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Agricultural and Environmental Chemistry

Design of a New Glutamine-Fipronil Conjugate with #-Amino Acid Function and its Uptake by A. thaliana Lysine Histidine Transporter 1 (AtLHT1) Xunyuan Jiang, Yun Xie, Zhanfu Ren, Ulrika Ganeteg, Fei Lin, Chen Zhao, and Hanhong Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02287 • Publication Date (Web): 26 Jun 2018 Downloaded from http://pubs.acs.org on June 27, 2018

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Page 1 of 37

Journal of Agricultural and Food Chemistry 1

1

Design of a New Glutamine-Fipronil Conjugate with α-Amino Acid

2

Function and its Uptake by A. thaliana Lysine Histidine Transporter 1

3

(AtLHT1)

4

Xunyuan Jiang,1§Yun Xie,1§ Zhanfu Ren,1 Ulrika Ganeteg,2 Fei Lin,1 Chen Zhao*1 and

5

Hanhong Xu*1

6

1

7

and Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education,

8

South China Agricultural University, Guangzhou 510642, China

9

2

10

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources

Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology,

Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden

11 12 13

Corresponding Authors

14

*Hanhong Xu and Chen Zhao Tel: +86-20-85285127. E-mail: [email protected];

15

[email protected].

16

Author Contributions

17

§

18

equally to this work. Xunyuan Jiang designed, synthesized the compounds and wrote the

19

manuscript. Yun Xie and Zhanfu Ren performed the biology experiments and HPLC data

20

analyses. All authors read and approved the manuscript.

All Authors conceived and designed the study. Xunyuan Jiang and Yun Xie contributed

21

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 37 2

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Abstract: Creating novel pesticides with phloem-mobility is essential for controlling

23

insects in vascular tissue and root, and conjugating existing pesticides with amino acid is

24

an effective approach. In order to obtain highly phloem-mobile candidate for efficient

25

pesticide, an electro-neutral L-glutamine-fipronil conjugate (L-GlnF) retaining α-amino

26

acid function was designed and synthesized to fit the substrate specificity of amino acid

27

transporter. Cotyledon uptake and phloem loading tests with Ricinus communis have

28

verified that L-GlnF was phloem mobile, and its phloem mobility was higher than its

29

enantiomer D-GlnF and other previously reported amino acid-fipronil conjugates.

30

Inhibition experiments then suggested that the uptake of L-GlnF was, at least partially,

31

mediated by active transport mechanism. This inference was further strengthened by

32

assimilation experiments with Xenopus oocytes and genetically modified Arabidopsis

33

thaliana, which showed direct correlation between the uptake of L-GlnF and expression

34

of amino acid transporter AtLHT1. Thus, conjugation with L-Gln appears to be a potential

35

strategy to ensure the uptake of pesticides via endogenous amino acid transport system.

36

Keywords: glutamine, fipronil, amino acid transporter, AtLHT1

37


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38

INTRODUCTION

39

Spray is the most common method to deliver pesticides to plants.1, 2 However, for

40

pesticides without capability to be uptake and translocated by plants, the means of spray

41

makes them almost impossible to control insects in vascular tissue and root.3-6 Thus,

42

development of systemic and phloem-mobile pesticides which are able to reach internal

43

tissues of plants has become urgent and necessary.

44

While developing a novel phloem-mobile pesticide is usually time-consuming and

45

expensive,7,

46

monosaccharide9-14 and amino acid4, 15-21) with an existing pesticide is a feasible and

47

efficient strategy to improve the phloem mobility of non-phloem mobile xenobiotic. For

48

example, in our previous work, a series of glycosyl-fipronil,9-12 glycinyl-fipronil,21 and

49

amino acid-chlorantraniliprole conjugates16 were developed, and have exhibited phloem

50

mobility in Glycine max or R. communis seedlings.

