Synthesis, Antiviral Activity, and Mechanisms of Purine Nucleoside

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Agricultural and Environmental Chemistry

Synthesis, antiviral activity, and mechanisms of purine nucleoside derivatives containing a sulfonamide moiety Fangcheng He, Jing Shi, Yanju Wang, Shaobo Wang, Jixiang Chen, Xiuhai Gan, Baoan Song, and De-Yu Hu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02681 • Publication Date (Web): 25 Jun 2019 Downloaded from pubs.acs.org on July 17, 2019

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Journal of Agricultural and Food Chemistry

Figure 1 Commercially available antiviral agents. 189x45mm (300 x 300 DPI)

ACS Paragon Plus Environment


Figure 2 Design ideas for target compounds. 149x54mm (300 x 300 DPI)


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Figure 3. Synthesis routes of compounds 1–25.



Figure 4. Compound 5 effects in tobacco leaves on Ca (A), Cb (B), and Ct (C). Straight sticks signify mean ± SD (n = 3).


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Figure 5. Compound 5 effects on tobacco leaves POD (A), PAL (B), and SOD (C) activity. Straight sticks signify mean ± SD (n= 3).



Figure 6. By RT-qPCR analysis related gene expression. Straight sticks signify mean ± SD (n= 3).


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Figure 7. Venn diagram shows that the proteome distribution between compound 5 and CK changes uniquely and shares proteins. 68x49mm (300 x 300 DPI)



Figure 8. Differentially expressed proteins were classified into molecular functions, biological processes, and cellular components between the control and test groups. 199x46mm (300 x 300 DPI)


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Figure 9. Differentially expressed proteins involved in photosynthetic pathway.



TOC 84x34mm (600 x 600 DPI)


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1

Synthesis, antiviral activity, and mechanisms of purine nucleoside

2

derivatives containing a sulfonamide moiety

3 4

Fangcheng He,† Jing Shi,† Yanju Wang,† Shaobo Wang,† Jixiang Chen,† Xiuhai Gan,†

5

Baoan Song,*,† Deyu Hu *, †

6

†

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Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering,

8

Ministry of Education, Guizhou University, Huaxi District, Guiyang 550025, China

9

*Corresponding author (Tel.:86-851-88292170; Fax: 86-851-88292170; E-mail:

10

State Key Laboratory Breeding Base of Green Pesticide and Agricultural

[email protected]; [email protected]

11 12 13 14 15 16 17 18 19 20 21 22 23 24

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ABSTRACT: Novel purine nucleoside derivatives containing a sulfonamide moiety

26

were prepared, as well as their antiviral activities against potato virus Y (PVY),

27

cucumber mosaic virus (CMV), and tobacco mosaic virus (TMV) were evaluated. The

28

antiviral mechanisms of the compounds were investigated. Results showed that most

29

of the compounds had good antiviral activities. Compound 5 at 500 μg/mL exhibited

30

excellent curative and protective activities of 52.5%, 60.0% and 52.0%, 60.2% to

31

PVY and CMV, respectively, which are higher than those of ningnanmycin (48.1%,

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49.6% and 45.3%, 47.7%), ribavirin (38.3%, 48.2% and 40.8%, 45.5%), and chitosan

33

oligosaccharide (32.5%, 33.8% and 35.1%, 34.6%). Moreover, compound 5 displayed

34

good inactivating activity against TMV, with an EC50 value of 48.8 μg/mL, which is

35

better than that of ningnanmycin (84.7 μg/mL), ribavirin (150.4 μg/mL) and chitosan

36

oligosaccharide (521.3 μg/mL). The excellent antiviral activity of compound 5 is

37

related to its immune induction effect which can regulate the physiological and

38

biochemical processes in plants, including defense-related enzyme activities,

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defense-related genes, and photosynthesis-related proteins. These results indicate that

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purine nucleoside derivatives containing a sulfonamide moiety are worthy of further

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research and development as new antiviral agents.

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KEYWORDS: sulfonamide moiety, purine nucleoside, potato virus Y, cucumber

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mosaic virus, tobacco mosaic virus, immune induction effect

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INTRODUCTION

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Potato virus Y (PVY), cucumber mosaic virus (CMV) and tobacco mosaic virus

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(TMV) are widespread plant viruses that can cause serious plant diseases and huge

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economic losses to agricultural production.1−3 For instance, the annual loss rate of

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Chinese tomato infected by CMV is 25%–50%.4 Few chemical agents for controlling

53

these viruses are currently available because they are absolutely parasitic in plants and

54

lack of a complete immune system in metabolic. As widely used antiviral agents,

55

ribavirin and ningnanmycin (Figure 1) show unsatisfactory field control effects.5−7 In

56

addition, ningnanmycin is not widely used in field trials because its photosensitivity

57

and water viscosity8 render the spread of viruses difficult to control effectively in the

58

wild.9−11 Chitosan oligosaccharide (COS, Figure 1) is an environment-friendly

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biological regulator that can induce plant immunity to viruses. However, it is less

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effective against plant virus diseases.12 Therefore, the development of high-efficiency,

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low-toxic, and environment-friendly antiviral drugs are the main task of current

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pesticide research.

