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Supplementary information for:

New Dual Functional Salts Based on Cationic

Derivative of Plant Resistance Inducer –

Benzo[1.2.3]thiadiazole-7-carbothioic Acid, S-

Methyl Ester

Marcin Smiglak,*,†, ‡ Rafal Kukawka,‡ Piotr Lewandowski,‡ Marta Budziszewska,§ Aleksandra

Obrępalska-Stęplowska,§ Krzysztof Krawczyk,§ Agnieszka Zwolińska,§ Henryk Pospieszny§

† Dr. M. Smiglak

Poznan Science and Technology Park

Adam Mickiewicz University Foundation

ul. Rubież 46, 61-612 Poznań, Poland

‡ MSc. R. Kukawka, MSc. P. Lewandowski

Faculty of Chemistry

Adam Mickiewicz University

ul. Umultowska 89b, 61-614 Poznań, Poland.

§ Prof. H. Pospieszny, Dr A. Obrępalska-Stęplowska, Dr. K. Krawczyk, Dr M. Budziszewska,

MSc. A. Zwolinska

Institute of Plant Protection – National Research Institute

ul. W. Węgorka 20, 60-318 Poznań, Poland

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Number of pages: 24

Number of figures: 3

Number of tables: 2

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Figures:

Figure S1. Thermal stability of [BTHCOOH(Me)]+ salts --▲-- (11), —△— (12), —◆— (13), --◆-- (14), —◼— (15), --◌-- (16)

Figure S2. Dissolution rate of [BTHCOOH(Me)]+ derivatives --▲-- (11), —△— (12), —◆— (13), —◼— (15),--◌-- (16)

Temperature [oC]

0 50 100 150 200 250 300 350 400 450 500

Wei

gh

t [%

]

0

10

20

30

40

50

60

70

80

90

100

BTHCOOH

[BTHCOOH(Me)][MeSO4]

[BTHCOOH(Me)][[NTf2]

[BTHCOOH(Me)][Doc]

[BTHCOO(Me)]+/-

[BTHCOOH(Me)][I]

Time [s]

0 500 1000 1500 2000 2500

A\b

sorb

an

ce [

%]

0

10

20

30

40

50

60

70

80

90

100

BTHCOOH

[BTHCOOH(Me)][Doc]

[BTHCOOH(Me)][NTf2]


[BTHCOO(Me)]+/-

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Figure S3. Phytotoxicity on tobacco plants after watering with 50 ml solution of [BTHMe][MeSO4] (2) at the concentration 20mg/l (left) and control (right)

Tables:

Table S1. MIC and MBC values of the tested salts (concentration in mg/l). The list of the salts is given in Table 1.

Strain (2) (3) (4) (5) (6) (7) (8) (9) (12) (13) (14) (15) (16)

Cocci

S. aureus MIC 125 — 1000 1000 175 253 — 1013 673 — — — 997

Rods MBC 125 — — — — 253 — 1013 673 — — — 997

E. coli MIC 667 — — — — 675 — — — — — — —

MBC 667 — — — — 675 — — — — — — —

P. carotovorum MIC 500 — — — 175 253 — — — — — —

MBC 500 — — — — 253 — — — — — —

P. syringae MIC 125 1000 1000 1000 175 253 — — 253 125 1000 80 997

MBC 125 1000 1000 1000 175 253 — — 253 125 1000 160 997

Maximal. non

phytotoxic

concentration

of the salts

tested

1000 1000 1000 1000 175 1012 1024 1013 1010 1002 1000 638 997

Legend: MIC (Minimal Inhibitory Concentration) - the lowest concentration of tested substances at which there was no visible bacterial growth (mg/l). MBC

(Minimum Bactericidal Concentration) - the lowest concentration of the salts supporting no bacterial colony formation (mg/l).

Symbol “— “ means that at maximum tested concentration (~1000 mg/l) the salts showed no antibacterial activity, hence pointing that its MIC and MBC at these

concentrations were not possible to be determined.

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Table 2. Characteristic wavelength (maximum

of absorbance) used for determination

dissolution rate

Compound Characteristic

wavelength [nm]

BTH (1) 338

[BTHMe][MeSO4] (2) 312

[BTHMe][I] (3) 292

[BTHMe][OTf] (4) 322

[BTHMe][Cl] (5) 285

[BTHMe][NTf2] (6)28 338

[BTHMe][Doc] (7)28 316

[BTHMe][MES] (8) 322

[BTHMe][LS] (9) 285

BTHCOOH (11) 316

[BTHCOO(Me)]+/- (12) 299


(13)

360

[BTHCOOH(Me)][I] (14) 292

[BTHCOOH(Me)][NTf2]

(15)

299

[BTHCOOH(Me)][Doc]

(16)

322

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Experimental Section

Synthesis and physicochemical properties of the precursors ([BTHMe][MeSO4] (2),

[Na][BTHCOO] (10) and BTHCOOH (11)), used to obtain new compounds, have been reported

in papers by Smiglak et al.[E1,E2] Furthermore, the methods for preparation, physical properties

and the level of induction of plant resistance caused by the compounds: [BTHMe][OTf] (4),

[BTHMe][NTf2] (6) and [BTHMe][Doc] (7) were reported in the earlier article.[E2] Data for those

compounds is incorporated in the article to present physical and biological properties of series of

BTH cationic derivatives and to expand research on antibacterial tests of these compounds.