51

8

combining the structure of an endogenous nutrient (such as

Several investigations have indicated that active transporters played important roles

52

in the uptake or phloem transport mechanism of nutrient-pesticide conjugates.10,

53

Uptake of glycosyl-fipronil conjugates by R. communis was proven to be mediated by

54

endogenous monosaccharide transporters.10, 22 Moreover, four genes relating to amino

55

acid transporters were found to be possible candidates involved in the uptake of

56

glycinergic-fipronil (GlyF) in R. communis seedlings.15 Therefore, it is possible to design

57

a highly phloem-mobile nutrient-pesticide conjugate according to the substrate specificity

58

of amino acid transporters directly.

59

15, 18

Compounds with the following two aspects were believed to be favorable by



Page 4 of 37 4

60

amino-acid transporters: 1) electroneutrality23, 24 and 2) α-amino acid function25-27. Thus,

61

in this study, an electro-neutral L-glutamine-fipronil conjugate (L-GlnF) with α-amino

62

acid function was designed and synthesized. We expect the new conjugate to be

63

recognized by amino acid transporters such as AtLHT1 and show improved

64

phloem-mobility compared to its parent compound. First, phloem mobility of the new

65

conjugate was determined in R. communis model system. Experiments on time course,

66

concentration dependence, pH dependence, and effector inhibition of the uptake process

67

of L-GlnF were then performed on R. communis seedlings to assess the uptake

68

mechanism. Finally, assimilation experiments were conducted with A. thaliana genotypes

69

differed in LHT1 expressions and Xenopus laevis oocyte heterologous expression system

70

to identify if AtLHT1 was involved in the uptake of L-GlnF. Considering that compounds

71

with L- configuration were usually preferred by amino acid transporters,24,

72

enantiomer of L-GlnF (D-GlnF) was also synthesized as a comparison.

73

MATERIALS AND METHODS

28

an

74

General Information for Synthesis. All reagents and solvents were used as

75

received from commercial sources unless otherwise stated. Analytical thin layer

76

chromatography (TLC) was performed using pre-coated plates (silica gel GF254), with

77

spots visualized using ZF-20D ultraviolet (UV) analyzer at 254 nm or by staining using

78

potassium permanganate. Silica gel (200-300 mesh) was used for flash column

79

chromatography. High resolution electro-spray ionization mass spectra (ESI-HRMS) were

80

obtained using an Agilent 6210 LC/MSD TOF instrument. All 1H NMR and

81

spectra were recorded using a Bruker AV-400 or AV-500 instruments. Chemical shifts


13

C NMR

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82

were expressed in parts per million (ppm, δ) with TMS used as an internal standard.

83

Coupling constants (J) were quoted in Hz. 1H NMR splitting patterns were designated as

84

singlet (s), doublet (d), triplet (t), quartet (q) and broad (br). Splitting patterns that could

85

not be interpreted or easily visualized were designated as multiplet (m).

86

(R)-2-Bromopentanedioic acid dimethyl ester (1a). (R)-2-bromoglutaric acid29, 30

87

was first prepared by gradually adding a solution of NaNO2 (55.6 g, 806.0 mmol) in water

88

(300 mL) over a period of 5 h to a mixture of D-glutamic acid (66.0 g, 448.0 mmol) and

89

NaBr (161.3 g, 1.6 mol) in 2 M HBr (400 mL) cooled at -5 °C. About 3 h after the

90

addition of NaNO2, concentrated H2SO4 (15 mL) was added. The mixture was extracted

91

with diethyl ether (200 mL). The combined organic phases were washed with brine, dried

92

over anhydrous Na2SO4, filtered and evaporated under reduced pressure.

93

The obtained un-purified (R)-2-bromoglutaric acid (yellow oil) was dissolved in a

94

solution of concentrated H2SO4 (3 mL) in methanol (100 mL). The mixture was then

95

heated to reflux for 2 h, with the resulting solution concentrated under reduced pressure.

96

Diethyl ether (100 mL) was added into the residue, and the organic phase was washed

97

with aqueous solution of NaHCO3 followed by brine, then dried over anhydrous Na2SO4.