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Figure 1

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Sulfonamides have wide-ranging biological activities, such as insecticidal,13

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antifungal,14 anti-HIV,15 influenza,16 and anti-cancer.17 Sulfonamides also exhibit

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good anti-plant virus activities.18,

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pharmacologists and chemists and have become an important research topic in

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medicine and pesticide chemistry. Purine is a significant endogenous biological

69

substance that plays an important role in the health of humans and plants. Natural

19

Therefore, they have attracted the attention of

3



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purine and its derivatives have always been valuable sources for lead discovery in

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medicinal and agricultural chemistry because of their novel scaffolds. Some purine

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drugs have been extensively used in medicine as antiviral agents,20 such as

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ganciclovir21 and abacavir.22 Purine derivatives also demonstrate good activities in

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regulating plant growth23, 24 and anti-plant virus.25, 26

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In our previous work, we found that benzenesulfonamide chalcone derivatives

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containing purine moiety (Figure 2) showed moderate antiviral activity and could be

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considered as a scaffold for the discovery of novel antiviral agents.27 However, these

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compounds lack satisfactory and broad-spectrum antiviral activities. Structural

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analysis revealed that ribavirin, ningnanmycin, and COS contain a sugar ring moiety

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with good water solubility. In the present study, purine in purine derivatives

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containing a sulfonamide was replaced with purine nucleoside (Figure 2) to obtain a

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series of novel purine nucleoside derivatives containing a sulfonamide moiety with

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improved water solubility. The antiviral activities of the derivatives were

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systematically tested. In addition, the plant defense response mechanism of compound

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5 was preliminarily studied by determining chlorophyll content, defense enzyme

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activity, and differential protein expression. Figure 2

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MATERIALS AND METHODS Chemicals. Analytical pure reagents without further drying and purification were used in the experiment. Instruments. The melting points of the target products were measured using a

4


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WRX-4 micro melting point apparatus (Shanghai Yice Apparatus & Equipment Co.,

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Ltd., China). 1H NMR and

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Ascend-400 spectrometer (Bruker, Germany) and a JEOL ECX-500 spectrometer

95

(JEOL, Tokyo, Japan) using DMSO-d6 as the solvent. High-resolution mass

96

spectrometry (HRMS) data were confirmed with a Thermo Scientific Q Exactive

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(Thermo, USA).

98 99

13C

NMR spectra were obtained on a Brookfield

Preparation of 2-(6-((4-aminophenyl)amino)-9H-purin-9-yl)-5-(hydroxymethyl) tetrahydrofuran-3,4-diol (A)

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In accordance with a previously reported method,28 triethylamine (7.67 mmol) was

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added to an ethanol solution of 6-chloropurine nucleoside (6.98 mmol) and

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p-phenylenediamine (20.93 mmol), and the reaction system was heated to 78 °C for 9

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h. Intermediate A was obtained, and its characterization data are as follows.

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Intermediate A. Yield 98%; gray solid; m.p. 178 °C–180 °C; 1H NMR (500 MHz,

105

DMSO-d6) δ 9.53 (s, 1H Ph-NH), 8.49 (s, 1H, Pu-8’H), 8.31 (s, 1H, Pu-2’H), 7.48 (d,

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J = 8.7 Hz, 2H, Ph-H), 6.60 (d, J = 8.6 Hz, 2H, Ph-H), 5.97 (d, J = 6.0 Hz, 1H,

107

ribose-2’CH), 5.52 – 5.40 (m, 2H, ribose 5’-CH2-), 5.24 (d, J = 4.6 Hz, 1H, ribose

108

3’-CH), 4.93 (s, 2H, Ph-NH2), 4.68 (q, J = 5.9 Hz, 1H, ribose 4’-CH), 4.21 (td, J = 4.7,

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2.8 Hz, 1H, ribose 4’-OH), 4.03 (d, J = 3.4 Hz, 1H, ribose 5’-CH), 3.74 (dt, J = 12.1,

110

4.2 Hz, 1H, ribose 3’-OH), 3.62 (ddd, J = 11.8, 7.3, 3.8 Hz, 1H, ribose 5’-OH).

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NMR (125 MHz, DMSO-d6) δ 152.94, 152.49, 149.19, 145.30, 140.47, 128.54,

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123.65, 120.35, 114.06, 88.34, 86.29, 73.92, 71.03, 62.04.

113

General methods for the synthesis of compounds 1–25.

5


13C


114

A reaction mixture of intermediate A (1.0 mmol) and triethylamine (1.1 mmol) in 5

115

mL of N, N-dimethylformamide (DMF) and 15 mL of dichloromethane (DCM) was

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stirred at 0 °C for 1 h. Then, different sulfonyl chlorides (1.3 mmol) were added at 0

117

°C and stirred for 4 h. The reaction mixture was washed with saturated saline and then

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extracted with 20 mL of dichloromethane for three times. The organic layer was

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concentrated in a vacuum to obtain a colorless liquid, which was then washed with

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saturated brine and extracted with 20 mL of ethyl acetate for three times. Ethyl acetate

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was removed, and compounds 1–25 were obtained by recrystallization with

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dichloromethane. The representative data for compound 1 is shown below.

