Synthesis and antiviral activities of novel chiral cyanoacrylate derivatives with (E) configuration

Article abstract of DOI:10.1016/j.bmc.2007.12.035

The intermediate 3 is converted by the nucleophilic attack of the appropriate aryl (heterocyclic) amine under microwave irradiation into the chiral (E) isomers of title compound 4. These compounds exhibited weak to good anti-TMV bioactivity with (R)-4p sh

Full text of DOI:10.1016/j.bmc.2007.12.035

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3076–3083
Synthesis and antiviral activities of novel chiral
cyanoacrylate derivatives with (E) configuration
Zhuo Chen, Xianyou Wang, Baoan Song,* Hua Wang, Pinaki S. Bhadury, Kai Yan,
Huiping Zhang, Song Yang, Linhong Jin, Deyu Hu, Wei Xue, Song Zeng and Jun Wang
Center for Research and Development of Fine Chemicals, Key Laboratory of Green Pesticide and Agricultural Bioengineering,
Ministry of Education, Guizhou University, Guiyang 550025, PR China
Received 24 November 2007; revised 14 December 2007; accepted 15 December 2007
Available online 21 February 2008
Abstract—The intermediate 3 is converted by the nucleophilic attack of the appropriate aryl (heterocyclic) amine under microwave
irradiation into the chiral (E) isomers of title compound 4. These compounds exhibited weak to good anti-TMV bioactivity with (R)-
4p showing significant enhancement of disease resistance in tobacco leaves.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
highly intricate relationship exists between the two
which complicates the strategic designs to control plant
viruses and the losses caused by them.6 Control pro-
grams depend on our understanding of the virus–host
relationship, and several conventional strategies to con-
trol virus infection have been explored but without
much success. In most of the modern approaches involv-
ing antiviral action, the induced resistance is very prom-
ising to a particular strain or group of viruses. PAL, the
key enzyme in the phenylpropanoid pathway, which is
associated with PR gene, is strongly induced after the
infection of tobacco plants with viral pathogens.7 In or-
der to extend our research work of cyanoacrylate as
antiviral agent, we designed and synthesized some novel
chiral cyanoacrylates with (E) configuration in which
the phenylamino group was replaced by 2-nitrophenyla-
mino, 3-nitrophenylamino, 2,4-dinitrophenylamino, 4-
methylbenzothiazol-2-amino, and benzylamino groups.
The synthetic route is shown in Scheme 1. The structures
of 4a–r were established by well-defined IR, NMR, and
elemental analysis. In the bioassay conducted by half-
leaf method, these new chiral compounds were found
to possess weak to good anti-TMV activities. Our previ-
ous and present studies have shown the effectiveness of
chiral cyanoacrylates in protecting tobacco plant against
disease caused by TMV. Their mode of action and cellu-
lar targets are however still unknown. The objective of
our study is to establish the role of (R)-4p in inducing
disease resistance in tobacco leaves against virus patho-
gen attack, and to study the activity of enzymes or
metabolites that might be involved for resistance devel-
Synthetic studies on cyanoacrylate derivatives have
recently received wide attention due to their herbicidal
action caused by disrupting photosynthetic electron
transport.1–3 Among them, (Z)-ethoxyethyl 2-cyano-3-
(4-chlorophenyl)-methylamino-3-isopropylacrylate (CPNPE)
has served as a model compound due to its excellent her-
bicidal activity.4 In our previous work, we designed and
synthesized some chiral cyanoacrylates with antiviral
activity by replacing methylthio moiety with (R)- or
(S)-1-phenylethylamine groups in some 2-cyano-3-meth-
ylthio-3-substituted-phenylaminoacrylates. The crystal
structure of the chiral product was determined by X-
ray single-crystal structure analysis which confirmed
the presence of an (E) configuration. The bioassay
showed that the chiral compound with 4-nitrophenyl
moiety had no herbicidal activities in vivo, but possessed
good antiviral activity.5 Viruses are unique in the decep-
tive simplicity of their structure. However, this simplic-
ity leads to a greater dependence on the host and a
Abbreviations: TMV, tobacco mosaic virus; CMV, cucumber mosaic
virus; PAL, L-phenylalanine ammonia-lyase; SOD, superoxide dismu-
tase; POD, peroxidase; SAR, systemic acquired resistance; PR, path-
ogenesis related proteins; PCR, polymerase chain reaction; BLAST,
basic local alignment search tool.