98

After careful removal of solvent under reduced pressure, the crude product was purified

99

by flash column chromatography (petroleum ether/ethyl acetate = 10:1, v/v) to offer

100

(R)-2-bromopentanedioic acid dimethyl ester (1a)31, 32 as a colorless oil (25.7 g, two steps:

101

24%). 1H NMR (500 MHz, Chloroform-d) δ 4.36 (dd, J = 8.5, 5.8 Hz, 1H), 3.77 (s, 3H),

102

3.67 (s, 3H), 2.56-2.46 (m, 2H), 2.41-2.34 (m, 1H), 2.30-2.23 (m, 1H).

103

MHz, Chloroform-d) δ 172.55, 169.89, 53.14, 51.93, 44.65, 31.36, 29.86.


13

C NMR (126


Page 6 of 37 6

104

(S)-2-Bromopentanedioic acid dimethyl ester (1b). Prepared with the same

105

method used for the synthesis of 1a except for using L-glutamic acid (66.0 g, 448.0 mmol)

106

as reactant. The final compound was obtained as a colorless oil (27.9 g, two steps: 26%).

107

1

108

3H), 2.55-2.45 (m, 2H), 2.40-2.33 (m, 1H), 2.30-2.22 (m, 1H).

109

Chloroform-d) δ 172.53, 169.87, 53.12, 51.92, 44.64, 31.33, 29.84.

110

H NMR (500 MHz, Chloroform-d) δ 4.36 (dd, J = 8.5, 5.8 Hz, 1H), 3.76 (s, 3H), 3.66 (s,

(S)-Dimethyl-2-azidopentanedioate

(2a).

Prepared

13

C NMR (126 MHz,

from

a

mixture

of

111

(R)-2-bromopentanedioic acid dimethyl ester (1a) (4.80 g, 20.0 mmol) and DMF (30 mL),

112

where sodium azide (6.50 g, 100.0 mmol) was added portion-wise during a 20-minute

113

period. The reaction mixture was stirred at room temperature until 1a was fully consumed

114

out as monitored by TLC. The reaction mixture was then diluted with diethyl ether (80

115

mL). The organic layer was washed with water (160 mL), dried over anhydrous Na2SO4,

116

and concentrated under reduced pressure to afford the product (S)-dimethyl

117

2-azidopentanedioate (2a) as a pale-yellow oil (4.10 g, 99%) without purification. 1H

118

NMR (500 MHz, Chloroform-d) δ 4.00 (dd, J = 8.3, 5.2 Hz, 1H), 3.78 (s, 3H), 3.67 (s,

119

3H), 2.50-2.40 (m, 2H), 2.20-2.13 (m, 1H), 2.03-1.96 (m, 1H).

120

Chloroform-d) δ 172.72, 170.50, 61.14, 52.79, 51.90, 29.91, 26.60.

13

C NMR (126 MHz,

121

(R)-Dimethyl-2-azidopentanedioate (2b). Produced using the same method as the

122

synthesis of 2a except for using (S)-2-bromopentanedioic acid dimethyl ester (1b) (4.80 g,

123

20.0 mmol) as reagent. The final product was obtained as a pale-yellow oil (3.80 g, 94%).

124

1

H NMR (400 MHz, Chloroform-d) δ 4.01 (dd, J = 8.7, 5.1 Hz, 1H), 3.80 (s, 3H), 3.69 (s,


Page 7 of 37


125

3H), 2.49-2.45 (m, 2H), 2.23-2.14 (m, 1H), 2.06-1.97 (m, 1H).

126

Chloroform-d) δ 172.77, 170.54, 61.20, 52.84, 51.95, 29.96, 26.65.

127

(S)-4-Azido-5-methoxy-5-oxopentanoic

acid

13

C NMR (126 MHz,

(3a).

