Design, Synthesis, Antiviral Bioactivity, and Defense Mechanisms of Novel Dithioacetal Derivatives Bearing a Strobilurin Moiety

Article abstract of DOI:10.1021/acs.jafc.8b01297

A series of dithioacetal derivatives bearing a strobilurin moiety were designed and synthesized on the basis of our previous work. The antiviral activities of these compounds against Potato virus Y (PVY), Cucumber mosaic virus (CMV), and Tobacco mosaic virus (TMV) were systematically evaluated. Bioassay results indicated that C14 elicited excellent curative and protective activities against PVY, CMV, and TMV. The former had 50% effective concentrations (EC₅₀) of 125.3, 108.9, and 181.7 μg/mL, respectively, and the latter had 148.4, 113.2, and 214.6 μg/mL, respectively, which were significantly superior to those of lead compound 6f (297.6, 259.6, and 582.4 μg/mL and 281.5, 244.3, and 546.3 μg/mL, respectively), Ningnanmycin (440.5, 549.1, and 373.8 μg/mL and 425.3, 513.3, and 242.7 μg/mL, respectively), Chitosan oligosaccharide (553.4, 582.8, and 513.8 μg/mL and 547.3, 570.6, and 507.9 μg/mL, respectively), and Ribavirin (677.4, 690.3, and 686.5 μg/mL and 652.7, 665.4, and 653.4 μg/mL, respectively). Moreover, defensive enzyme activities and RT-qPCR analysis demonstrated that the antiviral activity was associated with the changes of SOD, CAT, and POD activities in tobacco, which was proved by the related proteins of abscisic acid signaling pathway. This work provided a basis for further design, structural modification, and development of dithioacetal derivatives as new antiviral agents.

Full text of DOI:10.1021/acs.jafc.8b01297

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Agricultural and Environmental Chemistry
Design, Synthesis, Antiviral Bioactivity and Defense Mechanisms
of Novel Dithioacetal Derivatives Bearing a Strobilurin Moiety
Jin Chen, Jing Shi, Lu Yu, Dengyue Liu, Xiuhai Gan, Song Baoan, and De-Yu Hu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01297 • Publication Date (Web): 09 May 2018
Downloaded from http://pubs.acs.org on May 10, 2018
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Figure 1 Chemical structures of Chitosan oligosaccharide, Ribavirin, and Ningnanmycin.
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Figure 2. Design of the title compounds.
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Figure 3 Synthetic route of dithioacetal derivatives bearing strobilurin moiety.
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Figure 4 Effects of compound C14 on Ca (A), Cb (B), Ct (C), and Chlorophyll a/b (D) in tobacco leaves.
Vertical bars refer to mean ± SD (n = 3).
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Figure 5. Effects of compound C14 on CAT (A), POD (B), SOD (C), and PAL (D) activity in tobacco leaves.
Vertical bars refer to mean ± SD (n = 3).
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Figure 6. Gene expression analysis of the related genes by RT-qPCR. The genes expression levels of CAT-1,
ICS-1, and SOD were up-regulated by C14 treatment.
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Figure 8. Volcano plot of the relative protein abundance changes between control group and treatment
group. The red points are significant up-regulated proteins, while the green points are significant down-
regulated protein.
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Figure 9. The differential expression proteins between control and treatment group were classified based on
known cellular components (A), biological process (B) and molecular functions (C).
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Figure 10. The ABA signaling pathway in tobacco response to C14. Red color represent up-accumulated
proteins in this pathway, while the green color represent down-accumulated
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Design, Synthesis, Antiviral Bioactivity and Defense Mechanisms of
Novel Dithioacetal Derivatives Bearing a Strobilurin Moiety
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Jin Chen, Jing Shi, Lu Yu, Dengyue Liu, Xiuhai Gan, Baoan Song,* Deyu Hu*
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State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering,
Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Guizhou University, Huaxi District, Guiyang 550025, China
*Corresponding author (Tel.: 86ꢀ851ꢀ88292170; Fax: 86ꢀ851ꢀ88292170; Eꢀmail:
dyhu@gzu.edu.cn; songbaoan22@yahoo.com)
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ABSTRACT: A series of dithioacetal derivatives bearing a strobilurin moiety were
designed and synthesized on the basis of our previous work. The antiviral activities of
these compounds against Potato virus Y (PVY), Cucumber mosaic virus (CMV), and
Tobacco mosaic virus (TMV) were systematically evaluated. Bioassay results indicated
that C14 elicited excellent curative and protective activities against PVY, CMV, and TMV.