Methods

New BTH salts were characterized using Nuclear Magnetic Resonance and Infrared Spectroscopy.

The methods for obtaining thermal, spectroscopic and others data are described below.

NMR

H NMR spectra were recorded on a Varian XL 300 NMR (300 MHz) using d6-DMSO as solvent

with tetreamethylsilane as the internal standard. Proton chemical shifts are shown in parts per

million (δ ppm). C NMR spectra were obtained in the same instrument at 75 MHz.

Thermal analysis

Melting points were determined using DSC method in STARe System (Mettler Toledo). Samples

used were between 6 – 15 mg, closed in aluminum pans and stored under argon atmosphere (flow

rate: 20 ml/min). In the first heating cycle the heating ramp was set from 25 to maximum

temperature (160oC for compounds: BTH (1), [BTHMe][MeSO4] (2), [BTHMe][I] (3),

[BTHMe][NTf2] (6), [BTHMe][Doc] (7), [BTHMe][LS] (9) and 230oC for compounds:

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[BTHMe][OTf] (4), [BTHMe][Cl] (5), [BTHMe][MES] (8), [BTHCOO(Me)]+/- (12),

[BTHCOOH(Me)][MeSO4] (13), [BTHCOOH(Me)][I] (14), [BTHCOOH(Me)][NTf2] (15),

[BTHCOOH(Me)[Doc] (16)) and 270oC for compound: BTHCOOH (11)), with heating rate

10oC/min. At this temperature samples were held for a 5 minutes isotherm. In the next step samples

were cooled from the maximum temperature to -50oC with cooling rate 10oC /min, and then were

kept for 5 min at -50oC. The last step was heating sample from -50oC back to the maximum

temperature (as described above) with heating rate 10oC/min.

Thermogravimetric analyses were performed on TGA Q50 Texas Instrument. Samples between 5

and 10 mg were heated form 25oC to 500oC with heating rate of 10oC/min with a 10 min isotherm

at 80oC under nitrogen atmosphere. This isotherm step was intended to remove any remaining

water and possible volatile impurities present in the samples. Decomposition temperatures reported

for all materials were established as the onset temperature for decomposition of the first 5% of the

sample (T5%onset), and as the regular onset temperature for decomposition (Tonset), either for the

whole sample or for each of the consecutive steps in stepwise decomposition

Solubility analysis

Solubility in water was determined in two ways. Every compound (100 mg) was dissolved in 10

ml of water at room temperature during 1 hour time. The procedure was repeated three times. The

compounds which completely dissolved at that time were described as: soluble in water >10g/l. If

the compound did not dissolve in 1 hour time (e.g. [BTHMe][NTf2] (6) and

[BTHCOOH(Me)][NTf2] (15)), second experiment was performed to determine the maximum

solubility in water. 10 mg of compound was placed in flask with magnetic stirrer with 100 ml of

water for 24 hours. If all of the sample was dissolved, after one day next portion (10 mg) was

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added. Next portions were added until part of compound was no dissolved. Then the procedure was

repeated by dissolving just the determined maximum amount of a compound in water and next

adding new portion of the substance in an amount of 1 mg until no further dissolution was observed.

Dissolution rate

To determine dissolution rate the system for continuous flow measurement of absorbance of

tested substances was constructed and used. For every compound, characteristic wavelength (Table

S2 ) at which the absorbance of compound was at the maximum, was determined by UV-Vis.

Following this step, salt samples of the tested compounds (the mass of each sample between 4 and

10 mg, always below maximum solubility as determined in separate experiment) were placed in

the mashed cellulose bag in conical flask filed with 140 cm3 of distilled water with magnetic stirrer

(250 rpm). Using peristaltic pump the solution from the flask, where the dissolution experiment

was conducted, was transferred to the UV spectrometer and after passing through the UV cell, back

to the experiment flask in order to form a closed system. The absorbance of the solution was

measured constantly at the wavelength earlier determined as maximum absorption for particular

compound. Each experiment was carried until all tested sample was completely dissolved.

Dissolution rate of examined compounds was compared to the dissolution rates of neutral BTH and

BTHCOOH on relative scale. The relative dissolution rate was determined by dividing the amount

of time required for the absorbance to reached 95% of the maximum absorbance for investigated

compound vs starting material (BTH or BTHCOOH). The results are shown in graphs where x-

axis represents dissolution time and y-axis is % of absorbance of compound (100% absorbance is

absorbance of fully dissolved compound).