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N-(4-((9-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)

124

amino)phenyl)-4-methylbenzenesulfonamide (1). Yield 62%; gray solid; m.p. 184

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°C–186 °C; 1H NMR (500 MHz, DMSO-d6) δ 10.15 (s, 1H, -SO2-NH-), 9.89 (s, 1H,

126

Pu-8’H), 8.55 (s, 1H, Ph-NH), 8.37 (s, 1H, Pu-2’H), 7.79 (d, J = 8.7 Hz, 2H, Ph-H),

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7.68 (d, J = 8.0 Hz, 2H, Ph-H), 7.36 (d, J = 7.8 Hz, 2H, Ph-H), 7.08 (d, J = 8.5 Hz, 2H,

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Ph-H), 5.98 (d, J = 5.9 Hz, 1H, nucleoside 2’-CH), 5.56 (d, J = 5.8 Hz, 1H, nucleoside

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3’-CH), 5.36–5.29 (m, 2H, nucleoside 5’-CH2-), 4.65 (d, J = 5.9 Hz, 1H, nucleoside

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5’-CH), 4.22 (d, J = 5.0 Hz, 1H, nucleoside 3’-OH), 4.02 (d, J = 5.0 Hz, 1H,

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nucleoside 4’-CH), 3.75–3.67 (m, 1H, nucleoside 4’-OH), 3.60 (dd, J = 12.1, 6.4 Hz,

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1H, nucleoside 5’-OH), 2.35 (s, 3H, Ph-CH3).

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152.43, 149.82, 143.58, 141.17, 137.33, 136.59, 133.02, 130.15, 127.32, 122.13,

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121.67, 120.72, 88.34, 86.39, 74.22, 71.09, 62.06, 21.50. HRMS (ESI) calcd. for

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C23H24N6O6S ([M+H]+), cacld. 513.14780, found. 513.15411.

13C

NMR (125 MHz, DMSO-d6) δ

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Antiviral activity assay. Nicotiana tabacum cv. K326 leaves infected with CMV,

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PVY, and TMV were selected and purified to obtain CMV, PVY, and TMV in

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accordance with previously reported methods.29,

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compounds against PVY, CMV, and TMV were evaluated in vivo as previously

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described,28, 30 ningnanmycin, ribavirin, and COS are used as controls.

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The antiviral activities of title

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Physiological and biochemical determination. Experimental processing and

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sampling. Tobacco at the sixth leaf stage was selected and treated with COS (control

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drug), Compound 5, and CK (blank control). A 500 μg/mL solution of compound 5

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was uniformly sprayed on the entire blade. The blade was infected CMV after

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spraying for 12 h and fostered in a hothouse at 25 °C. Afterwards, 1-, 3-, 5-, and

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7-day tobacco tissue samples were gathered and used to test and calculate chlorophyll

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content and defense enzyme activity. All tests were repeated three times.

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Determination of chlorophyll content. Chlorophyll content was measured using

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chlorophyll assay kits (Suzhou Comin Bioengineering Institute, China) in accordance

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with the manufacturer’s instructions. The absorbance spectra of chlorophyll a (Ca),

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chlorophyll b (Cb), and total chlorophyll content (Ct) at 663 and 645 nm were

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determined.

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Defensive enzyme activity determination. The activities of superoxide dismutase

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(SOD), phenylalanine ammonia lyase (PAL), and peroxidase (POD) were calculated

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using enzyme assay kits in accordance with the manufacturer’s instructions (Corning

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Bioengineering Institute, Suzhou, China).

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Gene expression analysis. Total RNA of tobacco was extracted by a Trizol kit

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(TakaRa, Dalian, China), followed by reverse transcription using a cDNA kit

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(TakaRa). Experiments were performed using an iCycleriQ multicolor real-time PCR

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detection system (Bio-Rad, California, CA, USA) and SYBR Premix Ex TaqII

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(TakaRa) with a response bulk of 10 μL. The relative copy number of the gene was

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counted by the 2-ΔΔCt method.31 β-actin was used as an internal standard. The relative

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copy number of the gene was counted in accordance with a previously described

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method.12 The measurements were tested in triplicate.

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Analysis of differentially expressed proteins. Total protein extraction. Whole

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proteins of tobacco were extracted as previously described.32 Leaf samples (1.0 g)

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from different experimental conditions were rapidly ground with liquid nitrogen to

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homogenize at room temperature. Then, ice-cold extraction buffer (5 mL) containing

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0.1 M KCl, 0.7 M sucrose, 0.5 M Tris-HCl, 50 mM ethylenediaminetetraacetic acid,

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and 40 mM dithiothreitol, pH 7.5 was added. After shaking for 20 min, Tris-HCl (pH

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7.5) saturated phenol was added, and shaking was continued for 30 min at 4 °C.

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After centrifugation at 4500 rpm for 20 min, the upper phenol layer was collected

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with a fresh pipe, and the temperature was maintained at -20 °C before adding 100

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mM ammonium acetate methanol. After centrifugation again at 4500 rpm for 20 min

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at 4 °C, the deposition was gathered and washed three times with 80% precooling

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acetone under the same conditions. The precipitate was dried in a vacuum desiccator,

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and a rehydration solution (1000 μL, pH 8.5) containing 8 M urea, 0.1 M Tris, and

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10 mM DTT was added at 37 °C. The total protein concentration was then

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determined. Iodoacetamide (55 mM) was added to collect 100 μg of the protein

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solution, and the mixture was incubated in the dark for 1 h at room temperature. The

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mixture was then centrifuged with 3 kDa Millipore at 12,000 rpm for 20 min at 4 °C.

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The coarse protein was washed with a diluent rehydration solution for six times.

