effect of introducing amino acids into phenazine-1-carboxylic ......(thermo fisher scientific, ma,...

SUPPLEMENTARY MATERIAL

Effect of introducing amino acids into

phenazine-1-carboxylic acid on phloem mobility

Yongtong Xiong1, Xiang Zhu

1,2, Jinyu Hu

1, Yunping Wang

1, Xiaoying Du

1,2*, Junkai

Li1,2*

and Qinglai Wu1,2*

1School of Agriculture, Yangtze University, Jingmi Road 88, Jingzhou 434025, China

2Institute of Pesticides, Yangtze University, Jingmi Road 88, Jingzhou 434025, China

Yongtong Xiong, School of Agriculture, Yangtze University, [email protected];

Xiang Zhu, School of Agriculture, Yangtze University; Institute of Pesticides, Yangtze University,

[email protected];

Jinyu Hu, School of Agriculture, Yangtze University, [email protected];

Yunping Wang, School of Agriculture, Yangtze University, [email protected];

Xiaoying Du, School of Agriculture, Yangtze University; Institute of Pesticides, Yangtze

University, [email protected];

Junkai Li, School of Agriculture, Yangtze University; Institute of Pesticides, Yangtze University,

[email protected];

Qinglai Wu, School of Agriculture, Yangtze University; Institute of Pesticides, Yangtze

University, [email protected];

*Corresponding author. Tel/Fax: +86 716-8066314.

E-mail address: [email protected] (Xiaoying Du)


E-mail address: [email protected] (Junkai Li).


E-mail address: [email protected] (Qinglai Wu).

mailto:[email protected]



mailto:[email protected]%20%20(Qinglai

Abstract: To develop new phenazine carboxylic acid derivatives with better phloem

mobility, five novel 7-amino acid substituted phenazine-1-carboxylic acids were

synthesized by introducting amino acids into PCA at the 7-position. The phloem

mobility experiments in Ricinus communis seedlings showed that retaining the

carboxyl group of PCA and conjugating amino acids to its phenazine ring can also

endow PCA with phloem mobility. Comparing our previous research, we found the

amino acids substituted at 7-position on phenazine ring of PCA could clearly enhance

the phloem mobility of PCA than that of amino acids conjugated with carboxyl group.

Especially, the phloem transport concentration of the compound 7-L-isoleucine

substituted PCA (7d) was 21 times higher than PCA-L-isoleucine conjugate (8d).

These data suggest that the introduction of amino acids at different structural sites on

the phenazine ring could effectively enhance the phloem mobility of PCA and it is

worth a further study.

Keywords: Phenazine-1-carboxylic acid; amino acid; synthesis; phloem mobility

Experimental

1.1 General

All reagents and solvents were purchased from commercial suppliers. Melting

points were measured on by a WRR-Y melting point apparatus (Shanghai Yidian

Physical Optical Instrument Co., Ltd., Shanghai, China). Thin-layer chromatography

(TLC) was conducted on silica gel plates (GF254) (Qingdao Haiyang Chemical

Co.,Ltd., Qingdao, China), and spots can be seen on a ZF-I ultraviolet analyzer

(Shanghai Gucun Electro-optical Instrument Factory, Shanghai, China). Column

chromatography purification was carried out on silica gel (200–300 mesh) (Qingdao

Haiyang Chemical Co.,Ltd., Qingdao, China). NMR spectra were obtained using an

AVANCE III HD 400 NMR spectrometer (Bruker Corporation, Basel, Switzerland).

Mass spectrographic analysis was conducted on a Thermo Scientific Q ExactiveTM

(Thermo Fisher Scientific, MA, USA).

1.2 Synthesis of Intermediate 1

The intermediate 1 was prepared by the methods reported previously (Rewcastle

et al. 1987). A 100 mL round bottom flask was charged with

2-bromo-3-nitrobenzoicacid (40 mmol), p-toluidine (60 mmol), cuprous chloride (4.8

mmol), copper powder (0.15 mmol), N-ethylmorpholine (15 mL) and 2,3-butanediol

(25 mL). The mixture was stirred at 70°C for 15 h until the reaction was complete

(monitored by TLC). Then, 0.1 mol/L NH4OH aqueous solution (100 mL) was added

to the obtained solution, and filtered over celite. 2 mol/L HCl was added to the filtrate

until a yellow solid precipitated. Then filtered and dried to obtain intermediate 1.