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LogP determination

The LogP values were determined for all of reported compounds by using standard OECD

procedure utilizing reverse phase HPLC method.[E3] Samples were dissolved in mixture of HPLC

grade methanol and water (75:25 v/v) in concentration 0.6 mg/mL. Prepared samples were analyzed

using a Acquity UPLC BEH C18 1.7μm column by Waters with a mobile phase of methanol and

water (75:25 v/v) with a flow rate of 0.5 and 0.25 mL/min and detected by PDA detector. Due to

specificity of selected HPLC method for the determination of logP values of organic compounds

only values of logP between 0 and 6 can be determined. All compounds with logP values <0 exhibit

retention time on the column = T0 and thus could not be measured correctly. The standard curve

was generated by using materials of known logP values, nicotinic acid (0.07), butanone (0.30),

benzyl alcohol (1.10), methyl benzoate (2.10), ethyl benzoate (2.60) toluene (2.70) and naphthalene

(3.60) as references. Retention times were collected in triplicates for each of references and

analyzed salts. The eluent flow rate was set to 0.025 ml/min and the column temperature was set

at 30 oC. Retention times of reference compounds were adjusted for the T0 (359 s for 0.025

mL/min), and a standard curve of logP vs. retention time was generated (R2 = 0,998). Values of

logP for analyzed new salts, based on their the retention time, were calculated using equation of

the calibration curve.

pKa value determination

In order to calculate pKa values, the dissociation constant (Ka) of salts (13) - (16) containing

carboxylic acid group was determined using conductometric method with pH/conductivity meter

CPC-411 ELMEIRA with EC-60 ELMEIRA electrode. Conductance was measured for each

substance in prepared solutions with decreasing concentratios: 0.04, 0.02 0.01, 0.005 0.0025, 0.002

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and 0.001 [mol/l] for salts [BTHCOOH(Me)][MeSO4] (13), [BTHCOOH(Me)][I] (14), and

[BTHCOOH(Me)][Doc] (16). For [BTHCOOH(Me)][NTf2] (15) solutions were prepared in

concentrations: 0.01, 0.005, 0.0025, 0.002, 0.00125, 0.001, 0.0005 [mol/dm3] due to low solubility

in water. Due to low solubility in water the pKa was not determined for BTHCOOH (11). Prepared

solutions were thermostated at 25°C and conductance (G) was measured. Knowing the cell constant

(K = 0.95), the conductivity (κ) and molar conductivities (Λm) were calculated. The resulting

experimental data were plotted as y= 1/Λm i x = κ (R2 > 0.97). A and b parameters of linear

regression equation were calculated and used for calculation of dissociation constant (eq1).

(eq1)

pKa was calculated from Ka through mathematical conversion pKa=-logKa.

Synthesis

Preparation of salts with cations derived from benzo[1.2.3]thiadiazole-7-carbothioic acid, S-methyl

ester (BTH) and benzo[1.2.3]thiadiazole-7-carboxylic acid (BTHCOOH).

Reported new dual functional salts of BTH (1) and BTHCOOH (11) were prepared via alkylation

reaction of neutral BTH or BTHCOOH in order to form cationic derivatives (Figure 8 and 9) and

then, using one of three general methods for the anion exchange, total of 9 new products (out of 13

reported) was obtained. The anion exchange reactions were performed via (i) use of ion exchange

acidic resin, (ii) biphasic solvent extraction and (iii) by precipitation of product. Out of all reported

compounds, salts [Na][BTHCOO] (10), [BTHMe][OTf] (4), [BTHMe][MeSO4] (2),

[BTHMe][Doc] (7), [BTHMe][NTf2] (6) and neutral compound BTHCOOH (11) were already

𝐾𝑎 =1

𝑎(103 ∗ 𝐶𝑚𝑜𝑙)∗ 𝑏2

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described by us in earlier reports[1,2] but their physicochemical and biological properties needed

to be determined in order to present the whole series of derivatives and the trends in their changes.

Methylation reaction

3-methyl-benzo[1.2.3]thiadiazolium-7-carboxylic acid methyl sulfate,

[BTHCOOH(Me)][MeSO4], (13)

Three-neck flask equipped with reflux condenser was placed on the magnetic stirrer and

BTHCOOH (11) (0.5 g, 2.77 x 10-3 mol) was added and heated to 120oC while purging with argon.

After 3 minutes Me2SO4 (0.7 g, 5.5 x 10-3 mol) was added and all BTHCOOH immediately

dissolved in it. The reaction was stopped after 5 minutes and cooled to the room temperature. The

product was isolated from the solution as crystalline material by slowly cooling of the reaction

mixture and later by filtration. To the solid product acetone (10 ml) was added to wash out excess

of Me2SO4, unreacted BTHCOOH and other impurities. Such solution was stirred for 2 hours to

obtain powdery pink product. The IR, and NMR analysis confirmed obtaining of product 3-methyl-

benzo[1,2,3]thiadiazol-3-ium 7-carboxylic acid methyl sulfate with yield: 80 %.

m.p. 204.7oC; 1H NMR (300 MHz, [D6]DMSO, 25oC, TMS): δ/ppm = 9.05 (1 H, d, C(6)H), 8.58

(1 H, t, C(6)H), 8.31 (1 H, d, C(6)H), 5.00 (3 H, s, NMe); 13C NMR (75 MHz, [D6]DMSO, TMS):

δ/ppm = 166.8, 146.8, 144.7, 133.3, 132.9, 125.3, 122.9, 46.3; IR: ν/cm-1 = 3082, 3042, 2732, 2456,

1681, 1661, 1582, 1489, 1444, 1316, 1215, 1131, 1062, 1018, 874, 816, 765, 704, 592, 573.