Keywords: 2-Cyanoacrylates; (E) Configuration; Synthesis; Antiviral
bioactivity; Mechanism of action.
*
Corresponding author. Tel.: +86 851 362 0521; fax: +86 851 362
2211; e-mail: songbaoan22@yahoo.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.035

Z. Chen et al. / Bioorg. Med. Chem. 16 (2008) 3076–3083
3077
Scheme 1. Synthesis of novel chiral cyanoacrylate derivatives with (E) configuration.
opment. To the best of our knowledge, this is the
first report on the synthesis, antiviral activity of (E)-ethyl
3-[(R) or (S)-1-phenylethylamino]-3-(substituted phenyla-
mino)-2-cyanoacrylate derivatives, and the relationship
between the bioactivity of (R)-4p and the activity of
PAL, POD, and SOD enzymes and PR gene expression
in tobacco. Our studies suggest that (R)-4p possesses
antiviral activity by up-regulation of PR-1a and PR-5
gene and enhancing activity of some defensive enzyme
in defense response.
1639–1672 and 1539–1548 cmꢀ1, respectively. A set of
peaks near 2193–2212 cmꢀ1 and 1113–1165(s) were as-
signed to C@N and C–F vibrations, respectively. In
1H NMR data, all phenyl protons showed a multiplet
at d 6.94–7.55. The chemical shifts of CH2 ester gener-
ally appeared as a quartet in the region 4.22–4.37 ppm.
The NH proton of 3 appeared downfield as a broad sin-
glet around 9.02–11.12 ppm due to the existence of H-
bond interaction between ester carbonyl and NH of phe-
nylamino group. As for the 13C NMR data of 4, all the
carbon atoms were identified and the number of protons
1
calculated from the integration curve (in H NMR) was
consistent with the molecular formula.
2. Chemistry
2.1. Synthesis of novel chiral cyanoacrylate derivatives
2.2. Crystal structure analysis
Nucleophilic Michael attack by the amine at the olefinic
double bond followed by the expulsion of the leaving
group SCH3 from the conjugated system seems to gener-
ate compounds 3 and 4. To optimize the reaction condi-
tions, the synthesis of chiral compound (R)-4p was
carried out under several conditions. The effects of dif-
ferent solvents, reaction time, and the merit of using
microwave irradiation over classical reaction conditions
were investigated, and the results are shown in Table 1.
It revealed that the presence of microwave irradiation
could accelerate the reaction (entries 1–5). When the
reaction time was prolonged from 5 to 20 min under
microwave irradiation in refluxing ethanol at 78 ꢁC,
the yield of target chiral compound was increased from
39.4% to 52.5% (Table 1, entries 1–4). In classical meth-
od without microwave irradiation, the reaction was rel-
atively slow and when refluxed in ethanol for 300, 600,
900, and 1200 min, 41.9%, 47.0%, 52.0%, and 53.8%
yields were obtained, respectively (Table 1, entries 6–
9). The reaction under microwave irradiation gave com-
parable yield with significant reduction of reaction time
from 900 min to just 20 min, the average time ratio for
the two methods being 1:30 for similar yield.
It could be seen from the X-ray single crystal analysis that
chiral compound (R)-4b maintains a planar structure.
