsequently peracetylated
to obtain a syrupy 1,2,3,6-tetra-O-acetyl-4-azido-4-deoxy-α-D-glucose
(4a). 1,2,3,4-Tetraacetyl-6-O-p-tolylsulfonyl-D-glucopyranose
was prepared from D-glucose as previously described.17 Conven-
tional azidation in the presence of dimethylformamide resulted in
the formation of 1,2,3,4-tetra-O-acetyl-6-azido-6-deoxy-D-gluco-
pyranose (6a).25
specific peak at m/z 702.0135 [M + Na]+, from which the MW
was estimated at 702.0140 [M + Na]+. From these results, the
synthesized product was identified as 2-GTF.
Phloem Mobility. The castor bean seedling system is an
ideal biological model to evaluate the phloem systemicity of
xenobiotics because of its thin and highly permeable cuticle,
which facilitates molecule diffusion to and within the cotyledon
apoplast system.28 The system was adopted to test the phloem
mobility of GTF, 2-GTF, 3-GTF, 4-GTF, and 6-GTF. As
presented in a previous study,9 the plasma membrane potential of
the conjugates was measured via flow cytometric analysis with
DiBAC4(3) as the fluorescent dye indicator.29 In comparison to
the control sample, the relative fluorescence data did not have
any remarkable changes during the treatment period (Figure 1).
Synthesis and Characterization of GTF Conjugates. The
synthetic method of GTF conjugates was proposed by Yang
et al.9 To prepare these conjugates via click chemistry, tetra-
acetylglucosides containing the azide group were synthesized
according to the same method mentioned above (Scheme 1).
Propargyl was introduced into fipronil according to a previous
method by Yang et al.9 as a coupling partner. In Scheme 2, the
CuSO4·5H2O/ascorbate click reaction system was explored to
prepare the acetyl glucose-containing fipronil conjugates, which
were O-deacetylated via the Zemplen
́
procedure to obtain the
target products.
1
The H NMR spectra of these compounds are complicated
because fipronil has an asymmetrical sulfoxide and forms two
enantiomers.26,27 In addition, the α,β configuration of the
glucosyl group also exists. Their configurations were confirmed
on the basis of a chemical shift, coupling constants, and band
shape of H-1. For example, the NMR signals of product 2-GTF
were immediately measured after the dissolution of the glucosyl
conjugates in denatured methanol. A one-proton doublet was
observed at 5.28 and J1,2 = 3.2 Hz. These results indicate that the
substance was in the α-D-pyranose anomer form. The second
doublet, observed at 5.11 and J1,2 = 7.3 Hz, was assigned to H-1 of
the β-D-pyranose anomer. The characteristic peaks at 8.00 in the
1H NMR spectra and the peaks at 127.9 and 123.5 in the 13C
NMR spectra appeared after reaction. These results demonstrate
that 1,2,3-triazole was produced and the target conjugate was
obtained. The analysis via ESI−MS spectroscopy showed a
Figure 1. Plasma membrane potential in the protoplasts of R. communis
cotyledons after 6 h of treatment. (A) Fluorescent intensity histogram
marked DiBAC4(3) of the treatment of tested compounds and the
control (Con). (B) Relative fluorescence of the treatment of tested
compounds and the control (Con). The protoplasts were suspended in a
buffered solution at pH 5.5 with or without (control) tested compounds
(100 μM) for 5 h. The protoplasts were analyzed by flow cytometry for
DiBAC4(3) fluorescence. The data [mean standard error (SE); n = 3]
within a column are not significantly different, as shown by the Kruskal−
Wallis test (p > 0.05).
The results indicate that the five conjugates have no depolarizing
effects on the transmembrane potential. Therefore, the five
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Article
conjugates were not phytotoxic in the phloem mobility test
during the 5 h experimental period.
After a 5 h incubation (including the 1 h pre-incubation
period), the conjugates were clearly detected in the phloem sap.
The phloem sap determination results indicate that the
five conjugates (GTF, 2-GTF, 3-GTF, 4-GTF, and 6-GTF)
all exhibited moderate phloem mobility in Ricinus seedlings
(Figure 2). The GTF conjugates in the phloem sap were further
Figure 2. Phloem exudation collection of 4 sets of 12 castor bean
seedlings for each conjugate at 5 h (mean SE). The Kruskal−Wallis
tests at a 5% probability level were used to determine statistical
differences among treatments (p < 0.05).
identified with UPLC−MS (see the figure in the Supporting
Information). The UPLC−MS results indicated that the fipronil
parent molecule was not detected in the phloem sap.