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Then, 12.5 μg of trypsin digest was added, and the mixture was incubated at 37 °C

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for 16 h. The mixture was centrifuged at 12,000 rpm for 20 min at 4 °C, and the

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peptide solution was collected. After air-drying, 10 μL of 0.1% formic acid solution

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was added, and the peptide was analyzed by chromatography-tandem mass

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spectrometry (LC-MS/MS).

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Identification of proteins. Peptide samples were analyzed using a triple

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time-of-flight (TOF) 5600 mass (AB SCIEX, Foster City, CA, USA) in conjunction

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with the Nano LC-1 DTM plus system (Eksigent, Dublin, CA, USA). In addition, 8

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μL of the peptide sample was obtained using full-ring injection and desalted on a

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ChromXP Trap column (Nano LC TRAP column, 3 μm C18-CL, 120 Å, 350 μm×0.5

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mm, Foster City, CA, USA). A Nano LC-C18 reverse phase column (3C18-CL, 75

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μm×15 cm, Foster City, CA, USA) from mobile phase A (5% CAN, 0.1% FA) and

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mobile phase B (95% ACN, 0.1% FA) was used. The resulting linear gradient eluted

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each sample to the second analytical column at a flow rate of 300 nL/min for 120

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min. In data-dependent mode, the Triple TOF 5600 MS was operated, and between

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TOF-MS and product ion collection using Analyst (R) software (TF1.6) (AB SCIEX,

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Foster City, CA, USA) automatically switched. Elution was performed using

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β-galactosidase for 10 min and then identified for 30 min to adjust each two

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specimens.

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Analysis for proteomics data. LC-MS/MS data were analyzed by MaxQuant

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version 1.5.2.8 (Max Planck Institute, Munich, Germany).33 Data containing tobacco

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and CMV proteome data libraries downloaded from UniProt were searched. Variable

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modifications involving N-terminal acetylation, methionine oxidation, and

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immobilization of carbamoylmethylcysteine were also searched. The initial search

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parameters include a range of 20 ppm original mass tolerance and set quality

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recalibration.34 The iBAQ arithmetic was used to quantify proteins and classify for a

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single sample on the absolute abundance of DEP under the mistake check rate to

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0.01 for the recognition peptide.35 Difference in protein expression between two

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groups was compared using the normalized method. The eliminating identifications

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reverse database and common contaminants were used to filter protein tables. The

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treatment and control groups showed different protein accumulation as identified by

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performing an unpaired t-test two between sets of iBAQ information.

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Bioinformatics analysis. Annotation of differentially expressed proteins were

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dissected using Gene Ontology (GO) on the Kyoto Encyclopedia of Genes and

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Genomes (KEGG) and classified using Uniprot software. Projects in GO were found

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to be associated with biological processes (BP), cellular components (CC), and

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molecular functions (MF).36 For target lists with unlabeled proteomics results, the

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GO database was downloaded to generate a background list. Differentially expressed

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proteins

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(http://www.geneontology.org/), and the protein content of each GO term was

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calculated.

(>

1.5

expression

levels)

were

marked

10


to

the

GO

database

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RESULTS AND DISCUSSION

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Chemistry. As shown in Figure 3, ethanol was used as a solvent and triethylamine

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was used as a catalyst, 6-chloropurine nucleoside and p-phenylenediamine were used

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as raw materials and stirred under reflux for 10–12 h to obtain intermediate A. Then,

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intermediate A (1.0 mmol) was dissolved with 5 mL of DMF, and 15 mL of DCM

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triethylamine (1.1 mmol) was added and stirred at 0 °C. Then, different sulfonyl

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chlorides (1.3 mmol) were added and stirred for 4 h, and the target compounds were

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obtained after sulfonamide. Their structures were identified by 1H NMR,

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and HRMS (Supporting Information I).

13C

NMR,

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Antiviral activity. Anti-PVY activity in vivo. Table 1 shows the in vivo anti-PVY

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activities of compounds 1–25. Some target compounds exhibit significant anti-PVY

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activities at a concentration of 500 μg/mL. The curative activities of compounds 1, 2,

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5–7, 17, and 19 are 67.0%, 66.2%, 52.5%, 54.4%, 54.6%, 54.4%, and 53.7%,

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respectively, which are significantly higher than those of ningnanmycin (48.1%),

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ribavirin (38.3%), and COS (32.5%). The protective activities of compounds 5–7, 16,

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18, and 23 are 60.0%, 53.1%, 52.7%, 53.5%, 55.3%, and 52.5%, respectively, which

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are better than those of ningnanmycin (49.6%), ribavirin (48.2%), and COS (33.8%).

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Anti-CMV activity in vivo. As shown in Table 1, most of the compounds exert

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effective inhibitory effects. The curative activities of compounds 2, 3, 5, 11, 12,

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14–16, 18, and 23 are 50.0%, 53.2%, 52.0%, 51.6%, 54.4%, 50.9%, 59.5%, 55.5%,

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61.7%, and 53.3%, respectively, which are remarkably better than those of

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ningnanmycin (45.3%), ribavirin (40.8%), and COS (35.1%). The protective activities

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of compounds 1–5, 7, and 11 are 54.0%, 51.9%, 51.4%, 55.1%, 60.2%, 52.2%, and

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49.2%, respectively, which are more effective than those of ningnanmycin (47.7%),

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ribavirin (45.5%), and COS (34.6%). The EC50 values of these compounds are 363.7,

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382.0, 425.9, 349.7, 237.5, 412.8, and 490.0 μg/mL, respectively, which are better

250

than those of ningnanmycin (511.5 μg/mL), ribavirin (584.3 μg/mL), and COS (550.8

251

μg/mL).