A solution of intermediate 1 (40 mmol), Sodium borohydride (145 mmol) and

sodium ethoxide (1 mol) in absolute ethanol (500 mL) was heated under reflux until

the reaction was complete (monitored by TLC). The mixture was evaporated under

vacuum, and the residue is dissolved in H2O (200 mL). 2 mol/L HCl was added to the

mixture until a solid precipitated. Then filtered and dried to obtain intermediate 2.


Intermediate 2 (10 mmol) was dissolved in 60 mL of anhydrous CH2Cl2, then

oxalyl chloride (15 mmol) was slowly added. The reaction was heated under reflux

for 8 h. The reaction solution was evaporated under vacuum, and the residue is

dissolved in 30 mL anhydrous CH2Cl2, which used for the next reaction immediately.


To a 250 mL round bottom flask was added 100 mL of anhydrous methanol and

then stirred at 0 °C for 15 min. A solution of the intermediate 3 (10 mmol) in CH2Cl2

was slowly added dropwise to the above system. The mixture was stirred at 0C for

about 6 h until the reaction was complete (monitored by TLC). The reaction solution

was evaporated under vacuum. The residue was is dissolved in CH2Cl2 and washed

with a 5% sodium hydrogen carbonate solution. Then organic phase was dried over

anhydrous sodium sulfate, filtered and concentrated in vacuum. Finally, pure target

compound 4 was obtained by column chromatography (petroleum ether/ethyl acetate,

v/v= 4:1).


A solution of intermediate 4 (10 mmol), N-bromosuccinimide (11 mmol) and

dibenzoyl peroxide (1.8 mmol) in CCl₄ (40 mL) was heated under reflux for 4 h. The

reaction solution was evaporated under vacuum. The residue was is dissolved in ethyl

acetate and washed with H2O. Then organic phase was dried over anhydrous sodium

sulfate, filtered and concentrated in vacuum. Finally, pure target compound 5 was

obtained by column chromatography (petroleum ether/ethyl acetate, v/v= 30:1).


A solution of glycineethylester hydrochloride (12 mmol)

and N,N-diisopropylethylamine (30 mmol) in DMF (20 mL) was stirred at room

temperature. Then, a DMF solution of Intermediate 5 (10 mmol) was slowly added to

the above reaction system, and the reaction was stirred at room temperature for 1 h.

Next, the reaction was stirred at 60 °C for 4 h until the reaction was completed

(monitored by TLC). 100 mL of water was added to the reaction mixture, and the

mixture was extracted three times with 30 mL of ethyl acetate. The organic phase

was dried over anhydrous sodium sulfate, filtered and concentrated in vacuum.

Finally, pure target compound 6a was obtained by column chromatography

(petroleum ether/ethyl acetate, v/v= 4:1). Compounds 6b-6e were also synthesized by

this method.

1.8 General Synthesis Procedure for Compounds 7a-7e

To a solution of compound 6a (2 mmol) in H2O (10 mL) and 1,4-dioxane (10

mL), lithium hydroxide (10 mmol) was added dropwise, and the reaction mixture was

stirred at room temperature for 5 h until the reaction was complete (monitored by

TLC). The 1,4-dioxane and water was removed under vacuum, and the remaining

solid is dissolved with a small amount of water. The pH of the aqueous solution was

adjusted to 2 with 1 mol/L HCl. The solid precipitate was then filtered and dried to

obtain the pure target compound 7a. Compounds 7b-7e were also synthesized by this

method.

7-(((carboxymethyl)amino)methyl)phenazine-1-carboxylic acid (7a): Yellow solid;

yield: 82%; m.p. 230-231C; 1H-NMR (400 MHz, DMSO-d6) δ 8.60 – 8.47 (m, 3H,

Phenazine-H), 8.45 (d, J = 9.0 Hz, 1H, Phenazine-H), 8.18 (d, J = 8.2 Hz, 1H,

Phenazine-H), 8.12 (d, J = 7.0 Hz, 1H, Phenazine-H), 4.52 (s, 2H, Phenazine-CH2),

3.93 (s, 2H, CH2-COO); 13

C-NMR (101 MHz, DMSO-d6) δ 168.64, 167.04, 143.26,

143.06, 141.20, 140.48, 136.48, 134.37, 134.27, 133.81, 131.22, 129.72, 129.57,

49.96, 47.27; HRMS calcd for C16H13N3O4 [M+H]+ 312.0979, found 312.0984.