(S)-dimethyl

128

2-azidopentanedioate (2a) (3.10 g, 15.0 mmol) was dissolved in a mixed solvent of

129

MeOH (18 mL), THF (12 mL) and H2O (12 mL) followed by the addition of K2CO3 (2.28

130

g, 16.5 mmol). The mixture was stirred under room temperature until the starting material

131

was consumed out as tracked by TLC (about 3 h). The solvent was then evaporated in

132

vacuo, and the residue was washed with diethyl ether (25 mL) to remove byproducts. The

133

aqueous layer was neutralized with 1 N HCl and then extracted with ethyl acetate (50

134

mL). The combined organic phases were washed with brine, dried over anhydrous

135

Na2SO4,

136

(S)-4-azido-5-methoxy-5-oxopentanoic acid (3a)33 (yellow oil, 2.67 g, 95%) was then

137

obtained and applied in the next step without further purification. 1H NMR (500 MHz,

138

Chloroform-d) δ 7.89 (br, 1H), 4.10 (dd, J = 8.6, 5.0 Hz, 1H), 3.71 (s, 3H), 2.53-2.50 (m,

139

2H), 2.28-2.21 (m, 1H), 2.09-2.02 (m, 1H). 13C NMR (126 MHz, Chloroform-d) δ 175.11,

140

172.97, 60.91, 52.11, 29.94, 26.55.

filtered

and

evaporated

under

reduced

pressure.

The

crude

141

(R)-4-Azido-5-methoxy-5-oxopentanoic acid (3b). Prepared using the same

142

method as the synthesis of 3a except for using (R)-dimethyl 2-azidopentanedioate (2b)

143

(3.10 g, 15 mmol) as reactant. Crude compound 3b was obtained as a yellow oil (2.56 g,

144

91%). 1H NMR (400 MHz, Chloroform-d) δ 9.49 (br, 1H), 4.10 (dd, J = 8.7, 5.1 Hz, 1H),

145

3.71 (s, 3H), 2.54-2.50 (m, 2H), 2.28-2.20 (m, 1H), 2.10-2.01 (m, 1H).

146

MHz, Chloroform-d) δ 175.29, 173.02, 60.91, 52.11, 29.94, 26.53.


13

C NMR (126


Page 8 of 37 8

147

(2S)-Methyl-2-azido-5-{[3-cyano-1-(2,6-dichloro-4-trifluoromethyl)phenyl-4-(tri

148

fluoromethyl sulfinyl)-1H-pyrazol-5-yl]amino}-5-oxopentanoate (4a). A solution of

149

(S)-4-azido-5-methoxy-5-oxopentanoic acid (3a) (1.87 g, 10.0 mmol) in 20 mL dry

150

CH2Cl2 was mixed with oxalyl chloride (4.3 mL, 50.0 mmol) and a drop of anhydrous

151

DMF at 0 °C under inert atmosphere. The mixture was stirred for 2 h at room temperature

152

until the effervescence ceased. After removal of excess oxalyl chloride in vacuo, the

153

residue was then dissolved in anhydrous THF (15 mL) under argon and used immediately

154

in the next step.

155

At 0 °C under argon atmosphere, to a solution of fipronil (5.25 g, 12.0 mmol) in

156

anhydrous THF (75 mL) was gradually added NaH (60% dispersion in oil, 576 mg, 14.4

157

mmol) over a period of 20 min. The reaction mixture was then stirred at 0 °C for 1 h. The

158

crude acid chloride in THF solution from the last step was added dropwise via syringe,

159

and the resulting mixture was stirred for an additional 3 h at 0 °C before the reaction was

160

quenched with NH4Cl aq. dropwise. The mixture was extracted with ethyl acetate (80

161

mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was then

162

purified by flash column chromatography (petroleum ether/ethyl acetate = 8:1, v/v) to

163

produce the product as a white powder (5.39 g, 89%). 1H NMR (500 MHz, Chloroform-d)

164

δ 9.37 (s, 1H), 7.77 (dd, J = 32.3, 10.8 Hz, 2H), 4.15-4.11 (m, 1H), 3.67 (s, 3H),

165

2.41-2.26 (m, 2H), 2.19-2.10 (m, 1H), 2.01-1.95 (m, 1H).