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Anti-TMV activity in vivo. As shown in Table 2, the curative activities of

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compounds 2–5, 7 and 24 against TMV are 61.4%, 60.0%, 56.5%, 53.5%, 55.3%, and

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64.9%, respectively, which are similar to that of ningnanmycin (57.1%) and

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significantly superior to those of COS (34.6%) and ribavirin (41.7%). The protective

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activities of compounds 1, 4, 5, 7, 9, and 17 (63.6%, 60.1%, 52.9%, 65.2%, 62.8%,

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and 60.5%, respectively) against TMV are similar to that of ningnanmycin (57.1%)

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and significantly superior to those of COS (36.5%) and ribavirin (51.3%). The

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inactivating activities of compounds 4 and 5 against TMV (83.0% and 85.8%) are

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similar to that of ningnanmycin (82.6%) and better than those of COS (49.3%) and

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ribavirin (70.0%), with EC50 values of 67.3 and 48.8 μg/mL, respectively, which are

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preferred over those of ningnanmycin (84.7 μg/mL), ribavirin (150.4 μg/mL), and

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COS (521.3 μg/mL).

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SARs. In the purine nucleoside derivatives containing a sulfonamide moiety,

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when substituent on the phenyl ring is at the 4-position the curative activity of

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anti-TMV the electron supply group is superior to the electron-withdrawing group.

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For example, 2> 3 > 4 > 6 > 19 (4-butly-Ph > 4-Br-Ph > 4-Cl-Ph > 4-F-Ph >

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4-NO2-Ph). At the same time, the same rule exists in the anti-CMV protective activity

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2 > 3 > 6 > 19 (4-butly-Ph > 4-Br-Ph > 4-F-Ph > 4-NO2-Ph) except for compound 4

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(4-Cl-Ph). When the substituent are the same but the positions are different, the

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activity is different, such as 17 > 10 > 18 (2-position > 4-position > 3-position) in

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anti-TMV curative, protective activity and anti-PVY protective activity, 7 > 1 > 12

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(2-position>4-position>3-position) in anti-TMV protective activity and anti-PVY

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protective activity.

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Biochemical and physiological analyses. Analysis of chlorophyll contents.

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Chlorophyll is an important component of chloroplasts for photosynthesis. The

277

contents of chlorophyll a (Ca), chlorophyll b (Cb), and total chlorophyll (Ct) were

278

tested in this study. In the blank control group, Ca (Figure 4A), Cb (Figure 4B), and Ct

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(Figure 4C) contents decreased with time because of CMV infection. After treatment

280

with compound 5, the chlorophyll content of the leaves was significantly higher than

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that of the blank control group and the COS treatment group. Compound 5 can

282

significantly increase tobacco chlorophyll content and promote photosynthesis on

283

days 3, 5, and 7.

284

Influences of defense enzyme activity. Some major defense enzymes, such as POD,

285

PAL, and SOD, can significantly enhance the plant's own defense activity and induce

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plant disease resistance. The influence of compound 5 treatment on the defense

287

enzyme activity of tobacco was analyzed. Results showed that compound 5 can

288

increase the activities of POD, PAL, and SOD in tobacco and exerts the greatest

289

influence on POD activity (Figure 5A). The compound 5 treatment group only has the

13



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adjusted value on the first day, lower than that of the CK group. On days 3, 5, and 7,

291

activity values of 4286.97, 2629.36, and 4262.91 U/mg ptot are measured in the

292

compound 5 treatment group, respectively, which are 4.20, 2.96, and 5.70 times

293

higher than that of CK, respectively. This result indicates that compound 5 can

294

significantly increase the activity of POD. Moreover, compound 5 significantly

295

regulates the activity of PAL (Figure 5B) compared with the control group. On the

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first day, the activity value is 5.09 U/mg ptot, which is 1.27 times of CK. The effect

297

on the activity of SOD (Figure 5C) was compared with that of CK after spraying with

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compound 5. The activity of the compound 5 treatment group is higher than that of

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the CK group at days 1 and 7. The activity data are 14.46 and 9.59 U/mg ptot,

300

respectively, which are 1.32 and 1.31 times higher than that of CK. The results show

301

that compound 5 can enhance the defense enzyme activities, thereby enhancing the

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disease resistance of tobacco plants.

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Analysis of RT-qPCR. The sample at day 5 was used as a representative, and the

304

relative copy number of the gene was tested by RT-qPCR. Table 3 shows the primer

305

sequence information. As shown in Figure 6, the expression levels of ICS-1 and SOD

306

are 1.57 and 4.92 times higher in the compound 5 treatment group than in the CK

307

group, and the expression levels of PAL-4, PR-1, and NPR-1 are 2.50, 1.62, and 3.62

308

times higher in the compound 5 treatment group than in the COS treatment group,

309

respectively. The difference between SOD and NPR1 is significant, whereas other

310

genes are upregulated, but the differences are not significant.