(R)-7-(((1-carboxyethyl)amino)methyl)phenazine-1-carboxylic acid (7b): Yellow

solid; yield: 79%; m.p. 217-218C; 1H-NMR (400 MHz, DMSO-d6) δ 8.54 – 8.50 (m,

2H, Phenazine-H), 8.43 (d, J = 9.2 Hz, 2H, Phenazine-H), 8.15 (d, J = 9.0 Hz, 1H,

Phenazine-H), 8.12 (d, J = 7.0 Hz, 1H, Phenazine-H), 4.41 (s, 2H, Phenazine-CH2),

3.90 – 3.79 (m, 1H, CH-COO), 1.48 (d, J = 7.0 Hz, 3H, CH3); 13

C-NMR (101 MHz,

DMSO-d6) δ 172.55, 167.01, 143.21, 141.03, 140.65, 140.33, 134.34, 134.24, 133.91,

133.84, 131.14, 130.02, 129.48, 129.37, 55.62, 49.14, 16.18; HRMS calcd for

C17H15N3O4 [M+H]+ 326.1135, found 326.1142.

(R)-7-(((1-carboxy-2-methylpropyl)amino)methyl)phenazine-1-carboxylic acid (7c):

Yellow solid; yield: 74%; m.p. 192-193C; 1H-NMR (400 MHz, DMSO-d6) δ 8.33 –

8.08 (m, 4H, Phenazine-H), 8.02 – 7.91 (m, 2H, Phenazine-H), 4.14 (d, J = 15.0 Hz,

1H, Phenazine-CH2), 3.86 (d, J = 15.0 Hz, 1H, Phenazine-CH2), 2.91 (d, J = 5.8 Hz,

1H, CH-COO), 1.93 (dd, J = 13.4, J = 6.8 Hz, 1H, C-CH), 0.94 (dd, J = 6.8, J = 4.6

Hz, 4H, 2×CH3), 0.91 (d, J = 7.0 Hz, 1H, 2×CH3), 0.86 (d, J = 7.0 Hz, 1H, 2×CH3);

13C-NMR (101 MHz, DMSO-d6) δ 169.35, 166.53, 142.79, 142.64, 140.47, 139.92,

135.73, 134.89, 133.85, 131.88, 131.09, 128.87, 128.19, 64.79, 49.96, 28.86, 20.03,

17.40; HRMS calcd for C19H19N3O4 [M+H]+ 354.1448, found 354.1454.

7-((((1R)-1-carboxy-2-methylbutyl)amino)methyl)phenazine-1-carboxylic acid (7d):

Yellow solid; yield: 78%; m.p. 175-176C; 1H-NMR (400 MHz, DMSO-d6) δ 8.56 –

8.51 (m, 2H, Phenazine-H), 8.38 (d, J = 9.0 Hz, 1H, Phenazine-H), 8.30 (s, 1H,

Phenazine-H), 8.13 – 8.08 (m, 2H, Phenazine-H), 4.27 (d, J = 14.4 Hz, 1H,

Phenazine-CH2), 4.14 (d, J = 13.4 Hz, 1H, Phenazine-CH2), 3.40 (d, J = 9.8 Hz, 1H,

CH-COO), 1.89 – 1.80 (m, 1H, Methyl-CH2), 1.63 – 1.49 (m, 1H, Methyl-CH2), 0.91

(d, J = 6.6 Hz, 3H, CH3), 0.85 (d, J = 6.6 Hz, 3H, Methylene-CH3); 13

C-NMR (101

MHz, DMSO-d6) δ 171.28, 166.82, 143.45, 143.11, 142.94, 140.85, 140.27, 134.54,

133.84, 131.10, 130.69, 129.15, 127.56, 124.80, 62.81, 58.40, 49.17, 24.78, 23.40,

21.95; HRMS calcd for C20H21N3O4 [M+H]+ 368.1605, found 368.1613.