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3-methyl-benzo[1.2.3]thiadiazolium-7-carbothioic acid, S – methyl ester iodide, [BTHMe][I],

(3)

To the pressure tube with capacity of 20 ml with magnetic stirrer BTH (1) (0.5 g, 2.38 x 10-3 mol)

and methyl iodide (MeI) (0.69 g, 4.76 x 10 -3 mol) were added. The pressure tube was sealed and

heated to 100oC for 18 hours. BTH was dissolved in MeI at 80oC. During the reaction, solution

changed colour from light yellow to bottle green. After cooling to room temperature remaining

unreacted BTH immediately precipitated. After carefully opening reaction vessel, water was added

and mixture was vigorously shaken in order to separate product from water insoluble BTH. Water

phase was separated out and evaporated. Red crystals of 3-methyl-benzo[1.2.3]thiadiazolium 7-

carbothioc acid, S – methyl ester iodide with yield: 8% were obtained as confirmed by NMR.

m.p. 112.9oC; 1H NMR (300 MHz, [D6]DMSO, 25oC, TMS)) δ/ppm = 9.19 (1 H, d, C(6)H), 8.87

(1 H, t, C(6)H), 8.36 (1 H, d, C(6)H), 5.03 (3 H, s, NMe), 2.70 (3 H, s, SMe); 13C NMR (75 MHz,

[D6]DMSO, TMS) δ/ppm = 192.0, 148.2, 142.6, 134.1, 132.7, 130.4, 124.7, 47.0, 12.8; IR: ν/cm-1

= 2981, 2917, 1620, 1508, 1572, 1479, 1439, 1329, 1252, 1199, 1085, 1068, 997, 878, 845, 797,

725, 701, 673.

3-methyl-benzo[1.2.3]thiadiazolium-7-carboxylate (zwitterion), [BTHCOO(Me)]+/-, (12)

Into the three-neck flask under reflux condenser with magnetic stirrer sodium

benzo[1.2.3]thiadiazole-7-carboxylate ([Na][BTHCOO]) (10) (0.5 g, 2.77 x 10-3 mol), obtained as

described in literature[27] was added and heated to 120oC while purging with argon. Than Me2SO4

(0.7 g, 5.5 x 10-3 mol) was added and immediately all of [Na][BTHCOO] dissolved in it. After two

minutes the reaction mixture turn brown. The reaction was stopped and cooled to the room

temperature. While the solution was being cooled down, it crystalized. To the solid product dry

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methanol (10 ml) was added to precipitate the byproduct [Na][MeSO4]. Methanol phase was

evaporated and obtained product was again dissolved in methanol (5 ml). After that the mixture

was added dropwise to diethyl ether (30 ml), white powder precipitated and was separated. The IR

and NMR analysis confirmed obtaining product 3-methyl-benzo[1.2.3]thiadiazol-3-ium-7-

carboxylate with yield: 65 %.

m.p. 201.8oC; 1H NMR (300 MHz, [D6]DMSO, 25oC, TMS) δ/ppm = 8.7 (1 H, d, C(6)H), 8.2 (1

H, t, C(6)H), 8.1 (1 H, d, C(6)H), 4.9 (3 H, s, NMe); 13C NMR (75 MHz, [D6]DMSO, TMS) δ/ppm

= 165.9, 146.7, 144.5, 133.1, 132.9, 123.0, 46.0; IR: ν/cm-1 = 3125, 3082, 2832, 2452, 1681, 1667,

1582, 1474, 1412, 1316, 1261, 1214, 1172, 1131, 1052, 1018, 874, 849, 816, 765, 704, 592

Anion exchange reactions

Ion exchange resin

3-methyl-benzo[1.2.3]thiadiazolium-7-carbothioic acid, S-methyl ester chloride,

[BTHMe][Cl], (5)

In a round-bottom flask of ion exchange resin Lewait MonoPlus SP-112 (0.8 g) was regenerated at

room temperature using of 10% NaCl solution (3 ml). Then the solution was separated from the

resin and the resin was washed five times with distilled water (3 ml) until no chloride anions were

detected. In order to verify that all the chloride ions were absorbed on the resin test with an aqueous

solution of 1% AgNO3 was performed. No precipitate after adding AgNO3 solution meant that

chloride ions in washed water were not present. To the regenerated resin, an aqueous solution of

[BTHMe][MeSO4] (2) (165 mg, 4.9 x 10-4 mol [BTHMe][MeSO4], 3 ml) was added and stirred for

half an hour at 50°C. Slow discoloration of the solution was observed. After this time, the solution

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was separated off and the resin was washed once with distilled water. Then NaCl (3 g) was added

into water (3 ml) and such solution was added to resin and stirred for 10 minutes. After this time,

the solution was separated and NaCl (1 g) in water (3 ml) was added again to the resin. After 10

minutes and separating water phase NaCl solutions were combined and evaporated. Next in order

to separate product from NaCl dry acetone (5 ml) was added and intensively stirred causing NaCl

to precipitate. Afterwards acetone phase was separated and evaporated. To purify obtained product,

methanol (5 ml) was added to dissolve product and to separate any remaining solid residues. In the

last step, solvent was evaporated yielding product. Obtaining of the product was confirmed by