166

Chloroform-d) δ 172.89 (d, J = 14.1 Hz), 167.52, 140.89 (d, J = 14.8 Hz), 135.82 (d, J =

167

4.5 Hz), 136.06-134.36 (m), 134.84 (q, J = 34.6 Hz), 126.47 (d, J = 3.5 Hz), 126.42 (ddq,

168

J = 65.0, 32.1, 3.6 Hz), 125.09 (dq, J = 337.0, 2.6 Hz), 121.93 (q, J = 273.9 Hz), 109.87


13

C NMR (126 MHz,

Page 9 of 37


169

(d, J = 4.6 Hz), 108.96, 62.15 (d, J = 12.6 Hz), 52.10, 29.29 (d, J = 10.2 Hz), 26.95 (d, J =

170

13.0 Hz).

171

(2R)-Methyl-2-azido-5-{[3-cyano-1-(2,6-dichloro-4-trifluoromethyl)phenyl-4-(tri

172

fluoromethyl sulfinyl)-1H-pyrazol-5-yl]amino}-5-oxopentanoate (4b). Produced using

173

the same method as outlined for the synthesis of 4a except for using 3b (1.87 g, 10.0

174

mmol) as reactant. Compound 4b was obtained as a white powder (5.28 g, 87%). 1H

175

NMR (400 MHz, Chloroform-d) δ 9.37 (s, 1H), 7.77 (dd, J = 24.4, 8.8 Hz, 2H), 4.16-4.12

176

(m, 1H), 3.66 (s, 3H), 2.40-2.25 (m, 2H), 2.20-2.07 (m, 1H), 2.05-1.93 (m, 1H). 13C NMR

177

(126 MHz, Chloroform-d) δ 172.76 (d, J = 16.9 Hz), 167.47, 141.21 (d, J = 13.6 Hz),

178

136.00, 135.17 (dd, J = 71.2, 59.0 Hz), 134.95 (q, J = 34.6 Hz), 126.44 (d, J = 4.1 Hz),

179

126.41 (ddq, J = 69.4, 33.6, 3.9 Hz), 125.31 (qd, J = 336.7, 3.1 Hz), 121.95 (q, J = 273.9

180

Hz), 109.89 (d, J = 5.6 Hz), 108.49 (d, J = 4.5 Hz), 62.31 (d, J = 13.5 Hz), 52.08, 29.31 (d,

181

J = 11.6 Hz), 27.06 (d, J = 14.9 Hz).

182

(2S)-Methyl-2-amino-5-{[3-cyano-1-(2,6-dichloro-4-trifluoromethyl)phenyl-4-(tr

183

ifluoromethyl sulfinyl)-1H-pyrazol-5-yl]amino}-5-oxopentanoate (5a). A solution of

184

4a (4.85 g, 8.0 mmol) and tin(II) chloride dihydrate (3.60 g, 16.0 mmol) in MeOH (20

185

mL) was prepared and stirred at room temperature for 3 h.34 After removal of solvent in

186

vacuo, the residue was dissolved in ethyl acetate (30 mL). The solution was washed with

187

saturated Na2CO3 solution, and the aqueous layer was back-extracted using ethyl acetate

188

(50 mL). The combined organic phases were dried over anhydrous Na2SO4. After removal

189

of solvent, the residue was purified by flash column chromatography (CH2Cl2/MeOH =

190

20:1, v/v) to yield 5a (3.48 g, 75 %) as a white solid. 1H NMR (400 MHz, Methanol-d4) δ



Page 10 of 37 10

191

8.00-7.97 (m, 2H), 3.67-3.60 (m, 1H), 3.63 (s, 3H), 2.42-2.36 (m, 2H), 1.98-1.93 (m, 2H).