The former had 50% effective concentrations (EC ) of 125.3, 108.9, and 181.7 ꢁg/mL,
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respectively, and the latter had 148.4, 113.2, and 214.6 ꢁg/mL, respectively, which were
significantly superior to those of lead compound 6f (297.6, 259.6, and 582.4 ꢁg/mL and
281.5, 244.3, and 546.3 ꢁg/mL, respectively), Ningnanmycin (440.5, 549.1, and 373.8
ꢁg/mL and 425.3, 513.3, and 242.7 ꢁg/mL, respectively), Chitosan oligosaccharide
(553.4, 582.8, and 513.8 ꢁg/mL and 547.3, 570.6, and 507.9 ꢁg/mL, respectively), and
ribavirin (677.4, 690.3, and 686.5 ꢁg/mL and 652.7, 665.4, and 653.4 ꢁg/mL,
respectively). Moreover, defensive enzyme activities and RTꢀqPCR analysis demonstrated
that the antiviral activity was associated with the changes of SOD, CAT, and POD activity
in tobacco, which was proved by the related proteins of abscisic acid signaling pathway.
This work provided a basis for further design, structural modification, and development
of dithioacetal derivatives as new antiviral agents.
KEYWORDS: dithioacetal derivatives, strobilurin moiety, antiviral activity, potato virus
Y, cucumber mosaic virus, tobacco mosaic virus, plant resistance
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INTRODUCTION
Vegetables, which constitute an important food component for humans, are susceptible to
infection of various plant viruses, such as Potato virus Y (PVY), Cucumber mosaic virus
(CMV), and Tobacco mosaic virus (TMV), and Tomato chlorosis virus (ToCV). These
plant viruses are pathogenic and can infect almost all vegetables, including pepper,
tomato, eggplant, and cucumber. Vegetable viral diseases can cause massive economic
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losses because they are difficult to control. The annual losses of tomato infected with
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CMV in China account for 25%–50%. Chemical methods are the main solutions to
control vegetable viral diseases. Ningnanmycin and Ribavirin (Figure 1) are typically
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used to control vegetable viral disease but have unsatisfactory control effect. Chitosan
oligosaccharide (COS; Figure 1) is an biological regulator with environmentally friendly
that can induce plant immunity, but its control effect on vegetable viral diseases is not
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ideal. Considerable efforts have been devoted to developing new and effective plant
virucides, but their agricultural use is restricted because of an unsatisfactory antiviral
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activity.
Therefore, new antiviral agents with increased efficiency are needed to be
developed.
Dithioacetal and its derivatives have been extensively pharmacologically investigated
because of their extensive biological activities, such as antibacterial, antileishmanial,
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antiviral, and antifungal. In our previous work, the novel dithioacetal derivative 6f
(Figure 2) exert markedly increased curative and protective activities against and PVY
CMV. However, the compound exhibits moderate or even decreased activity against TMV.
Structure–activity relationships (SARs) analysis have revealed that the benzyl
etherification of 4ꢀOH was beneficial to increase the antiviral activities and halogen atom
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substituted benzyl ether compounds exhibited better antiviral activities than others,
especially, chlorine atomꢀsubstituted benzyl ether. About different substitutions of
disulfide on dithioacetal, aryl group compound is more suitable for antiviral activities
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than the alkyl group. Therefore, the introduction of a unique pharmacophore through
benzyl etherification can be used to develop antiviral agents based on lead compound 6f
with increased effectiveness and spectrum width.
Strobilurins are important fungicidal compounds containing βꢀmethoxyacrylate
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pharmacophore (Figure 2). Certain strobilurin fungicides have been commercialized
because of their excellent characteristics, such as unique action mechanism, wide
fungicidal spectrum, low toxicity toward mammalian cells, and environmental
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nonreactiveness. Strobilurins exhibit other biological properties, including improved
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crop physiology, insecticidal, acaricidal, and herbicidal effects, and induced
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resistance. Strobilurin as an essential pharmacophore, the modification of strobilurin
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side chains is an effective approach to obtain new highly active analogs.
We proposed to introduce the increasedꢀspectrum bioactive strobilurin group to
dithioacetal to obtain a series of novel dithioacetal derivatives bearing a strobilurin
moiety (Figure 2) and to further investigate the antiviral activities of dithioacetal
derivatives. The antiviral activities of these derivatives against PVY, CMV, and TMV
were systematically evaluated. Bioassay results showed that several title compounds
showed more fullꢀscale and better antiviral activity than commercially available
Ningnanmycin, COS, and ribavirin. Compared with lead compounds 6f, compound C14
not only has better antiviral activity against PVY and CMV, but also can be used to
control TMV. Moreover, the plant defense response mechanisms of C14, including
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chlorophyll content, enzyme activities, and differential protein expression were
researched. For all as our knowledge, this work first demonstrated that C14 could
enhance resistance of tobacco to virus. Thus, C14 could be considered as a novel plant
virucide.