311

Analysis of Proteomics As shown in Figure 7, 2286 proteins were found and

14


Page 24 of 38

Page 25 of 38


312

quantified by searching for peptide results using version 1.5.2.8 MaxQuant (Max

313

Planck Institute, Munich, Germany). Protein-specific expression of tobacco leaves in

314

the compound 5 treatment and CK groups were measured. A total of 2286 tobacco

315

proteins were identified, including 2009 proteins of the CK group and 1905 proteins

316

of the compound 5 treatment group. Among them, 103 proteins are up-regulated and

317

97 proteins are downregulated.

318

Analysis of Bioinformatics Integrated Discovery 6.8, Database for Annotation

319

Difference, and Visualization were used to annotate the expressed proteins. On the

320

basis of the GO category, differentially expressed proteins were analyzed and

321

classified into cell component (Figure 8A), molecular function (Figure 8B), and

322

biological process (Figure 8C) in the treatment and control groups.37 The differential

323

proteins induced by compound 5 were compared (p ≤ 0.05) by GOSlim analysis. On

324

the basis of the 5+CMV vs. CK+CMV differentially expressed proteins discrepancy,

325

the GO annotation map showed that the cell components are significantly enriched in

326

10 GO-terms after treatment with compound 5. The terms include a membrane, a

327

cytoplasm, a photosystem, a mitochondrion, a chloroplast, and so on. The molecular

328

functions are significantly enriched in 10 GO terms, including RNA binding and

329

DNA binding, protein binding, kinase activity, and signal transducer activity. The

330

differentially expressed proteins in biological processes are significantly enriched in 8

331

GO terms, including photosynthesis, defense response, response to stress, response to

332

oxidative stress, generation of precursor metabolites and energy, and lipid metabolism

333

biological process. The proteins involved are mainly about heat shock protein 70,

15



334

SOD, POD, and glutathione reductase. This result indicates that compound 5 can

335

significantly alter the physiological and biochemical characteristics of tobacco, such

336

as immune response and photosynthesis-related biological processes, from plant cell

337

components, key molecular functions, and photosynthetic immune response and

338

metabolism.

339

KEGG Classification. The role of compound 5 triggers the reaction of the study

340

with KEGG and determines the mode of action of compound 5. As shown in Figure 9

341

and Table 4, electrons transferred from cell fluid to NADP+ during photosynthesis are

342

regulated by four classes of protein components of Photosystem I (PSI), Photosystem

343

II (PSII), cytochrome (Cytob6f) complex, and ATP synthase. Compound 5

344

upregulates chloroplast-releasing oxygen regulatory proteins (PsbO, PsbO1, PsbP,

345

PsbP4, and PsbF) in PSII, promotes the production of HO by water decomposition,

346

and thus enhances the reaction of the PSII center. Among them, PsbO is a

347

manganese-stabilized protein that involves the transport and accumulation of

348

manganese in PSII. PsbO can also protect the functional proteins CP43 and CP47 of

349

plant protein hydrolysis to improve its stability. The above results indicate that after

350

treatment with compound 5, the function of activating host disease resistance is

351

finally realized from the regulation of chlorophyll content, related defense enzyme

352

activity, and protein content of photosynthetic physiological process, which is related

353

to chlorophyll content and photosynthesis test results.

354

In summary, 25 novel purine nucleoside derivatives containing a sulfonamide

355

moiety were designed and synthesized, and their antiviral activities were evaluated

16


Page 26 of 38

Page 27 of 38


356

systematically. Compound 5 shows excellent and broad-spectrum antiviral activity.

357

The excellent antiviral activity is due to the immune induction effect of compound 5,

358

including chlorophyll content, defense-related enzyme activities, defense-related

359

genes, and photosynthesis. This study provides a solid foundation for the research and

360

development of purine nucleoside derivatives containing a sulfonamide moiety as

361

novel antiviral agents.

362

CONTENT ASSOCIATED

363

Supporting Information

364

The 1H NMR and 13C NMR spectra of intermediate A and title compounds 1−25 are

365

shown in Supplementary Information I, and all identified proteins are shown in

366

Support Information II, including the differential protein expression in the treatment

367

group compared with the control group and GO term abundance analysis proteins.

368

AUTHOR INFORMATION

369

Corresponding Authors

370

*E-mail: [email protected].; [email protected].

371

*Phone: 86-851-88292170. Fax: 86-851-88292170.

372

ORCID

373

Baoan Song: 0000-0002-4237-6167

374

Deyu Hu: 0000-0001-7843-371X

375

Funding

376

We gratefully acknowledge the assistance from the National Key Research

377

Development Program of China (2018YFD0200100) and the National Natural

17



378

Science Foundation of China (21562013).

379

Notes

380

The authors declare no competing financial interest.

381

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382

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508

Information of FIGURE

509

Figure 1. Commercially available antiviral agents.

510

Figure 2. Design ideas for target compounds.

511

Figure 3. Synthesis routes of compounds 1–25.

512

Figure 4. Compound 5 effects in tobacco leaves on Ca (A), Cb (B), and Ct (C). Straight

513

sticks signify mean ± SD (n = 3).

514

Figure 5. Compound 5 effects on tobacco leaves POD (A), PAL (B), and SOD (C)

515

activity. Straight sticks signify mean ± SD (n= 3).

516

Figure 6. By RT-qPCR analysis related gene expression. Straight sticks signify mean

517

± SD (n= 3).

518

Figure 7. Venn diagram shows that the proteome distribution between compound 5

519

and CK changes uniquely and shares proteins.

520

Figure 8. Differentially expressed proteins were classified into molecular functions,

521

biological processes, and cellular components between the control and test groups.