192

13

193

153.52 (d, J = 19.9 Hz), 138.86 (d, J = 8.8 Hz), 137.39-137.02 (m), 134.61 (q, J = 34.2

194

Hz), 127.58 (qd, J = 339.1, 3.0 Hz), 127.04 (p, J = 3.6 Hz), 126.77 (d, J = 2.6 Hz), 122.67

195

(q, J = 273.7 Hz), 113.44 (d, J = 6.9 Hz), 104.64 (dd, J = 19.2, 2.3 Hz), 55.60, 52.22,

196

30.38, 27.81. HRMS (ESI): calcd for C18H14Cl2F6N5O4S (M+H)+ 580.0042, found

197

580.0035.

198

C NMR (126 MHz, Methanol-d4) δ 174.61 (d, J = 5.1 Hz), 172.36 (d, J = 19.4 Hz),

(2R)-Methyl-2-amino-5-{[3-cyano-1-(2,6-dichloro-4-trifluoromethyl)phenyl-4-(t

199

rifluoromethyl sulfinyl)-1H-pyrazol-5-yl]amino}-5-oxopentanoate (5b). Produced

200

using the same method as outlined for the synthesis of 5a except for using 4b (4.85 g, 8.0

201

mmol) as reagent. The final product 5b was obtained as a white powder (3.68 g, 79%). 1H

202

NMR (400 MHz, Methanol-d4) δ 8.16-8.13 (m, 2H), 4.19 (ddd, J = 14.9, 7.0, 4.9 Hz, 1H),

203

3.69 (d, J = 2.3 Hz, 3H), 2.46-2.36 (m, 2H), 2.17-2.01 (m, 2H).

204

Methanol-d4) δ 173.87 (d, J = 19.4 Hz), 169.16 (d, J = 10.9 Hz), 140.88 (d, J = 39.8 Hz),

205

137.51-136.50 (m), 136.22 (qd, J = 34.5, 5.6 Hz), 135.90 (d, J = 6.1 Hz), 128.22-127.80

206

(m), 127.60 (d, J = 6.5 Hz), 127.51 (qd, J = 338.4, 2.7 Hz), 123.40 (q, J = 273.4 Hz),

207

113.11 (d, J = 44.9 Hz), 111.38 (d, J = 5.0 Hz), 53.71 (d, J = 16.4 Hz), 52.61 (d, J = 3.7

208

Hz), 29.75 (d, J = 7.8 Hz), 27.24. HRMS (ESI): calcd for C18H14Cl2F6N5O4SNa (M+Na)+

209

601.9862, found 601.9863.

210

13

C NMR (126 MHz,

(2S)-2-Amino-5-{[3-cyano-1-(2,6-dichloro-4-trifluoromethyl)phenyl]-4-(trifluor

211

omethyl sulfinyl)-1H-pyrazol-5-yl]amino}-5-oxopentanoic.HCl (L-GlnF). A solution

212

of 5a (2.90 g, 5.0 mmol) in dioxane (15 mL) mixed with 1 N HCl (20 mL) was heated to


Page 11 of 37


213

reflux. TLC was used to track the reaction until the disappearance of starting material

214

(about 7 h). After the solvent was evaporated, diethyl ether (40 mL) was added to wash

215

the residual mixture. The diethyl ether layer was then back-extracted with 1 N HCl (80

216

mL). The combined aqueous layer was extracted with ethyl acetate (30 mL), and the

217

organic phases were dried over anhydrous Na2SO4, filtered, and evaporated under

218

reduced pressure to give the final product L-GlnF (white powder, 2.05 g, 68%). 1H NMR

219

(400 MHz, Methanol-d4) δ 8.15-8.11 (m, 2H), 4.17-4.11 (m, 1H), 2.47-2.31 (m, 2H),

220

2.14-1.98 (m, 2H). 13C NMR (126 MHz, Methanol-d4) δ 175.22 (d, J = 6.3 Hz), 169.43 (d,

221

J = 9.4 Hz), 141.54 (d, J = 32.7 Hz), 137.45-136.62 (m), 136.45 (qd, J = 34.7, 5.5 Hz),

222

136.09 (d, J = 6.2 Hz), 128.15-127.89 (m), 127.64 (d, J = 5.1 Hz), 127.03 (qd, J = 338.0,

223

1.6 Hz), 123.46 (qd, J = 273.2, 2.4 Hz), 112.76 (d, J = 50.6 Hz), 111.50 (d, J = 5.2 Hz),

224

53.92 (d, J = 16.2 Hz), 29.92 (d, J = 9.9 Hz), 27.44. HRMS (ESI): cald for

225

C17H12Cl2F6N5O4S (M+H)+ 565.9886, found 565.9887.