NMR where disappearance of the peak of the anion [MeSO4]- was indicative of the reaction

completion.

m.p. 124.9oC; 1H NMR (300 MHz, [D6]DMSO, 25oC, TMS) δ/ppm = 9.2 (1 H, d, C(6)H),

8.9 (1 H, t, C(6)H), 8.4 (1 H, d, C(6)H), 5.0 (3 H, s, NMe), 2.7 (3 H, s, SMe), 13C NMR (75 MHz,

[D6]DMSO, TMS) δ/ppm = 190.6, 147.4, 133.9, 130.3, 129.8, 124.5, 42.16, 12.2; IR: ν/cm-1 =

3071, 2991, 2949, 2924, 1628, 1586, 1449, 1327, 1245, 1201, 1119, 1069, 997, 843, 798, 751

Extraction method

3-methyl-benzo[1.2.3]thiadiazolium-7-carboxylic acid docusate, [BTHCOOH(Me)][Doc],

(16)

[BTHCOOH(Me)][MeSO4] (13) (1g, 3.27 x 10-3 mol) was dissolved in water in a beaker.

Meanwhile) of sodium docusate (1.23 g, 2.8 x 10-3 mol, 0.85 molar eq) was dissolved in water in

another beaker. Sodium docusate is used in the reaction in the deficiency in respect to

[BTHCOOH(Me)][MeSO4] due to easier later separation of product (dissolved in organic phase)

from the excess [BTHCOOH(Me)][MeSO4] (well soluble in water). After accurately stirring two

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mixtures, the solution of sodium docusate was added to the solution of [BTHCOOH(Me)][MeSO4]

and such solution was placed in separation funnel. After that, dichloromethane (10 ml) was added

and two-phase system was shaken vigorously. After phases separation, dichloromethane (DCM)

phase was separated. The organic phase was washed two times with water in order to remove

[Na][MeSO4] and possible unreacted substrate [BTHCOOH(Me])[MeSO4]. Next DCM phase was

evaporated to obtain brown-yellow oil which was also washed two times with water and one time

with hexane due to the fact that [Na][Doc] is well soluble in hexane and water. The structure of

obtained product [BTHCOOH(Me)][Doc] (3-methyl-benzo[1.2.3]thiadiazolium 7-carboxylic acid

docusate) was confirmed by NMR. In the IR spectrum peaks from both ions were identified. Also

the disappearance of the peak from the anion [MeSO4]- was observed in the NMR. Yield: 90%,

dark yellow, low melting wax, no clear melting point.

m.p. no clear m.p.; 1H NMR (300 MHz, [D6]DMSO, 25oC, TMS) δ/ppm = 9.1 (1 H, d, C(6)H),

8.6 (1 H, t, C(6)H), 8.3 (1 H, d, C(6)H), 5.0; (3 H, s, NMe), 3.9 (2 H, d, CHO), 3.6 (2 H, d, CHO),

3.3 (1 H, t, CHSO3), 2.9 (2 H, d, CH), 2.8 (3 H, s, SMe), 1.3-1.5 (16 H, m, alkyl), 0.8-0.9 (12 H,

m, alkyl); 13C NMR (75 MHz, [D6]DMSO, TMS) δ/ppm = 171.0, 168.6, 166.0, 146.7, 144.5, 133.1,

132.9, 123.5, 66.1, 63.0, 46.1, 41.4, 34.3, 29.8, 29.7, 29.6, 28.6, 28.3, 23.0, 22.6, 22.4, 22.3, 13.9,

13.8, 10.8, 10.7; IR: ν/cm-1 = 3462, 2958, 2930, 2860, 1731, 1687, 1586, 1460, 1413, 1380, 1329,

1222, 1157, 1034, 898, 850, 798, 762, 709, 600.

3-methyl-benzo[1.2.3]thiadiazolium-7-carbothioc acid, S – methyl ester dodecyl sulfate,

[BTHMe][LS], (9)

[BTHMe][MeSO4] (2) (1 g, 4.76 x 10-3 mol) and sodium lauryl sulfate (1.38 g, 4.76 x 10-3 mol)

were dissolved in water in separate vessels. After accurately stirring, and combining into one

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solution, water was evaporated. After drying on high vacuum, methanol (5 ml) was added.

Immediately white precipitate (identified on IR as inorganic byproduct) was observed and

separated. Methanol phase was evaporated and white powder was obtained. The IR spectrum

proved presence of the peaks from two substrate ions. In the NMR spectra the disappearance of the

peak from the anion [MeSO4]- was observed indicating obtaining of the product. Product 3-methyl-

benzo[1.2.3]thiadiazolium 7-carbothioc acid, S – methyl ester lauryl sulfate was obtained as white

powder. Yield of reaction: 65%


8.9 (1 H, t, C(6)H), 8.4 (1 H, d, C(6)H), 5.0 (3 H, s, NMe), 3.7 (2 H, t, CHSO3), 2.7 (3 H, s, SMe),

1.4 (2 H, t, CH), 1.2 (18 H, m, alkyl), 0.8 (3 H, t, Me); 13C NMR (75 MHz, [D6]DMSO, TMS)

δ/ppm = 190.3, 147.0, 141.2, 133.3, 131.7, 129.2, 123.9, 65.4, 60.7, 46.1, 32.5, 31.3, 29.0, 28.9,

25.5, 22.0, 13.9, 11.7; IR: ν/cm-1 = 3079, 2954, 2920, 2850, 1625, 1586, 1248, 1216, 1199, 1152,

1077, 1055, 968, 834, 751.