MATERIALS AND METHODS
General Information. All of the reagents were purchased from commercial suppliers
and used without further purification. All of the solvents were used without further
purification and drying before use. Thinꢀlayer chromatography with UV detection was
conducted on silica gel GF . The melting points of the products were determined with a
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WRXꢀ4 microscopic melting point meter (Shanghai Yice Apparatus & Equipment Co.,
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Ltd., China) with an uncorrected thermometer. H, C, and F nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker Ascendꢀ400 spectrometer (Bruker,
Germany) and JEOL ECXꢀ500 spectrometer (JEOL, Tokyo, Japan) in CDCl or DMSOꢀ
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d6 solution with tetramethylsilane as internal standard. Highꢀresolution mass spectral
(HRMS) data were determined with Thermo Scientific Q Exactive (Thermo, USA).
General Procedure for Preparation of Intermediates A1–A2 and B1–B12
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Intermediates A1–A2 were prepared on the basis of a previously described method. A
solution of intermediates A1–A2 (8.74 mmol) and substituted 4ꢀhydroxybenzaldehyde
(8.74 mmol) in acetonitrile (35 mL) was heated to reflux for 8–12 h in K CO (100
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mol.%) and KI (5 mol.%). After the reaction was completed, the solvent was removed by
vacuum. Then residue was extracted using ethyl acetate to obtain intermediates B1–B12.
The representative data for B1 are shown below.
(E)ꢀmethylꢀ2ꢀ(2ꢀ((4ꢀformylꢀ2ꢀmethoxyphenoxy)methyl)phenyl)ꢀ2ꢀ(methoxyimino)acetate
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(B1). Yield: 3.00 g (96%); yellow solid; m.p. 78 °C–80 °C; H NMR (400 MHz, CDCl )
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δ 9.83 (s, 1H, ꢀCH=O), 7.54 (d, J = 7.2 Hz, 1H, ArꢀH), 7.45–7.38 (m, 3H, ArꢀH), 7.35 (dd,
J = 8.4, 2.0 Hz, 1H, ArꢀH), 7.20 (dd, J = 7.2, 0.8 Hz, 1H, ArꢀH), 6.89 (d, J = 8.4 Hz, 1H,
ArꢀH), 5.11 (s, 2H, ꢀOCH ꢀ), 4.04 (s, 3H, =NꢀOCH ), 3.94 (s, 3H, ꢀCOꢀOCH ), 3.86 (s,
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3H, ArꢀOCH ); C NMR (101 MHz, CDCl ) δ 190.94 (1C), 163.23 (1C), 153.29 (1C),
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150.03 (1C), 149.16 (1C), 134.42 (1C), 130.40 (1C), 129.78 (1C), 129.09 (1C), 128.49
(1C), 127.99 (1C), 127.35 (1C), 126.59 (1C), 112.35 (1C), 109.27 (1C), 69.00 (1C), 63.90
+
(1C), 56.04 (1C), 53.07 (1C); HRMS (ESI) m/z for C H O NNa [M+Na] calcd.
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380.11046, found. 380.10980.
General Procedure for the Synthesis of Dithioacetal Derivatives Bearing
Strobilurin (C1–C28). ZrCl (0.01 mmol) was added to a roundꢀbottomed flask
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containing intermediates B1–B12 (1.40 mmol) and substituted mercaptane (2.80 mmol)
in THF solvent (3 mL). And then mixture was stirred at room temperature for 10–60
minutes. Vacuum removal of solvent after the reaction was completed. The mixture was
added to water and extracted with ethyl acetate thrice. The collected organic extracts were
dried, concentrated, and purified through silica gel column chromatography with a 3:1
(v/v) mixture of petroleum ether and ethyl acetate to obtain products C1–C28. The
representative data for C1 are shown below.