The configuration of target compound (R)-4b was estab-
lished as (E) by X-ray diffraction. The bond length of
˚
C(4)–C(6) (1.418 A) is longer than normal C@C
(1.34 A), C(3)–O(2) (1.361 A) is shorter than normal sin-
˚
˚
˚
˚
gle C–O (1.44 A), C(3)–C(4) (1.436 A) and C(4)–C(5)
˚
(1.413 A) are shorter than normal C–C (1.54 A), C(6)–
˚
˚
N(2) (1.341 A) and C(6)–N(3) (1.362 A) bonds are shorter
˚
˚
than the normal C–N single bond (1.49 A), suggesting the
existence of an electron density delocalization among
N(3)–C(6)–C(4)–C(3)–O(1), C(5), and N(2). As shown
in the packing diagram of the title compound (Figs. 1
and 2), there is an intramolecular hydrogen bond interac-
tion within N(2)–H(2)ꢁ ꢁ ꢁO(1), in which N(2)–H(2) =
˚
˚
0.86 A, H(2)ꢁ ꢁ ꢁO(1) = 2.04, N(2)ꢁ ꢁ ꢁO(1) = 2.692(3) A,
N(2)–H(2)ꢁ ꢁ ꢁO(1) = 132.1ꢁ (ꢀx,ꢀy + 1/2,ꢀz + 1/2). The
N–Hꢁ ꢁ ꢁO type intramolecular interaction leading to a
six-membered stable cyclic structure perhaps plays a
major role in stabilizing the molecules in the unit
cell.
3. Antiviral activity
The structures of all the compounds were confirmed by
1H NMR, 13C NMR, elemental analysis, IR, and mass
spectrometry. IR spectra of compound 4 show absorp-
tion band in the region 3220–3379 cmꢀ1, indicating the
presence of NH. The characteristic C@O stretching
and strong C@C vibrations appeared in the region
3.1. Preliminary antiviral activity assay
The results of bioassay in vivo against TMV are pro-
vided in Table 2. Ningnanmycin was used as reference
antiviral agent. The results indicated that, the antiviral

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Z. Chen et al. / Bioorg. Med. Chem. 16 (2008) 3076–3083
Table 1. Results at different reaction times for the synthesis of (E)-ethyl-3-[(R)-1-(4-fluoro phenylethylamino)]-3-benzylamine-2-cyanoacrylate [(R)-
4p]
Entry
Power (w)
Temperature (ꢁC)
Time (min)
Solvent
Amount of the solvent (ml)
Yieldc (%)
1a
2a
3a
4a
5a
6b
7b
8b
9b
50
50
50
50
50
—
—
—
—
78
78
78
78
78
78
78
78
78
5
10
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
10
10
10
10
10
10
10
10
10
39.4
44.2
49.5
52.5
53.1
41.9
47.0
52.0
53.8
15
20
30
5 · 60
10 · 60
15 · 60
20 · 60
a The reaction was conducted in 10 ml solvent under microwave irradiation, power 50 W.
b The reaction was conducted at 78 ꢁC in 10 ml solvent under stirring without microwave.
c Isolated yields.
As shown in Figure 3B, it could be found that SOD
activity in (R)-4p-treated tobacco gradually decreased
from 1st day to the start of the 3rd day and then started
to increase by the end of the 3rd day after inoculation.
Similar trend in the change of SOD activity was ob-
served with water-treated tobacco and not much differ-
ence was noticed within and among groups. It was
found that the SOD activity in TMV gradually increased
after the first day of inoculation, but the level was lower
than that in water-treated by the first day (*P < 0.05).
As shown in Figure 3C, it could be seen that POD activ-
ity of (R)-4p increased and reached a peak on the 5th
day after the inoculation, then gradually decreased
(Fig. 3). POD contents in (R)-4p-treated tobacco leaf
were 8.42 times as large as that (*P < 0.05) in water-trea-
ted tobacco leaf on the 1st day of inoculation. The POD
activity of water and TMV treatments both reached
their peaks on the 3rd and 1st day after the inoculation,
but the peak value of POD activity corresponding to the
control was lower than that of (R)-4p-treated one.
Figure 1. The molecular structure of compound (R)-4b.
3.3. Gene expression analysis of PR-1a and PR-5 in (R)-
4p-treated tobacco leaf
activity is greatly dependent upon the nature of substit-
uents. When R1 was fluoro and R2 was benzyl, the title
compound (R)-4p showed higher curative rate (64.0%)
than that of the reference (57.8%) and comparable inac-
tivation rate (49.5%) with the standard Ningnanmycin
(50.0%) against TMV at 500 lg/ml. It should be noted
that no protection effect was observed on (R)-4p. The
other compounds have a relatively lower antiviral activ-
ity than that of (R)-4p.
The results of product of PCR in PR-1a and PR-5 and
its sequence identification, see Supporting information.