3-methyl-benzo[1.2.3]thiadiazolium-7-carboxylic acid bis(trifluoromethanesulfon)imide,

[BTHCOOH(Me)][NTf2], (15)

[BTHCOOH(Me)][MeSO4] (13) (1 g, 3.27 x 10-3 mol) was dissolved in water (1 ml) in test-tube.

When all of the substrate was completely dissolved in water solution of

bis(trifluoromethanesulfon)imide lithium salt (LiNTf2, 80% solution in water) (0.8 g, 2.8 x 10-

3mol, 0.85 molar eq) was added. The solution was evaporated and methanol was added. White,

inorganic powder ([Li][MeSO4]) was obtained and filtrated from the solution. The methanol

solution was evaporated. Product 3-methyl-benzo[1.2.3]thiadiazolium 7-carboxylic acid

bis(trifluoromethanesulfon)imide was obtained as white powder with yield 58%.

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8.9 (1 H, t, C(6)H), 8.4 (1 H, d, C(6)H), 5.0 (3 H, s, NMe); 13C NMR (75 MHz, [D6]DMSO, TMS)

δ/ppm = 165.4, 145.9, 145.0, 132.9, 131.5, 123.6, 121.2, 120.7, 118.2, 45.8; IR: ν/cm-1 = 3085,

1702, 1588, 1438, 1322, 1317, 1188, 1140, 1077, 1051, 934, 831, 798, 468, 740, 712, 609, 567,

512.

Precipitation method

3-methyl-benzo[1.2.3]thiadiazolium--7-carbothioic acid, S-methyl ester 2-(N-

morpholino)ethanesulfonate, [BTHMe][MES], (8)

[BTHMe][MeSO4] (2) (1 g, 4.76 x 10-3 mol) and 2-(N-morpholino)ethanesulfonate sodium salt

([Na][MES]) (1.04 g, 4.76 x 10-3 mol) were dissolved in water. After accurately stirring, and

combining into one solution, water was evaporated. After drying on high vacuum, methanol (1 ml)

was added. Immediately formation of white precipitate (identified on IR as inorganic byproduct

[Na][MeSO4]) was observed. Precipitate was separated and then methanol phase was evaporated.

White powder was obtained. The IR spectrum allowed to identify peaks from both of substrate

ions. In the NMR spectra the disappearance of the peak of the anion [MeSO4]- was observed.

Product 2-(N-morpholino)ethanesulfonic acid 3-methyl-benzo[1.2.3]thiadiazolium 7-carbothioc

acid salt as yellow powder was obtained. Yield of reaction: 65%


H, t, C(6)H), 8.4 (1 H, d, C(6)H), 5.0 (3 H, s, NMe), 3.4 (4 H, t, OCH), 3.1 (4 H, t, NCH), 2.9 (2

H, t, CHSO3), 2.9 (2 H, t, NMe), 2.7 (3 H, s, SMe); 13C NMR (75 MHz, [D6]DMSO, TMS) δ/ppm

= 191.1, 147.1, 141.2, 133.3, 131.7, 129.2, 123.9, 63.4, 51.2, 46.1, 44.9, 11.7; IR: ν/cm-1 = 3062,

3028, 1625, 1583, 1442, 1262, 1242, 1166, 1121, 1080, 1062, 1037, 995, 977, 910, 846, 764, 545.

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3-methyl-benzo[1.2.3]thiadiazolium -7-carbothioic acid, S-methyl ester iodide, [BTHMe][I],

(3)

To round bottom flask [BTHMe][MeSO4] (2) (1 g, 4.76 x 10-3 mol) was dissolved in methanol (3

ml) and sodium iodide (0.714 g, 4.76 x 10-3 mol ) in methanol (10 ml) was added. After steering

overnight white crystals of [Na][MeSO4] precipitated and were than filtered. Crystal were

separated and methanol phase was collected and evaporated to obtain product as orange powder

with yield: 99%.


H, t, C(6)H), 8.4 (1 H, d, C(6)H), 5.0 (3 H, s, NMe), 2.7 (3 H, s, SMe); 13C NMR (75 MHz,

[D6]DMSO, TMS) δ/ppm = 192.0, 148.2, 142.6, 134.1, 132.7, 130.4, 124.7, 47.0, 12.8; IR: ν/cm-1

= 2981, 2917, 1620, 1508, 1572, 1479, 1439, 1329, 1252, 1199, 1085, 1068, 997, 878, 845, 797,

725, 701, 673.