(E)ꢀmethylꢀ2ꢀ(2ꢀ((4ꢀ(bis(2ꢀhydroxyethyl)dithioacetal)ꢀ2ꢀmethoxyphenoxy)methyl)phenyl)
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ꢀ2ꢀ(hoxyimino)acetate (C1). Yield: 502 mg (72%); white solid; m.p. 61 °C–63 °C; H
NMR (400 MHz, CDCl ) δ 7.54 (t, J = 7.6 Hz, 1H, ArꢀH), 7.44−7.35 (m, 2H, ArꢀH), 7.18
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(dd, J = 7.2, 1.2 Hz, 1H, ArꢀH), 7.03 (d, J = 2.0 Hz, 1H, ArꢀH), 6.85 (dd, J = 8.4, 2.0 Hz,
1H, ArꢀH), 6.70 (d, J = 8.4 Hz, 1H, ArꢀH), 5.02 (s, 1H, ꢀSCHSꢀ), 4.99 (s, 2H, ArꢀCH Oꢀ),
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4.04 (s, 3H, =NꢀOCH ), 3.89 (s, 3H, ꢀCOꢀOCH ), 3.85 (s, 3H, ArꢀOCH ), 3.72 (t, J = 5.9
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Hz, 4H, ꢀCH ꢀ), 2.85–2.77 (m, 2H, ꢀSCH ꢀ), 2.74–2.67 (m, 2H, ꢀSCH ꢀ), 2.51 (s, 2H, ꢀ
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OH); C NMR (101 MHz, CDCl ) δ 163.29 (1C), 149.90 (1C), 149.29 (1C), 147.87(1C),
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135.26 (1C), 132.90 (1C), 129.67 (1C), 129.02 (1C), 128.30 (1C), 127.70 (1C), 127.50
(1C), 119.96 (1C), 113.24 (1C), 110.89 (1C), 69.02 (1C), 63.88 (1C), 61.36 (2C), 56.05
+
(1C), 52.97 (1C), 35.67 (2C); HRMS (ESI) m/z for C H O NNaS [M+Na] calcd.
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518.12776, found. 518.12756.
Antiviral Activity Assay. Purification of virus. The Nicotiana. tabacum cv. K326
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leaves inoculated with PVY, CMV and TMV were selected, and PVY, CMV and
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TMV were purified with the reported methods.
Curative activities of the title compounds. The leaves of and Chenopodium
amaranticolor and N. tabacum L. growing at the same ages were selected to evaluate the
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antiꢀPVY , antiꢀCMV and antiꢀTMV activities . The virus was dipped and inoculated on
the whole leaves, which were scattered with silicon carbide beforehand. After 1 h, the
leaves were washed with water and then dried. The compound solution was smeared on
the left side, and the solvent was smeared on the right side. The local lesion numbers were
recorded and antiviral activities were counted. Three repetitions were conducted for each
compound.
Protective activities of the title compounds. The compound solution was smeared on
the left side of the growing C. amaranticolor and N. tabacum L. leaves. The solvent was
smeared on the right side. The leaves were inoculated with virus after 1 day. Then, the
leaves were washed with water after inoculation for 2 h. The number of local lesions was
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counted and inhibition rate of the compound was calculated.
Three repetitions were
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conducted for each compound.
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Physiology and Biochemistry of Tobacco. Compound Treatments and Sampling.
Nicotiana tabacum cv. K326 of equally grown at the fourth leaf stage was selected. The
three treatments were CK, COS, and C14. CK was solvent and COS was used as negative
and positive controls, respectively. 500 ꢁg/mL of C14 solution were smeared on whole
leaves. After 12 h of spraying, the leaves of all of the plants were evenly inoculated with
CMV and cultured in a greenhouse. After the plants were inoculated with the virus, tissue
samples were collected 1, 3, 5, and 7 days after the treatment, then test and calculate the
content of chlorophyll and the activity of defense enzyme. All tests were repeated three
times.
Chlorophyll Content Test. Chlorophyll content was determined with chlorophyll assay
reagent kits in accordance with the manufacturer’s instructions (Suzhou Comin
Bioengineering Institute, China). Absorbance spectra were obtained at 663 and 645 nm
for chlorophyll a (C ) and chlorophyll b (C ), respectively. C , C , total chlorophyll
a
b
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b
content (C ), and chlorophyll a/b were then calculated.
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Determination of Defensive Enzyme Activities. Using enzyme assay reagent kits from
the manufacturer’s instructions (Suzhou Comin Bioengineering Institute, China)
calculated the activities of catalase (CAT), peroxidase (POD), superoxide dismutase
(SOD), and phenylalanine ammonia lyase (PAL).
Gene Expression Analysis. The total RNA was extracted of the tobacco with a Trizol
reagent kit (TakaRa, Dalian, China) and was reverseꢀtranscribed using a cDNA kit
(TakaRa). The experiment was executed with reaction volume of 10 µL and SYBR
Premix Ex TaqII (TakaRa) by using an iCycleriQ multiꢀcolor realꢀtime PCR detection
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system (BioꢀRad, California, CA, USA). Calculating the relative copy numbers of the
ꢀꢂꢂCt
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genes by the 2
method.
Differentially Expressed Proteins Analysis. Total Protein Extraction. According to
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slightly modified reported methods, the total proteins of tobacco were extracted.