In vitro synthesized single-stranded cDNA from RNA
samples was isolated from leaf in water-treated tobacco
and the TMV-treated tobacco and the (R)-4p- and
TMV-treated tobacco. The differential expression analy-
sis of the PR-1a and PR-5 gene was determined by the
semi-quantitative PCR and the relative quantification
real-time PCR analysis. The mRNAs of PR-1a and
PR-5 gene accumulated to detectable levels in (R)-4p-
and TMV-treated tobacco leaf, while no-detectable lev-
els were reached in water-treated tobacco and the TMV-
treated tobacco. The mRNA content of (R)-4p-treated
tobacco leaf for PR-1a gene started to increase after
12 h and reached a peak at the end of the 2nd day before
falling to the normal level. In contrast, in TMV-treated
tobacco leaf, no significant increase in the levels of gene
expression was noticed (Figs. 4A and 5A). The expres-
sion levels of PR-5 gene in (R)-4p-treated tobacco rap-
idly increased and reached a peak within 12 h after the
inoculation and then started to decrease gradually. As
3.2. Effect of (R)-4p treatment on PAL, POD, and SOD
activity in tobacco
As shown in Figure 3A, PAL levels in (R)-4p-treated to-
bacco rapidly increased by the end of the first day after
the inoculation and reached 5.88 EU/mg protein on the
7th day, 37.93 times more as on the first day (*P < 0.05)
after inoculation. In contrast, in control and TMV, no
significant increase in PAL activity was measured
(Fig. 3). The PAL activity in tobacco (control and
TMV) increased marginally and reached a peak at the
end of the fifth day after inoculation before starting to
fall gradually thereafter.

Z. Chen et al. / Bioorg. Med. Chem. 16 (2008) 3076–3083
3079
Figure 2. Packing diagram of the unit cell of compound (R)-4b.
Table 2. The protection, inactivation, and curative effect of the chiral new compounds to TMV in vivo
Agents
Concentration (mg/L)
Protection effect (%)
Inactivation effect (%)
Curative effect (%)
(S)-4a
(R)-4b
(S)-4c
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
0
0
0
0
0
0
0
19.7
0
44.8 4*
38.1 4*
10.0 4*
(R)-4d
(S)-4e
(R)-4f
2.7
16.7 4*
2
7.5
4
38.7 8*
21.7 4*
9.6 2*
20.0 5*
0
0
*
(S)-4g
(R)-4h
(S)-4i
7.8 4*
48.1 4.*
11.0 2*
51.9 7*
0
23.2
32.3 7*
11.0
16.8 3*
4
(R)-4j
(S)-4k
(R)-4l
12.3 4*
15.4
0
0
3.8
1
28.6 4*
0
(S)-4m
(R)-4n
(S)-4o
(R)-4p
(s)-4q
17.4 3*
5.4 4.2
46.4 5*
65.7 8*
22.6 3*
25.0 4*
100 7**
4.2
2
14.3 3*
46.5 4*
49.5 5*
32.6 4*
0
34.5 4*
64.0 5**
55.5 5**
36.9 4*
57.8 6**
(R)-4r
Ningnanmycin
5.3
50.0 4**
1
All results are expressed as means SD; n = 3 for all groups; *P < 0.05, **P < 0.01.
depicted in Figures 4B and 5B, (R)-4p-treated tobacco
leaves showed significant enhancement in the levels of
4. Discussion
gene expression, almost twice as large as compared to
TMV-treated tobacco leaves within 12 h after the inocu-
lation. In contrast, in TMV-treated tobacco leaf, no sig-
nificant increase in the levels of gene expression was
observed Figs. 4 and 5.
Although the action mechanism of anti-TMV has not
yet been elucidated, some studies show that there exists
requisite association between the defense enzymes of to-
bacco and salicylic acid-mediated signal transduction
pathway regarding pathogenesis related proteins. PAL

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Z. Chen et al. / Bioorg. Med. Chem. 16 (2008) 3076–3083
Figure 3. Effect of (R)-4p treatment on PAL and SOD and POD activity in Nicotiana tabacum. Nicotiana tabacum inoculated by TMV was treated
with (R)-4p at 500 ppm for 1, 3, 5, and 7 day. Leaf sample extracts using homogenized and centrifuged methods were used to determine the PAL and
SOD and POD activities as described in Section 6. Water treatment and TMV treatment were used for negative and positive controls. Data are
expressed as means SD (n = 3) of three individual experiments. One-way ANOVA revealed significant difference (*P < 0.05).