226

(2R)-2-Amino-5-3-{[cyano-1-(2,6-dichloro-4-trifluoromethyl)phenyl-4-(trifluoro

227

methyl sulfinyl)-1H-pyrazol-5-yl]amino}-5-oxopentanoic·HCl (D-GlnF). Prepared via

228

the same procedure as the synthesis of 6a except for using 5b (2.90 g, 5.0 mmol) as

229

reactant. The final product D-GlnF was obtained as a white powder (2.11 g, 70%). 1H

230

NMR (400 MHz, Methanol-d4) δ 8.14-8.10 (m, 2H), 4.19-4.11 (m, 1H), 2.47-2.28 (m,

231

2H), 2.14-2.02 (m, 2H).

232

169.21 (d, J = 13.4 Hz), 140.81 (d, J = 39.8 Hz), 137.34-136.55 (m), 136.30 (qd, J = 34.6,

233

6.6 Hz), 135.89 (d, J = 8.5 Hz), 128.19-127.90 (m), 127.64 (d, J = 6.2 Hz), 126.97 (q, J =

234

338.6 Hz), 123.41 (qd, J = 273.4, 5.2 Hz), 113.21 (d, J = 51.3 Hz), 111.36 (d, J = 4.6 Hz),

13

C NMR (126 MHz, Methanol-d4) δ 175.18 (d, J = 8.0 Hz),



Page 12 of 37 12

235

53.80 (d, J = 14.5 Hz), 29.89 (d, J = 9.4 Hz), 27.39. HRMS (ESI): calcd for

236

C17H10Cl2F6N5O4S (M-H)- 563.9740, found 563.9746.

237

Plant/Animal Materials. Castor bean seeds (R. communis L.) no. 9 were purchased

238

from the Agricultural Science Academy of Zibo (Shandong, China) and were cultured as

239

previously described.35 After 6 days of growth, seedlings with hypocotyls of about 20 mm

240

length were selected for experiments.

241

Wild-type Arabidopsis. thaliana L. (Columbia-0), the amino acid transporter mutants

242

(lht1-5), and the LHT1 overexpressor (35SLHT1-2) were grown under hydroponic

243

conditions.28, 36 After 21 days of growth, seedlings of average size were used for root

244

uptake experiments.

245

African Xenopus laevis were purchased from the Shanghai Institute of Biochemistry

246

and Cell Biology (Shanghai, China). Mature female Xenopus laevis were selected to be

247

incubated in a clean glass tank and fed on Tianbangmeiwa 2 material three times per

248

week and chopped liver once per month. cRNA of target gene was obtained using

249

mMESSAGE mMACHINETM T7 Transcription Kit (Thermo Fisher Scientific, Vilnius,

250

Lithuania), and 18.4 nL of purified cRNA was injected into each oocyte using a Nanoliter

251

2000 syringe (World Precision Instruments, Sarasota, FL, U.S.A.). The injected oocytes

252

were then incubated at 16 °C for 1 day before the uptake experiments were initiated.39

253

Uptake by R. communis cotyledons. A recently described method was used for

254

cotyledon uptake experiments with minor modifications.10, 22 Cotyledons of R. communis

255

seedlings with removed endosperm were floated in pre-incubation buffer (20 mM MES,

256

0.25 mM MgCl2, and 0.5 mM CaCl2) at pH 5.6. Every six cotyledons were tested as a


Page 13 of 37


257

single set. L-GlnF was added into the buffer solution for time course and concentration

258

dependency tests. To investigate the effect of pH on the uptake of 0.05 mM L-GlnF, the