3-methyl-benzo[1.2.3]thiadiazolium-7-carboxylic acid iodide, [BTHCOOH(Me)][I], (14)

[BTHCOOH(Me)][MeSO4] (13) (2 g, 6.5x10-3 mol) and (0.98 g, 6.5x10-3 mol) sodium iodide (NaI)

were each dissolved separately in dry methanol (5 ml). When substrates were completely dissolved

the solutions were mixed. Immediately, white powder precipitated. After filtration methanol phase

was evaporated and dried on vacuum pump. Orange powder of 3-methyl-benzo[1.2.3]thiadiazole-

3-ium-7-carboxylic acid iodide was obtained with yield: 90%.


H, t, C(6)H), 8.4 (1 H, d, C(6)H), 5.0 (3 H, s, NMe); 13C NMR (75 MHz, [D6]DMSO, TMS) δ/ppm

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= 166.2, 147.1, 144.1, 133.1, 125.2, 122.9, 46.4; IR: ν/cm-1 = 3602, 3406, 3060, 1687, 1583, 1492,

1438, 1316, 1241, 1158, 1135, 1065, 1010, 899, 849, 756, 726, 696, 616, 602, 593.

Antibacterial activity tests

Microorganisms used in this studies were: Staphylococcus aureus ATCC 6538, Escherichia coli

ATCC 25922, Pectobacterium carotovorum ATCC 15713, and Pseudomonas syringae ATCC

19508. Strains were supplied by American Type Culture Collection (ATCC)

The antibacterial activity was determined using the dilution method.[E4] Bacteria strains were

cultured on a Mueller-Hinton broth for 24 h. A suspension of the bacteria at a concentration of 106

cfu/cm-3 (cfu = colony forming units), was prepared from each culture. Then each dilution of the

tested salt was inoculated with a broth medium suspension, as described above, in a 1:1 ratio.

Additionally one concentration in 2:1 ratio (salt: culture broth medium suspension) was made and

tested. Growth of (or the lack of) the microorganisms was determined visually after incubation for

24 h at 37ºC for S. aureus and E. coli, and in 27ºC for P. carotovorum and P. syringae. The lowest

concentration of tested substances at which there was no visible growth (turbidity) was taken as

the MIC (Minimal Inhibitory Concentration). Then, an aliquot taken from each tube in a sample

loop was cultured in an agar medium with inactivates (0.3% lecithin, 3% polysorbate 80 and

0.1% ysteine L) and incubated for 48 h at the temperatures mentioned above. The lowest

concentration of the salt supporting no colony formation was defined as the MBC (Minimum

Bactericidal Concentration). Studies were conducted on four strains of bacteria. Due to a

phytotoxic effect of the tested salts, its highest concentration tested was also concluded as the

highest non phytotoxic concentration. That is why in some cases it was not possible to define both

MIC and the MBC values.

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Phytotoxicity test

The N. tabacum var Xanthi plants were watered with water solutions (50 ml) containing active

substance in concentration of 20 mg/l (0.36 mg/l for [BTHMe][NTf2] (6); 12.6 mg/l for

[BTHCOOH(Me)][NTf2] (15)). Several days after treatment visual effects of used compounds on

the plants were analyzed. The symptoms of phytotoxicity were observed as necrotic spots,

yellowing of part or all leaves, retarded leaf growth, or general plant growth inhibition.

Direct influence of BTH derivatives on the virus infectivity

To assess direct impact of tested BTH derivatives on the infectivity of virus particles, compounds

were dissolved in water at concentration: 50 mg/l or in 5% aqueous methanol solution in case of

([BTHMe][NTf2] (6) and [BTHCOOH(Me)(NTf2)] (15) at concentrations 0.9 mg/l and 31.5 mg/l

respectively. Initially, the tobacco mosaic virus (TMV) was incubated for about 30 minutes in

solution of BTH derivatives, and then N. tabacum var. Xanthi leaves were mechanically infected.

In the control, tobacco leaves were infected with a mixture of TMV virus which was previously

incubated only in distilled water. After 4 days post the infection the necrotic spots on tobacco plant

infected with virus from control and treated before in BTH and BTHCOOH derivatives solution

were counted and compared.

SAR induction activity

In the following experiment, plants of N. tabacum var Xanthi, at the stage of three-developed

leaves, were watered with 50 ml solutions of salts in water in concentration: 20 mg/l (except for

([BTHMe][NTf2] (6) and [BTHCOOH(Me)(NTf2)] (15) at concentrations 0.9 mg/l and 31.5 mg/l

respectively) and the control with distilled water that was used for the preparation of solutions of

S21

salts. Seven days later, the treated leaves were infected mechanically with Tobacco mosaic virus.

After the next 4-5 days a local necrotic spots, as a result of a viral infection, were counted and

compared between the number of spots on the leaves treated with salt and distilled water (control).

Reduction in the number of necrotic spots on the leaves treated with salt, in comparison with the

control, shows inhibition of viral infection by induction of plant resistance through the use of salts.

Moreover, aside from the reduction in the number of local necrotic spots, in tobacco plants treated

with salts reduction in the size of local necrotic spots formed on leaves was observed.