Figure 4. Semi-quantitative RT-PCR analysis of RNA isolated from tobacco leaf. The figure shows that 20 cycles were performed and the amplified
products were resolved on a 1.5% agarose gel by following the RT. Parts A and B in the figures indicate result of PR-1a and PR-5 gene expression
treated by (R)-4p compound at 500 ppm for 0.5, 1, 2, and 5 day, respectively. Lanes 1–4 represent the amount of PCR amplification at period four. b-
Actin gene served as an internal reference gene.
Figure 5. Real-time PCR analysis of RNA isolated from tobacco leaf. Letters A and B in the figures indicate the results of PR-1a and PR-5 gene
expression ratio through CT value formula DDCT = DCT(test) ꢀ DCT(calibrator). Relative values of real-time PCR between target gene and b-actin gene
were calculated, each value represents the mean SD (n = 4) and each experiment was performed in triplicate sets. The data were analyzed by one-
way ANOVA.

Z. Chen et al. / Bioorg. Med. Chem. 16 (2008) 3076–3083
3081
is a rate-limiting enzyme in the activation of the phenyl-
propanoid pathway, and an increase in PAL activity is
associated with biosynthesis of active metabolites, such
as salicylic acid, which is used for molecular signal in
plant SAR defense pathways.8 POD is also a very
important enzyme in the plant defense reaction and is
involved in the scavenging of reactive oxygen species.
The increase in POD activity promotes the cross-link
of lignin, strengthens the structure barrier, and enhances
disease resistance.9–12 Rasmussen has shown that SA
could enhance the activity of POD and PAL.13 PR-1a
and PR-5 are regulation proteins of downstream in SA
signal pathway that play a significant role in antiviral,
antifungal, and wound repair.14–16 Our studies have
shown that PR-1a and PR-5 gene was induced to up-
regulation by (R)-4p compound at 500 mg/ml on the
1st day after inoculation. Meanwhile, the activity of
PAL and POD were increased from 1st day to 5th day
after inoculation. These results show that there is signif-
icant association between curative effect and up-regula-
tion of PR-1a and PR-5 gene against anti-TMV by
(R)-4p to enhance defense enzymes activity level. Future
research would be focused on studying the action mech-
anism between gene regulation and anti-TMV, and the
knockdown technique to evaluate the antiviral effect
against target gene.
analytical reagent grade or chemically pure. All solvents
were dried, deoxygenated, and redistilled before use. 2-
Cyano-3,3-dimethylthioacrylate was prepared according
to the literature method.19
6.1. General procedure for the preparation of intermedi-
ates 3a–d
A solution of ethyl 2-cyano-3,3-dimethyl thioacrylate
(0.4 mmol) in EtOH (20 ml) was stirred, followed by the
addition of [(S)-1-(4-fluorophenylethyl]amine] or [(R)-1-
(4-fluorophenylethyl) amine] (0.4 mmol). The mixture
was irradiated in the microwave oven at 78 ꢁC and 50 W
for 10 min. The solvent was removed under reduced pres-
sure. The crude product was purified by column chroma-
tography on a silica gel (eluent, ethyl acetate/petroleum
ether, 4:1, v/v) to give the intermediates 3a–d. The struc-
ture was confirmed by 1H NMR, 13C NMR, IR, and ele-
mental analysis (see Supporting information).
6.2. General procedure for the preparation of title chiral
compounds 4
A solution of ethyl intermediate 3 (0.72 mmol) in EtOH
(10 ml) was stirred, followed by the addition of substi-
tuted phenylamine or 2-aminobenzothiazole (0.72 mmol).
The resulting mixture was irradiated in the microwave at
78 ꢁC, 60 W for 20 min. The course of the reaction was
followed by TLC. The mixture was poured into ice
water (100 ml), and acidified with 10% HCl to a pH of
6–7, and filtered. The residue was purified by column
chromatography on silica gel (eluent, ethyl acetate/
petroleum ether, 1:4, v/v) to give title compounds 4
(see Supporting information).