Figure 3. Prediction of phloem mobility of tested compounds (GTF, 2-
GTF, 3-GTF, 4-GTF, and 6-GTF) using the Kleier map (log Cf as a
function of log Kow and pKa) according to Kleier;3,5 plant parameters are
for a short plant.3 Log Kow and pKa were calculated by the ACD
Laboratories Percepta program, version 14.0, and log Kow and pKa values
were classic values.
The “rule of five”, which is widely adopted in the
pharmaceutical industry, can also be used to predict the diffusion
of endogenous molecules or xenobiotics through plant and
animal membranes.30 According to this rule, the passive
absorption of small molecules is more likely to occur when
their MW, logP, number of hydrogen bond donors, and number
of hydrogen bond acceptors are less than 500 Da, 5, 5, and 10,
respectively. Given the physical properties of the five conjugates
(Table 1), their diffusion through the membranes should be
very low. Models based on the physicochemical properties of
pesticides are widely used to predict their phloem mobility. The
Kleier model is widely used to predict the phloem systemicity of a
compound based on the physicochemical properties (pKa and
log Kow) of xenobiotics.14,16,28 According to Kleier’s prediction
model, the physical properties of the five conjugates (Table 1) in
terms of log Kow and pKa are found in non-mobile areas (Figure 3).
In general, based on the “rule of five” and the Kleier prediction
model, the five conjugates should have a low diffusion through the
membrane and no phloem mobility should be observed. However,
the experimental results of the phloem sap analysis violated the “rule
of five” and the Kleier model. Specific carrier processes were
probably involved in the phloem transport, as demonstrated in the
phloem transport of GTF in Ricinus seedlings, which involves an
active carrier-mediated mechanism that effectively contributes to
GTF phloem loading.16
have very similar properties with each other (Table 1). Therefore,
we speculate that sugar carrier systems are involved in the phloem
loading of 2-GTF, 3-GTF, 4-GTF, and 6-GTF. Moreover, glucosyl
derivatives, which are conjugated at different carbons of glucose,
have a significant influence on affinities for sugar carriers. Among the
O-methylsulfonyl derivatives of D-glucose, mesylation at C-4 and C-
6 of glucose resulted in a slightly diminished affinity for the GLUT1,
whereas mesylation at C-3 led to the complete loss of affinity.33
Therefore, their phloem mobility difference of glucosyl conjugates
might be caused by their different affinities for sugar carriers.
In conclusion, four new conjugates containing both fipronil
and glucose moieties were synthesized via click chemistry. The
phloem mobility tests in the 5 h experimental period demonstrated
that the conjugates could enter into the phloem and exhibit
moderate phloem mobility without being degraded. Results show
that the phloem mobility was closely related to the position of the
glucose. Crop protection and production technology have given
increasing attention to the effective transport of pesticides through
the phloem. Therefore, understanding the influence of the positions
of glucose on phloem mobility provides a foundation for synthesis of
excellent phloem-mobile sugar pesticides.
Among these conjugates, the analysis result showed that
linking the fipronil to C-2, C-3, C-4, and C-6 of glucose affected
phloem mobility of GTF conjugates. The concentrations of
GTF, 2-GTF, 3-GTF, 4-GTF, and 6-GTF in phloem sap were
32.35 1.51, 37.37 1.44, 35.25 1.32, 26.87 1.07, and
35.10 1.19 μM, respectively (Figure 2). In a previous work,
sugar transporters have been shown to transport many glucose
derivatives and glucose conjugates across the membrane. 2-NBDG
is incorporated into mammalian cells through glucose transporters
in a time-, concentration-, and temperature-dependent manner.31
The uptake of IPNG into tobacco cells involves an active transport
process.32 Specifically, GTF can be mediated by sugar carrier
systems during its phloem loading. The glucose conjugates
(2-GTF, 3-GTF, 4-GTF, and 6-GTF) are isomers of GTF and
ASSOCIATED CONTENT
* Supporting Information
UPLC−MS indicating that the GTF conjugates were not degraded
in the phloem sap during the 5 h experiment. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Telephone: +86-20-85285127. Fax: +86-20-38604926. E-mail:
hhxu@scau.edu.cn.
■
Author Contributions
†Zhiwei Lei and Jie Wang contributed equally to this work.
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Article
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(19) Cagnoni, A. J.; Varela, O.; Gouin, S. G.; Kovensky, J.; Uhrig, M. L.
Synthesis of multivalent glycoclusters from 1-thio-β-D-galactose and
their inhibitory activity against the β-galactosidase from E. coli. J. Org.
Chem. 2011, 76, 3064−3077.
(20) Alper, P. B.; Hung, S.; Wong, C. Metal catalyzed diazo transfer for
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Funding
This work was supported by the National Natural Science
Foundation of China (Grant 31171886) and The Doctor Science
Research Foundation of the Education Ministry of China (Grant
20114404110020)
Notes
The authors declare no competing financial interest.
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