The
leaf sample (1.0 g) from different treatment groups were milled to homogenization in
liquid nitrogen, and iceꢀcold extraction buffer (5 mL) was added at room temperature, it
includes KCl of 0.1 M, TrisꢀHCl of 0.5 M, crose of 0.7 M, ethylene diamine tetraacetic
acid of 50 mM, dithiothreitol [DTT] of 40 mM, and pH was 7.5. Then the TrisꢀHCl (pH
7.5) saturated Phenol was added after 20 min of oscillation, continue to shake for 30 min
at 4 °C. The upper phenol layer was centrifuged for 20 min at 4500 rpm and collected
with a new tube, then maintain the temperature at −20 °C overnight after added the
methanol containing 100 mM ammonium acetate. The precipitate was collected after
centrifuged at 4500 rpm for 20 min at 4 °C and washed thrice under the same conditions
with 80% iceꢀcold acetone. Afterward, drying the precipitate in a vacuum drier,
rehydration solution (1000 ꢁL) were added at 37 °C, it contains urea of 8 M, Tris of 0.1
M, DTT of 10 mM and pH was 8.5. Thereafter, Bradford method was used to determine
the total protein concentration. 100 ꢁg of protein solution was collected and
iodoacetamide (55 mM) was added, the mixture was incubated at room temperature for
1 h in the dark, and centrifuged at 12,000 rpm for 20 min at 4 °C with 3 kDa Millipore.
The crude protein was washed six times using diluent rehydration solution. Then the
trypsin digest with 12.5 ꢁg trypsin from Sigma of USA was incubated at 37 °C for 16 h.
Peptide solution was collected after the mixture was centrifuged at 4 °C for 20 min at
12,000 rpm. And then air dried, acidified by 10 µl of formic acid for further
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chromatography–tandem MS (LC–MS/MS) analysis.
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Protein Identification. Peptide samples were analyzed using a Nano LCꢀ1DTM plus
system (Eksigent, Dublin, CA, USA) combined with triple timeꢀofꢀflight (TOF) 5600
mass (AB SCIEX, Foster City, CA, USA). Furthermore, 8 ꢁL of peptide samples was
obtained using a fullꢀloop injection and desalted on a ChromXP Trap column (Nano LC
TRAP Column, 3 ꢁm C18ꢀCL, 120 Å, 350 ꢁm × 0.5 mm, Foster City, CA, USA). Each
sample was eluted into a second analytical columnꢀNanoLCꢀC18 reversedꢀphase column
(3C18ꢀCL, 75 ꢁm × 15 cm, Foster City, CA, USA) by a linear gradient formed by
mobile phase A (5% ACN, 0.1% FA) and mobile phase B (95% ACN, 0.1% FA) for 120
min at a flow rate of 300 nL/min. TripleTOF 5600 MS was operated in a dataꢀdependent
mode to automatically switch between TOF–MS and product ion acquisition by utilizing
Analyst (R) software (TF1.6) (AB SCIEX, Foster City, CA, USA). The use of βꢀ
galactosidase digestion for 10 minutes elution and identification of 30 min to calibrate
every two samples.
Proteomic Data Analysis. Analysis of LCꢀMS/MS data using MaxQuant version
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1.5.2.8 (Max Planck Institute, Munich, Germany). The data were searched on the basis
of a database consisting of the tobacco and CMV proteomes and on the basis of the
tobacco proteome downloaded from UniProt. The search parameters contained an initial
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search with initial mass tolerance of 20 ppm, it set for quality recalibration. The search
also involved an Nꢀterminal acetylation, mutabie modification of methionine oxidation,
and fixed modification of carbamidomethyl cysteine. To identify the peptides, the error
detection rate was set to 0.01. To determine the standardized protein intensity,
differences in protein expression levels between the two groups were compared with
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labelꢀfree quantification and at least two ratio counts. iBAQ algorithm is a protein
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quantitative method, it used to sort the absolute abundance of DEPs in a single sample.
Filtered protein tables eliminate the identification of reverse database and common
pollutants. To identify the accumulation of different proteins between treatment and
control groups, unpaired t test of iBAQ data of two groups was carried out.
Bioinformatics analysis. Classification of differently expressed proteins was analyzed
with gene ontology (GO) annotation on Kyoto Encyclopedia of Genes and Genomes
(KEGG) using Uniprot software. Items in GO are related of cellular component (CC),
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biological process (BP), and molecular function (MF). Differentially expressed
proteins (expression level > 1.5ꢀfold) were mapped to the GO database
(http://www.geneontology.org/). Calculate the amount of proteins per GO term. the
labelꢀfree proteomics results as a target list, and generate a background list by
downloading the GO database.