259

pH of incubation buffer was adjusted from 5 to 8. To assess the inhibition of cotyledon

260

uptake, cotyledons were pre-incubated for 30 minutes with either 0.05 mM CCCP

261

(carbonyl cyanide 3-chlorophenylhydrazone)28, 10 mM L-glutamine (L-Gln), 10 mM

262

L-glutamic acid (L-Glu), or 10 mM L-phenylalanine (L-Phe) prior to the addition of 0.05

263

mM L-GlnF or D-GlnF. At the end of each uptake experiment, cotyledons were harvested

264

and washed for six times with buffer solution to remove the remaining reagents on the

265

surface. The cotyledons were then dried using paper towel, weighed, frozen with liquid

266

nitrogen, grounded, suspended in 10 mL of methanol, and ultrasonicated for 30 min. The

267

extract solutions were centrifuged at 14000 g for 10 min and then filtered with 0.22 μm

268

filters. The final extract solutions were analyzed by HPLC.15

269

Phloem Sap Collection. Phloem sap was collected as previously described with

270

modifications.10 Cotyledons of R. communis seedlings with removed endosperms were

271

incubated in buffer solution containing 0.05 mM of L-GlnF or D-GlnF, with or without

272

the addition of 10 mM L-Gln, 10 mM L-Glu, or 10 mM L-Phe. Roots of the seedlings

273

were remained in soil. After 1 hour of incubation, hypocotyls were severed at the hook

274

region for phloem exudation. The interval between phloem sap collections was 1 h and

275

the sap was collected for a duration of 5 h. The collected phloem sap was diluted with

276

pure water (phloem sap/pure water = 1:4, v/v) and analyzed by HPLC.

277

Xenopus oocyte uptake. Each set of the experiment contains ten oocytes. Xenopus

278

oocytes were injected with cRNA of AtLHT1 or with water as positive control. Oocytes



Page 14 of 37 14

279

that had not received any injection were used as negative control. The oocytes were put

280

into 2 mL Eppendorf tubes with 0.5 mL Kulori buffer (90 mM NaCl, 1 mM KCl, 1 mM

281

CaCl2, 1 mM MgCl2 and 5 mM MES, pH 5.6)37 containing 0.05 mM L-GlnF or D-GlnF.

282

After incubated for 1 h, oocytes were washed for four times using Kulori buffer, then 0.1

283

mL of 10% SDS solution was added into each tube, which was then ultrasonicated to

284

dissociate oocytes. The extract solutions were dried by Eppendorf concentrator to

285

evaporate all solvent and then reconstituted with 0.1 mL methanol. The final solutions

286

were analyzed by HPLC.

287

A. thaliana root uptake. Roots of A. thaliana seedlings were put into 5 mL

288

Eppendorf tubes and submerged in J medium38 containing 0.05 mM L-GlnF or D-GlnF.

289

After 1 hour, the roots were washed for six times with J medium, wiped, and separated.

290

The roots were then weighted, frozen with liquid nitrogen, ground with 3 mL methanol,

291

and ultrasonicated for 30 min. The extract solutions were dried with Eppendorf

292

concentrator to evaporate all solvent and then reconstituted with 0.1 mL methanol. The

293

final solutions were centrifuged at 14000 g for 10 min and the liquid supernatants were

294

collected for HPLC analysis.

295

Analytical methods. Content of L- or D-GlnF was quantified using Agilent 1260

296

HPLC system. Separations were performed with a SB C18 reversed-phase column (5 μm,

297

250×4.6 mm inner diameter, Agilent Co., Santa Clara, CA, U.S.A.) at 30 °C. The

298

injection volume was 10 μL, and the flow rate was 1 mL/min. The elution system

299

consisted of acetonitrile and water (50:50, v/v) with 0.1% trifluoracetic acid. Statistical

300

analysis was performed using ANOVA, followed by Dunnett’s test to identify the


Page 15 of 37


301

differences among the means of different experimental groups and the control group (P

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