(R)-7-(((1-carboxy-2-phenylethyl)amino)methyl)phenazine-1-carboxylic acid (7e):

Yellow solid; yield: 76%; m.p. 151-152C; 1H-NMR (400 MHz, DMSO-d6) δ 8.50 (d,

J = 8.4 Hz, 2H, Phenazine-H), 8.39 – 8.33 (m, 2H, Phenazine-H), 8.10 (dd, J = 8.8, J

= 7.0 Hz, 2H, Phenazine-H), 7.33 – 7.25 (m, 5H, Ar-H), 4.37 (t, J = 11.0 Hz, 2H,

Phenazine-CH2), 3.94 (d, J = 2.6 Hz, 1H, CH-COO), 3.23 (s, 1H, Ar-CH2), 3.13 –

3.08 (m, 1H, Ar-CH2); 13

C-NMR (101 MHz, DMSO-d6) δ 170.82, 166.86, 143.12,

143.01, 140.88, 140.25, 139.60, 137.51, 136.20, 135.92, 134.50, 133.87, 131.09,

130.80, 129.90, 129.54, 129.15, 128.88, 128.72, 127.49, 61.17, 49.76, 36.08; HRMS

calcd for C23H19N3O4 [M+H]+ 402.1448, found 402.1457.

1.9 Plant materials

Seeds of Ricinus communis L. were provided by the Zibo Agricultural Science

Research Institute. The selected Ricinus communis seeds were soaked in water for 12

h and then placed on wet cotton for germination at 28 ± 1°C. After 6 d of planting in

the substrate soil, seedlings of uniform size were used for the next experiment.

1.10 Phloem sap collection

The method of collecting phloem sap was similar to that reported in previous

literature (Hsu et al. 1995; Niu et al. 2017). The 6-day-old seedling endosperm was

carefully removed and the entire plant was washed clean. The cotyledons were

cultured in buffered solution containing 0.2 mmol/L test compounds and roots were

cultured in 0.5 mmol/L CaCl2 solution. After 2 h of culture, the hypocotyls were

excised for phloem exudation. The phloem sap was collected every hour and collected

a total of five times.

1.11 Analytical methods

The phloem sap was diluted with pure water (phloem sap/pure water, v/v=1:9),

and analyzed by UHPLC-MS (Thermo UltiMate 3000 TSQ-Quantis, MA, USA). A

C18 reversed-phase column (3 um, 100 2.1 mm, Thermo Fisher Scientific Co., Ltd.,

MA, USA) was used for separations at 30°C. The mobile phase was made of

methanol and water containing 0.1% formic acid with an isocratic elution

(methanol/water containing 0.1% formic acid, v/v=70:30) at a flow rate of 0.4

mL/min. And the injection volume was 2 μL. The optimization parameters of the

mass spectrometer were as follows: ion source type, heated ESI; positive ion spray

voltage, 3500 V; sheath gas, 30 Arb; aux gas, 5 Arb; ion transfer tube temp, 350C;

vaporizer temp, 400C.

Results

1.1 Table S1 and Figures S1 and S2

Table S1. Physicochemical properties of compounds 7a-7e and PCA.

Compound Molecular Formula Molecular Weight (g/mol) Log Kow pKa

7a C16H13N3O4 311.29 0.00 2.21

7b C17H15N3O4 325.32 0.56 2.17

7c C19H19N3O4 353.37 1.20 2.23

7d C20H21N3O4 367.40 1.67 2.36

7e C23H19N3O4 401.41 1.78 2.07

PCA C13H8N2O2 224.21 1.59 2.34

Notes: The “Log Kow” was calculated by the ALOGPS 2.1 program; The

“pKa” was calculated by the ACD Log D v 6.00 software.

Figure S1. The phloem sap was collected from the second hour and ended at

the fourth hour. For the column chart, n=12. The column chart marked with *

indicates a significant difference between the amino acid conjugating at the

7-position and the carboxyl group of PCA., determined by the

Mann-Whitney U test (p < 0.05).

Phloem mobility scale (log Cf).

< -4.0 (Non-mobile)

-4.0-0.5 (Possibly mobile)

-0.5-1.0 (Moderately mobile)

>1.0 (Very mobile)

Figure S2. Prediction of phloem mobility of compounds 7a-7e and PCA

using the Kleier map (log Cf as a function of pKa and Log Kow).