RESULTS AND DISCUSSION
Synthetic Chemistry of the Title Compounds. Acetonitrile and K CO were used as a
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solvent and
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catalyst, respectively. The corresponding substituted 4ꢀ
hydroxybenzaldehyde and intermediates A1–A2 were stirred for 8–12 h under reflux to
obtain intermediates B1–B12, and the corresponding title compounds C1–C28 were
produced by using ZrCl as a catalyst and by condensing B1–B12 with substituted
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mercaptane (Figure 3). Their structures were identified through H NMR, C NMR, and
HRMS (Supporting Information).
Antiviral Activity. In Vivo AntiꢀPVY Activity. The antiviral activities of title
compounds C1–C28 against PVY are shown in Table 1. Several title compounds
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exhibited notable activities against PVY in vivo at 500 ꢁg/mL. The curative activities of
C13–C15, C17–C19, C21, C23, and C25–C27 (66.0%, 70.4%, 67.5%, 68.9%, 60.3%,
61.8%, 59.4%, 60.9%, 66.9%, 69.6%, and 68.6%, respectively) against PVY were
significantly higher than those of 6f (58.9%), Ningnanmycin (50.6%), COS (36.3%), and
ribavirin (40.0%). The protective activities of C13–C15, C17–C19, and C25–C27
(60.4%, 67.6%, 62.9%, 63.6%, 58.9%, 59.8%, 62.6%, 69.4%, and 62.9%, respectively)
against PVY were more potent than those of 6f (57.2%), Ningnanmycin (51.3%), COS
(37.2%), and ribavirin (41.2%).
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To confirm the potential inhibitory capacity of these compounds against PVY, we
further evaluated EC of several title compounds against PVY on the basis of previous
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bioassays. In Table 2, the curative and protective activities of C13–C15, C17, and C25–
C27 (EC of 209.2, 125.3, 173.8, 156.7, 189.1, 130.4, and 179.6 ꢁg/mL and 214.2, 148.4,
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189.7, 170.1, 221.3, 152.9, and 198.7 ꢁg/mL, respectively) against PVY were higher than
those of 6f (297.6 and 281.5 ꢁg/mL), Ningnanmycin (440.5 and 425.3 ꢁg/mL), COS
(553.4 and 547.3 ꢁg/mL), and ribavirin (677.4 and 652.7ꢁg/mL).
In Vivo AntiꢀCMV Activity. The evaluation of antiꢀCMV activity proved that most of
the title compounds exerted potent inhibitory effects. In Table 2, the curative and
protective activities of C13–C15, C17, and C25–C27 (EC of 204.0, 108.9, 181.4, 139.2,
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211.5, 124.7, and 209.3; and 220.1, 113.2, 190.7, 142.4, 228.1, 140.8, and 213.4 ꢁg/mL,
respectively) against CMV were significantly superior to those of 6f (259.6 and 244.3
ꢁg/mL), Ningnanmycin (549.1 and 513.3 ꢁg/mL), COS (582.8 and 570.6 ꢁg/mL), and
ribavirin (690.3 and 665.4 ꢁg/mL).
In Vivo AntiꢀTMV Activity. The antiꢀTMV activities of C1–C28 are shown in Table 2.
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The curative activities of C13–C15, C17, and C25–C27 (EC of 257.6, 181.7, 217.6,
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207.8, 282.5, 229.4, and 241.4 ꢁg/mL, respectively) against TMV were significantly
superior to those of 6f (582.4 ꢁg/mL), Ningnanmycin (373.8 ꢁg/mL), COS (513.8 ꢁg/mL),
and ribavirin (686.5 ꢁg/mL). The protective effects of C14 and C17 (EC of 214.6 and
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224.7 ꢁg/mL, respectively) against TMV were higher than those of 6f (546.3 ꢁg/mL),
Ningnanmycin (242.7 ꢁg/mL), COS (507.9 ꢁg/mL), and ribavirin (553.4 ꢁg/mL).
SARs. In the dithioacetal derivatives containing a strobilurin moiety, compounds with
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R ꢀbearing ethyl were more favorable to antiviral activity than R ꢀbearing aromatics
(C2 > C3, C4), 2ꢀhydroxyethyl (C2 > C1 and C9 > C8 and C14 > C12, and C17 > C16),
and other alkyls (C14 > C13, C15, and C17 > C18, C19). Halogen atoms substituted at
the 2ꢀposition of aromatic ring were more favorable to antiviral activity than electronꢀrich
groups, especially the chlorine atom C14 > C17 > C2, C6, C8, C9, and C11. This finding
was contradictory to those with substituent groups at the 3ꢀposition of the aromatic ring:
C25 > C23 > C21. Moreover, the introduction of carbon atoms instead of nitrogen atoms
caused the relatively decreased inhibitory activities against plant viruses as confirmed by
the following pattern: C26 < C14 and C27 < C15 and C28 < C17.