5. Conclusion
In conclusion, a series of novel (E)-ethyl-3-[(R)- or (S)-1-
phenyl-ethylamino]-3-(substituted phenylamino)-2-cya-
no-acrylate derivatives were synthesized by the treat-
ment of chiral intermediate 3 with aryl (heterocyclic)
amine under microwave irradiation. This method is
easy, rapid, and moderate-yielding for the synthesis of
title chiral compounds 4. Their structures were verified
by spectroscopic data. The results of bioassay showed
that these title compounds exhibited weak to good
anti-TMV bioactivity. Title compounds (R)-4p showed
better biological activity than their structurally related
analogues 4a–o, 4q–r. Preliminary studies showed that
treatment by compound (R)-4p can significantly en-
hance disease resistance of tobacco leaf and substitute
for antiviral agent control of TMV diseases in tobacco.
6.3. Crystal structure determination
In order to study the single-crystal structure (Figs. 1 and
2), X-ray intensity data were recorded on a Rigaku Rax-
is-IV diffraction meter using graphite monochromatic
˚
MoKa radiation (k = 0.71073 A). In the range
2.14ꢁ 6 h 6 25.01ꢁ, 3741 independent reflections were ob-
tained. Intensities were corrected for Lorentz and polari-
zation effects and empirical absorption, and all data were
corrected using SADABS program.22 The structure was
solved by direct methods SHELXS-97 program.21 All
the non-hydrogen atoms were refined on F2 anisotropi-
cally by full-matrix least squares method. The hydrogen
atoms were located from the difference Fourier map,
but their positions were not refined. The contributions
of these hydrogen atoms were included in structure-factor
calculations. The final least-square cycle gave
wR = 0.1307, R = 0.0539 for 9478 reflections with
6. Experimental
The melting points of the products were determined on a
XT-4 binocular microscope (Beijing Tech Instrument
Co., China). The IR spectra were recorded on a Bruker
VECTOR 22 spectrometer in KBr disk. 1H and 13C
NMR (solvent CDCl3) spectra were recorded on a
JEOL-ECX 500 NMR spectrometer at room tempera-
ture using TMS as an internal standard. Elemental anal-
ysis was performed on an Elementar Vario-III CHN
analyzer. Microwave reaction was performed on a Fo-
cused Microwave Synthesizer, DiscoverTM LabMate
(with a power of 50 W), according to Huang’s and
Zhou’s methods.17,18 Analytical TLC was performed
on silica gel GF254. Column chromatographic purifica-
tion was carried out using silica gel. All reagents were of
I > 2r(I); the weighting scheme, w ¼ 1=½r2ðF o2Þþ
2
ð0:0:0852PÞ þ 0:000Pꢂ, where P ¼ ½ðF 2oÞ þ 2F c2ꢂ=3. The
max and min difference peaks and holes were 0.198 and
ꢀ0.263 e Aꢀ3, respectively, s = 1.107. Atomic scattering
factors and anomalous dispersion corrections were taken
from International Table for X-ray Crystallography.20
Crystallographic data (excluding structure factors) for
the structure have been deposited with the Cambridge
Crystallographic Data Center as supplementary publica-
tion No. CCDC-299719.

3082
Z. Chen et al. / Bioorg. Med. Chem. 16 (2008) 3076–3083
Table 3. Sequences of gene-specific primers used in RT-PCR analysis
Gene family
Accession No.
50 primer
30 primer
b-Actin
PR-1a
PR-5
U60495
X12737
X03913
50-gacatgaaggaggagcttgc-30
50-caatacggcgaaaacctagctga-30
50-gcttccccttttatgccttc-30
50-atcatggatggctggaagag-30
50-cctagcacatccaacacgaa-30
50-cctgggttcacgttaatgct-30
6.4. Protection and inactivation and cure effect of
compound against TMV in vivo
6.8. The relative quantification real-time PCR for
expression of the target gene
Purification of TMV was assessed by Gooding’s meth-
od, as described in Supporting information.23 The bio-
activity assay for protection and inactivation and cure
effect was assessed according to Li’s method24 (see Sup-
porting information).