RNA isolation and cDNA synthesis

Total RNA was extracted from collected plant material [N. tabacum] i) treated with BTH (1), ii)

treated with cationic derivative of [BTHMe][Doc] (7), and iii) treated only with water, using TRI-

Reagent (ThermoFisher Scientific, Waltham, MA) according to manufacturer’s instruction. The

pellet of total RNA was dissolved in 20-40 μl of RNase-free water, quantified by

spectrophotometry, and stored at -70°C. Reverse transcription (RT) was performed using

RevertAid First Strand cDNA Synthesis Kit (ThermoFisher). In this aim, 1μg of total RNA was

combined with 200 ng of random hexamer primers and incubated at 65°C for 5 min, then the

mixture was cooled on ice and 4 µl of 5x Reaction Buffer, 2 µl of 10 mM dNTPs, 20 U/μl RiboLock

RNase Inhibitor and 200 U/μl of RevertAid M-MuLV Reverse Transcriptase (Thermo Scientific)

were added. The reaction was carried out in a total volume of 20 μl following the thermal profile:

25°C for 10 min, 60 min at 42°C, and terminated by heating at 70°C for 5 min. The obtained cDNA

samples were used as template for real-time PCR reactions.

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Real-time PCR

The effectiveness of treatments with BTH (1) and analyzed cationic derivative [BTHMe][Doc]

(7) on the expression levels of defense related genes - PR1a and PAL in relation to control (plants–

treated only with water) was determined by real-time PCR approach. The PR-1 gene was amplified

with specific primers designed previously.[E5] PR-1F 5′-

CATAACACAGCTCGTGCAGATGTAG-3′ and PR-1R 5′-

AACCACCTGAGTATAGTGTCCACAC-3′, the gene of defense-related PAL enzyme was

amplified with primers palF 5’AGAGGATCCGTTTCGTGAAG 3′, palR

5′TGAGGCTGCTGCTATTATGG 3′ ).[E6] For normalization, the level of the N. tabacum EF1a

gene transcription was used as a reference. For the EF1a amplification following specific primers

were used: EF1aF 5′ TGTGATGTTTTTGTTCGGTCTTTAA 3′, EF1R 5′

TCAAAAGAAAATGCAGACAGACTCA 3′.[E7] The real-time PCR reaction was carried out in

a Roche LightCycler 480 Real-Time PCR System and universal ‘‘FAST’’ cycling conditions,

which were as follows: for EF1a gene amplification (5 min 95˚C, 40 cycles of 15 s at 95˚C and 60

s at 60˚C) and for PR1a and PAL genes (5 min 95˚C, 40 cycles of 15 s at 95˚C, 20 s at 58˚C and

20 s at 72˚C) followed by the generation of a dissociation curve to determine the accuracy of the

reaction. The reaction contained 1x iTaq™ universal SYBR Green supermix (Biorad) with primers

at a final concentration of 0.5 μM each, 1 μl of cDNA obtained in the previous step, and water up

to 10μl of total volume. All reactions were performed for four biological replicates in each

conditions. An analysis and interpretation of the results was carried out using Relative Expression

Software Tool V2.0.13 (Qiagen) based on the ΔΔCt method.[E8]

S23

References for supplementary information

[E1] Lewandowski, P.; Kukawka, R.; Smiglak, M.; Pospieszny, H. Bifunctional quaternary

ammonium salts based on benzo[1,2,3]thiadiazole-7-carboxylate as plant systemic

acquired resistance inducers, New J. Chem. 2014, 38, 1372-1375.

[E2] Smiglak, M.; Kukawka, R.; Lewandowski, P.; Pospieszny, H. Cationic derivatives of the

plant resistance inducer benzo[1,2,3]thiadiazole-7-carbothioic acid S-methyl ester (BTH)

as bifunctional ionic liquids, Tetrahedron Lett. 2014, 55, 3565-3568.

[E3] OECD Paris, Partition Coefficient, 1981, Test Guideline 107, Decision of the Council

C(81) 30 final.

[E4] Cieniecka-Rosłonkiewicz, A.; Pernak, J.; Kubis-Feder, J.; Ramani, A.; Robertson, A.

Seddon, R. Synthesis, anti-microbial activities and anti-electrostatic properties of

phosphonium-based ionic liquids, Green Chem. 2005, 7, 855-862.

[E5] Kulye, M.; Liu, H.; Zhang, Y.; Zeng, H.; Yang, X.; Qiu, D. Hrip1, a novel protein elicitor

from necrotrophic fungus, Alternaria tenuissima, elicits cell death, expression of defence-

related genes and systemic acquired resistance in tobacco, Plant Cell Environ. 2002, 35,

2104–2120.

[E6] Chang, Y. H.; Yan, H. Z.; Liou, R. F. A novel elicitor protein from Phytophthora parasitica

induces plant basal immunity and systemic acquired resistance, Mol. Plant Pathol. 2015,

16, 123-136.

[E7] Lochman, J.; Mikes, V. Ergosterol treatment leads to the expression of a specific set of

defence-related genes in tobacco, Plant Mol. Biol. 2006, 62, 43–51.

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[E8] Pfaffl, M. W.; Horgan, G. W.; Dempfle, L. Relative expression software tool (REST©) for

group-wise comparison and statistical analysis of relative expression results in real-time

PCR, Nucleic Acids Res., 2002, 30, e36-e36.

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