Physiological and Biochemical Analysis of Tobacco. Effect on Chlorophyll Contents.
Chlorophyll is a core chloroplast component that plays a key role in photosynthesis. The
chlorophylls comprised C , C , C , and C was tested in this study. The chlorophyll
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content decreased gradually after tobacco plants were inoculated with CMV (Figure 3).
However, from day 1 to day 7, the chlorophyll content had increased, and reached the
maximum on day 5 after the CMVꢀinoculated tobacco leaves were treated with C14. The
trend of C14 therapy was better than that of COS treatment. Hence, C14 might enhance
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the resistance of the plant host to diseases by increasing the chlorophyll content.
Effect on Defensive Enzyme Activities. CAT, POD, SOD, and PAL are several important
defense enzymes, whose enhanced activities are significantly related to inducible
resistance. Antioxidant defense machinery can protect plant from oxidative damage
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caused by reactive oxygen species (ROS). In this study, the defensive enzyme activities
of tobacco after C14 treatment were analyzed. CAT can decomposed into water and
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oxygen by catalyzing hydrogen peroxide to protect host from oxidative damage to ROS.
The CAT activities of C14 treatment groups were 1.90, 1.31, 1.27, and 1.99 times as
much as that of the control groups after 1, 3, 5, and 7 days, respectively. From 1day to 5
day, the values were increased and reached the maximum on 5 day (Figure 5A). POD can
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reduce the hydrogen peroxide content in the plant body. The POD activities of C14
treatment groups were 1.05, 2.01, 2.38, and 1.38 times as much as that of the control
group on day 1, 3, 5, and 7, respectively, and the maximum were obtained on day 5
(Figure 5B). The SOD activities of C14 treatment groups were 1.78, 1.95, and 1.05 times
that of the control groups after 3, 5, and 7 days, respectively (Figure 5C). The PAL
activities after C14 treatment was administered did not significantly change (Figure 5D).
The results demonstrated that C14 could improve the resistance of tobacco to the virus
through defense response induced by the enhanced activities of enzymes, especially POD
and SOD.
RT-qPCR analysis. As representatives of the sample on day 5, to study the mechanism
of C14 response in CMVꢀinfected tobacco, the relative expression levels of the defense
genes of NPRꢀ1, CATꢀ1, PALꢀ1, isochorismate synthase 1 (ICSꢀ1), enhanced disease
susceptibility 1 (EDSꢀ1), and SOD were investigated through reverse transcription (RTꢀ
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qPCR). In Figure 6, the relative expression levels of CATꢀ1, ICSꢀ1, and SOD of C14
treatment groups were higher than the CK and COS control groups. The relative
expression of SOD in the treatment group was about 4.00 and 3.36 times more that of CK
and COS. The relative expression of CATꢀ1 and ICSꢀ1 increased by approximately 1.32
and 1.57 times and by 2.00 and 2.07 times, respectively. These results demonstrated that
C14 could improve the disease resistance of tobacco by inducing defenseꢀrelated proteins,
namely, CATꢀ1, ICSꢀ1, and SOD.
Analysis of Proteomics MaxQuant version 1.5.2.8 (Max Planck Institute, Munich,
Germany) was used to search for peptide results, and 2088 proteins were found and
quantified. As showed of Figure 7, a total of 1706 and 1676 proteins were identified in
the control and treatment groups, respectively. In the total proteins, 412 (19.7%) and 382
(18.3%) were specifically expressed in CK and C14 groups, respectively. A volcanic map
was plotted to understand the differential protein expression levels (Figure 8). Of the total
number of identified differentially expressed proteins, 412 were downregulated (blue dots)
and 382 were upregulated (red dots) (fold change > 1.5, p < 0.05) in the treatment and
control groups (Supporting InformationꢀII).
Bioinformatics Analysis of the Control and Treatment Groups. The expressed
proteins were annotated using the Database for Annotation Differentially, Visualization,
and Integrated Discovery 6.8 (DAVID 6.8). On the basis of the GO categories, we
analyzed the differentially expressed proteins identified in CC, BP, and MF in the
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treatment and control groups. Figure 9 shows the GO term enrichment analysis of
differentially expressed proteins (DEPs, P < 0.05). The DEPs in the cytoplasm,
membrane, nucleus, photosystem, chloroplast, mitochondrion, cytosol, extracellular
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region, and plasma membrane were grouped according to their CC (Figure 9A). The
DEPs involved in the generation of precursor metabolites and energy, photosynthesis,
transport, signal transduction, response to oxidative stress, defense response, lipid
metabolism, and response to stress were categorized on the basis of their BP (Figure 9B).