522

Figure 9. Differentially expressed proteins involved in photosynthetic pathway.

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524

Table 1 Activities of target compounds against PVY and CMVa

525

CMV Curative Protective Curative Protective EC50 for activity (%) activity (%) activity activity protective (%) (%) activity (μg/mL) 1 67.0 ± 2.3 48.4± 2.4 44.8 ± 2.5 54.0 ± 1.8 363.7 ± 4.3 2 66.2 ± 3.8 32.6 ± 1.1 50.0 ± 2.3 51.9 ± 2.8 382.0 ± 3.1 3 45.5 ± 4.4 47.8 ± 3.4 53.2 ± 1.9 51.4 ± 3.3 425.9 ± 4.9 4 47.8 ± 1.0 39.5 ± 2.8 41.6 ± 1.9 55.1 ± 3.2 349.7 ± 7.0 5 52.5 ± 3.1 60.0± 3.1 52.0 ± 2.6 60.2 ± 2.9 237.5 ± 4.5 6 54.4 ± 2.6 53.1 ± 2.0 46.8 ± 2.7 39.3 ± 2.8 793.7 ± 5.4 7 54.6 ± 3.7 52.7 ± 2.3 43.4 ± 1.3 52.2 ± 2.1 412.8 ± 7.8 8 41.2 ± 2.3 44.8 ± 3.1 41.0± 1.4 31.1 ± 2.9 1555.8 ±13.1 9 33.3 ± 2.7 43.7 ± 4.2 38.5 ± 3.1 40.8 ± 4.1 849.6 ± 2.2 10 46.0 ± 2.5 48.4 ± 1.6 47.5 ± 2.7 48.4 ± 1.6 512.9 ± 8.8 11 42.6 ± 3.8 47.2 ± 3.1 51.6 ± 1.4 49.2 ± 1.0 490.0 ± 9.2 12 44.1 ± 2.5 44.3± 1.0 54.4 ± 3.1 43.9 ± 3.0 646.1 ± 4.3 13 48.0 ± 1.4 41.4± 3.3 44.2 ± 1.0 32.0 ± 2.8 1126.9 ± 7.5 14 51.0 ± 2.3 47.4 ± 1.9 50.9 ± 1.5 40.1 ± 1.0 835.1 ± 3.7 15 29.5 ± 3.5 27.7 ± 2.4 59.5 ± 3.1 20.6 ± 2.9 / 16 50.4 ± 1.2 53.5 ± 2.6 55.5 ± 3.1 35.2 ± 2.3 1264.7 ± 9.7 17 54.4 ± 3.2 25.4 ± 4.0 15.5 ± 1.0 29.7 ± 3.1 / 18 38.3 ± 1.0 55.3 ± 2.7 61.7 ± 1.0 42.0 ± 3.3 739.9 ± 4.2 19 53.7 ± 3.2 29.0 ± 3.0 47.1 ± 3.3 32.8 ± 2.5 1554.3 ± 7.6 20 45.8 ± 3.7 36.0 ± 1.9 30.3 ± 1.1 26.4 ± 1.3 / 21 49.9 ± 3.7 26.4 ± 3.4 45.9 ± 3.1 35.5 ± 2.8 1164.5 ± 10.3 22 51.2 ± 3.9 45.7 ± 2.7 40.7 ± 4.0 42.7 ± 2.9 689.5 ± 7.1 23 43.0 ± 3.0 52.5 ± 3.9 53.3 ± 1.9 45.2 ± 3.1 624.6 ± 8.2 24 38.6 ± 1.0 20.2 ± 3.4 48.6± 3.1 35.5 ± 3.9 1008.0 ± 4.4 25 50.9 ± 2.4 41.6 ± 3.1 43.5 ± 1.2 24.5 ± 2.5 / COS b 32.5 ± 1.2 33.8 ± 2.4 35.1± 1.3 34.6 ± 2.3 550.8 ± 8.3 Ribavirinc 38.3 ± 1.9 48.2 ± 2.1 40.8 ± 1.3 45.5 ± 3.0 584.3 ± 5.7 Ningnanmycin d 48.1 ± 1.0 49.6 ± 1.9 45.3 ± 1.6 47.7 ± 2.8 511.5 ± 3.7 aAverage of three replicates, bChitosan oligosaccharide, cRibavirin, and dNingnanmycin were used

526

as controls.

PVY

Compound

25



528

Page 36 of 38

Table 2 Anti-TMV activities of target compoundsa Compound

Curative activity (%)

Protective activity (%)

Inactivating activity (%)

EC50 of inactivating activity (μg/mL)