The relative quantification real-time PCR was carried
out with iCycler IQ according to manufacturer’s pro-
tocol with primer of b-actin serving as an internal ref-
erence gene. Precautions were taken to ascertain
reliable quantitative results: Log-linear dilution curves
were performed with primers for the target gene as
well as with primers for the b-actin. Reactions per-
formed without reverse transcriptase or without tem-
plate did not result in any product. By following
PCR, 110 steps for melt curve analysis were completed
in 10 s at temperature ranging from 40 to 95 ꢁC. The
amplification efficiency was 95–99% for PR-1a and
PR-5 gene, respectively (the standard curve figure is
not shown). Each target gene peak was assigned an
arbitrary quantitative value correlated to the b-actin
gene peak, according to the formula DDCT =
DCT(test) ꢀ DCT(calibrator), CT being the cycle threshold.
Rates of stimulation of RNA expression were
calculated from the DCT values at various time
points.31
6.5. Determination of PAL and POD and SOD activity
The leaf samples were homogenized in the 2-mercap-
toethanol-boric acid buffer (5 mM, pH 8.8) on the ice
bath and centrifuged. The supernatant was used for
experiment. PAL and POD and SOD activities were
determined by He’s method,25 Polle’s method,26 and
Beauchamp’s method,27 respectively (see Supporting
information).
6.6. RT-PCR assay
Trizol kit was used according to the standard protocol
for total RNA isolation. Prior to RT-PCR, the total
RNA samples were treated with DNase I for 10 min
and quantified by spectrophotometry and identified by
agarose gel electrophoresis.28 cDNA was synthesized
with oligo (dT)18 at the 30 end of mRNA as a primer.
Total RNA (1 lg) was used for template of first-strand
cDNA synthesis using extend reverse transcriptase. Re-
verse transcription was carried out at 37 ꢁC for 1 h. The
single-stranded DNA mixture was used as template in
PCR. The primers for PCR amplification are shown in
Table 3. The PCR was performed in Tris–HCl buffer
(10 mM, pH 8.3), KCl (50 mM), MgCl2 (1.8–2.0 ll),
dNTPs (0.02 lM), primers (0.04 lM), and DNA poly-
merase (1 U). PCR amplification steps consisted of a
preliminary denaturation step at 94 ꢁC for 1 min, fol-
lowed by 35 cycles at 94 ꢁC for 40 s, at 58 ꢁC for 40 s,
and at 72 ꢁC for 50 s on icycle of Bio-Rad. PCR prod-
ucts were separated on 1.5% agarose gel in 0.5· TBE
buffer and visualized under UV light after staining with
ethidium bromide.29
6.9. Statistical analysis
All statistical analyses were performed with SPSS 10.0.
Data were analyzed by one-way analysis of variance
(ANOVA). Mean separations were performed using
the least significant difference method (LSD test).
Each experiment had three replicates and all experi-
ments were run three times with similar results. Mea-
surements from all the replicates were combined and
treatment effects analyzed.
Acknowledgments
The authors thank the National Key Program for Ba-
sic Research (No. 2003CB114404), the Cultivation
Fund of the Key Scientific and Technical Innovation
Project,
Ministry
of
Education
of
China
(No.705039), and National Basic Research Priorities
Program (No. 2005CCA01500), the National Natural
Science Foundation of China (No.20672024) for the
financial support.
6.7. Semi-quantity PCR for expression of gene
In order to assess relative expression levels of target-
gene in water-treated tobacco and (R)-4p- and TMV-
treated tobacco and tobacco by inoculated TMV only,
semi-quantity PCR consisting of 20 cycles (within the
logarithmic range of amplification of gene) with putting
primer of b-actin serving as an internal reference gene
was employed for the study. The amplified products
were analyzed on a 1.5% agarose gel by the method of
Mohamed.30
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2007.12.035.

Z. Chen et al. / Bioorg. Med. Chem. 16 (2008) 3076–3083
3083
17. Huang, W.; Yang, G. F. Bioorg. Med. Chem. 2006, 14,
8280–8285.
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