1.2 Spectra of target compounds

Figure S3. 1H-NMR Spectrum of compound 7a in DMSO-d6

Figure S4. 13

C-NMR Spectrum of compound 7a in DMSO-d6

Figure S5. HRMS Spectrum of compound 7a

XYT-6 #241 RT: 2.36 AV: 1 NL: 8.44E8T: FTMS + p ESI Full ms [100.0000-1500.0000]

308 310 312 314 316 318 320 322

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

312.0984

313.1010

Figure S6. 1H-NMR Spectrum of compound 7b in DMSO-d6

Figure S7. 13

C-NMR Spectrum of compound 7b in DMSO-d6

Figure S8. HRMS Spectrum of compound 7b

Figure S9. 1H-NMR Spectrum of compound 7c in DMSO-d6


322 324 326 328 330 332 334 336

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

326.1142

327.1168

Figure S10. 13

C-NMR Spectrum of compound 7c in DMSO-d6

Figure S11. HRMS Spectrum of compound 7c


350 352 354 356 358 360 362 364

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

354.1454

355.1479

Figure S12. 1H-NMR Spectrum of compound 7d in DMSO-d6

Figure S13. 13

C-NMR Spectrum of compound 7d in DMSO-d6

Figure S14. HRMS Spectrum of compound 7d

Figure S15. 1H-NMR Spectrum of compound 7e in DMSO-d6


364 366 368 370 372 374 376

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

368.1613

369.1637

Figure S16. 13

C-NMR Spectrum of compound 7e in DMSO-d6

Figure S17. HRMS Spectrum of compound 7e


398 400 402 404 406 408 410 412

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

402.1457

403.1483

7a-A

7a-B

7a-C

Figure S18. The phloem sap analysis by UHPLC-MS. 7a-A: standard sample of 7a

(RT: 5.78 min); 7a-B: control, the cotyledons were incubated in the standard medium;

7a-C: treated set, the cotyledons were incubated in the same solution with 7a (RT:

5.78 min) at 0.2 mmol/L concentration.

7b-A

7b-B

7b-C

Figure S19. The phloem sap analysis by UHPLC-MS. 7b-A: standard sample of 7b

(RT: 5.85 min); 7b-B: control, the cotyledons were incubated in the standard medium;

7b-C: treated set, the cotyledons were incubated in the same solution with 7b (RT:


7c-A

7c-B

7c-C

Figure S20. The phloem sap analysis by UHPLC-MS. 7c-A: standard sample of 7c

(RT: 6.09 min); 7c-B: control, the cotyledons were incubated in the standard medium;

7c-C: treated set, the cotyledons were incubated in the same solution with 7c (RT:


7d-A

7d-B

7d-C

Figure S21. The phloem sap analysis by UHPLC-MS. 7d-A: standard sample of 7d

(RT: 6.46 min); 7d-B: control, the cotyledons were incubated in the standard medium;

7d-C: treated set, the cotyledons were incubated in the same solution with 7d (RT:


7e-A

7e-B

7e-C

Figure S22. The phloem sap analysis by UHPLC-MS. 7e-A: standard sample of 7e

(RT: 6.69 min); 7e-B: control, the cotyledons were incubated in the standard medium;

7e-C: treated set, the cotyledons were incubated in the same solution with 7e (RT:


PCA-A

PCA-B

PCA-C

Figure S23. The phloem sap analysis by UHPLC-MS. PCA-A: standard sample of

PCA (RT: 7.52 min); PCA-B: control, the cotyledons were incubated in the standard

medium; PCA-C: treated set, the cotyledons were incubated in the same solution with

PCA at 0.2 mmol/L concentration.

References

Edgington LV. 1981. Structural requirements of systemic fungicides. Annu. Rev. Phytopathol. 19,

107-124.

Hsu FC, Sun K, Kleier DA, Fielding MJ. 1995. Phloem mobility of xenobiotics VI. A

phloem-mobile pro-nematicide based on oxamyl exhibiting root-specific activation in

transgenic tobacco. Pestic Sci. 44:9-19.

Niu JF, Nie DY, Yu DY, Wu QL, Yu LH, Yao ZL, Du XY, Li JK. 2017. Synthesis, fungicidal

activity and phloem mobility of phenazine-1-carboxylic acid-alanine conjugates. Pest

Biochem Physiol. 143:8-13

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