1

40.8 ± 2.6

63.6 ± 3.0

72.7 ± 1.2

149.8 ± 2.8

2

61.4 ± 1.0

47.2 ± 2.6

76.4 ± 3.9

141.8 ± 3.0

3

60.0 ± 1.1

57.6 ± 3.5

74.5 ± 3.7

147.4 ± 2.3

4

56.5 ± 3.3

60.1 ± 2.9

83.0 ± 2.2

67.3 ± 5.8

5

53.5 ± 2.6

52.9 ± 2.8

85.8 ± 2.1

48.8 ± 3.7

6

32.7 ± 1.1

50.3 ± 3.0

64.8 ± 0.4

203.0 ± 4.1

7

55.3 ± 3.0

65.2 ± 3.1

48.7 ± 2.5

521.7 ± 4.5

8

48.8 ± 1.3

59.0 ± 2.5

74.6 ± 3.3

143.3 ± 5.8

9

54.1 ± 1.4

62.8 ± 3.9

44.5 ± 1.7

651.2 ± 2.9

10

35.9 ± 0.7

52.5 ± 1.9

67.6 ± 0.6

179.5 ± 5.3

11

48.9 ± 2.7

57.8 ± 1.6

72.2 ± 3.8

121.9 ± 2.1

12

57.5 ± 3.1

51.8 ± 3.3

52.8 ± 2.7

392.8 ± 4.6

13

41.4 ± 2.9

54.3 ± 2.7

59.6 ± 2.5

255.7 ± 4.3

14

31.0 ± 1.3

55.2 ± 2.5

71.2 ± 1.7

126.9 ± 2.8

15

59.2 ± 3.7

56.4 ± 3.6

76.9 ± 2.7

109.2 ± 6.3

16

41.6 ± 3.7

57.5 ± 1.5

61.6 ± 2.9

267.0 ± 3.4

17

55.5 ± 1.9

60.5 ± 2.3

62.0 ± 2.6

237.7 ± 4.1

18

24.7 ± 2.4

30.8 ± 2.1

71.0 ± 1.0

146.4 ± 5.6

19

21.6 ± 3.6

55.6 ± 1.7

62.8 ± 2.6

238.9 ± 4.1

20

57.6 ± 1.9

38.3 ± 1.2

50.4 ± 2.6

456.8 ± 2.4

21

48.2 ± 2.2

31.9 ± 1.8

54.9 ± 2.4

359.6 ± 2.9

22

51.9 ± 2.6

29.8 ± 2.9

69.1 ± 2.8

171.9 ± 4.1

23

32.5 ± 3.9

57.3 ± 3.7

80.5 ± 1.6

81.5 ± 2.6

24

64.9 ± 3.1

46.7 ± 2.0

65.6 ± 2.9

194.5 ± 7.0

25

29.1 ± 1.9

54.5 ± 3.2

46.1 ± 2.7

585.3 ± 5.2

cosb

34.6 ± 2.1

36.5 ± 3.1

49.3 ± 2.7

521.3± 4.2

Ribavirinc

41.7 ± 2.5

51.3 ± 2.3

70.4 ± 2.2

150.4 ± 1.8

Ningnanmycind

57.4 ± 3.3

63.6 ± 1.6

82.6 ± 2.5

84.7 ± 5.8

529

aAverage

of three replicates, bChitosan oligosaccharide, cRibavirin, and dNingnanmycin were used

530

as controls.

26


Page 37 of 38

532


Table 3. Primer sequences of RT-qPCR. Gene Name

Forward Primer

Reverse Primer

NPR1 PR1

5′-GGCGAGGAGTCCGTTCTTTAA-3′ 5′-ATGGGATTTGTTCTCTTTTCACA-3′

5′-TCAACCAGGAATGCCACAGC-3′ 5′-TTAGTATGGACTTTCGCCTCT-3′

PAL4

5′-CTCGGCCCTCAGATCGAA-3′

5′-CCGAGTTGATCTCCCGTTCA-3′

ICS1

5′-CAGCGCTGGCCTTGGA-3′

5′-GGAGGTGGGTTGGATTTCAA-3′

EDS1

5′-GGCTCGAGTATGCCCTGAAG-3′

5′-CTTGCCCAGAAACATGATTCC-3′

SOD

5′-CGACACTAACTTTGGCTCCCTAGA-3′

β-actin

5′-AGGGTTTGCTGGAGATGATG-3′

5′-GGTTCCTCTTCTGGGAATAGACGT3′ 5′-CGGGTTAAGAGGTGCTTCAG-3′

533 534 535

NPR1: Nonexpressor of rathogenesis-related genes; PR1: pathogenesis-related genes 1; PAL4: phenylalanine ammonia lyase 4; ICS1: isochorismate synthase 1; EDS1: enhanced disease susceptibility 1; SOD: superoxide dismutase.

27



537

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Table 4. Differentially expressed proteins involved in photosynthetic pathway ID

Protein name

Gene name

Regulated

P27496 A0A1L2BNP0 I0B7J8 Q84QE8 A0A1S4CFG3 A0A1S4CFV4 A0A1S3ZIT1 A0A1S4AFD5 A0A1S3ZVN0 A0A1S4AQ72 A0A1S4AA25 A0A1L2BNL5 A0A1L2BNM5 O24136

Chlorophyll a-b binding protein 50 Cytochrome b559 subunit beta Chloroplast PsbP4 Oxygen evolving complex 33 kDa photosystem II protein Chloroplast PsbO1 Chloroplast photosynthetic oxygen-evolving protein 33 kDa subunit psbP domain-containing protein 4 Photosystem II reaction center Psb28 protein photosystem II D1 precursor processing protein PSB27-H2 psbP domain-containing protein 1 ATP synthase delta chain Photosystem I P700 chlorophyll a apoprotein A1 Cytochrome f CP12 (Chloroplast protein 12)

CAB50 psbF psbP4 PsbO psbO1 psbO psbP psb28 psb27 psbP atpF-delta psaA petA CP12

up up up up up up up up up up up down down down

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Synthesis, Antiviral Activity, and Mechanisms of Purine Nucleoside

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