The differently expressed proteins involved in RNA binding, DNA binding, enzyme
regulator activity, kinase activity, protein binding, nuclease activity, transporter activity,
ATPase activity, and signal transducer activity were classified on the basis of their MF
(Figure 9C).
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Functional Classification by KEGG. KEGG was used to research the response to the
action of C14 and determine the way of action triggered by C14. In Figure 10 and Table 3,
several specific proteins, including ABC transporter F family member 1 (ABC proteins),
calciumꢀdependent protein kinase 2ꢀlike (CDPKs), and superoxide dismutase (SOD, EC
1.15.1.1) were upregulated. These proteins play key roles in the abscisic acid (ABA)
signaling pathway. Few studies have shown that ABA levels in a virally infected host
plant can increase, thereby demonstrating the involvement of ABA in antivirus defense of
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plants.
A previous study revealed that ABC proteins are key transmembrane
transporters, which can transport ABA from the outer compartment to the inner
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compartment to participate in ABA signaling. Afterward, PP2Cs function as negative
regulators to repress ABA signaling and bind to PYR/PYL/RCAR receptors. The function
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of SnRK2s is dependent on PYR/PYL/RCAR receptors in ABA signaling pathway.
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main regulators of Ca ꢀmediated ABA and stress responses essential for plant survival.
CDPKs can regulate ABAꢀmediated gene expression though ABFs, which are ABAꢀ
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responsive transcription factors that serve as substrates of several CDPKs. The present
study demonstrated that SOD was upregulated in the treatment groups. A previous study
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indicated that ABA causes an increase in the sustained SOD. CDPKs are involved in
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ABAꢀinduced upregulation of SOD activity and expression. The disproportionation of
superoxide to molecular oxygen and hydrogen peroxide can be catalyed by SOD, which
constitute an antioxidant defense system with defense enzymes, such as CAT and POD.
The antioxidant defense system protects plants against oxidative damage by scavenging
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ROS.
ABA accumulation can increase the generation of ROS and the induction of
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several antioxidant enzymes. In the present study, heat shock proteins (HSPs) were
downregulated in the treatment groups. The expression of HSPs is ABA dependent, and
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they negatively regulate ABA response.
On the basis of the broad bioactivity of strobilurins and the lead compound 6f, we
designed and synthesized 28 novel dithioacetal derivatives bearing a strobilurin moiety.
Bioassay results indicated that the antiviral potency of C14 against CMV and PVY were
superior to those of 6f, Ningnanmycin, COS, and ribavirin. Meanwhile, compound C14
can be used to control TMV. This antiviral activity was associated with an increase in the
content of the chlorophyll and the enhanced activity of defensive enzyme in tobacco
treated with C14. RTꢀqPCR analysis revealed the relative expression levels of SOD in the
C14 treatment groups were higher than those of the CK and COS control groups. This
discovery has been confirmed by the specific expression of the stress responses and
related proteins of abscisic acid signaling pathway. In this paper, we demonstrated that
C14 can enhance the tolerance of plants to CMV infection by inducing the accumulation
of ABA. Therefore, C14 can be considered as a new type of antiviral agent.
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ASSOCIATED CONTENT
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Supporting Information
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Characterization data, H and C NMR spectra, and HRMS for intermediates B1–B12
and title compounds C1–C28 are provided. Supplementary data associated with this
article can be found in the online version at http://pubs.acs.org.
All of the identified proteins, GO term enrichment analysis, and differential protein
expression in the treatment group compared with that in the control group are listed in
Supporting InformationꢀII.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the National Natural Science Foundation of China
(No, 21562013)
Notes
The authors declare no competing financial interest.
REFERENCES
(1) Tomlinson, J. A. Epidemiology and control of virus diseases of vegetables. Ann. Appl.
Biol. 1987, 110, 661−681.
(2) Kennedy, J. S. A biological approach to plant viruses. Nature 1951, 168, 890−894.
(3) Harrison, B. D.; Mayo, M. A.; Baulcombe, D. C. Virus resistance in transgenic plants
that express cucumber mosaic virus satellite RNA. Nature 1987, 328, 799−802.
(4) Scholthof, K. B. G.; Adkins, S.; Czosnek, H.; Palukaitis, P.; Jacquot, E.; Hohn, T.;
Hohn, B.; Saunders, K.; Candresse, T.; Ahlquist, P.; Hemenway, C.; Foster, G. D. Top
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