Identification of racemic and chiral carbazole derivatives containing an isopropanolamine linker as prospective surrogates against plant pathogenic bacteria: In vitro and in vivo assays and quantitative proteomics

Article abstract of DOI:10.1021/acs.jafc.9b02036

Recent observations on the emergence of drug-resistant plant pathogenic bacteria have highlighted and elicited an acute campaign to develop novel, highly efficient antibiotic surrogates for managing bacterial diseases in agriculture. Thus, a type of racemic and chiral carbazole derivative containing an isopropanolamine pattern was systematically synthesized to discover low-cost and efficient antibacterial candidates. Screening results showed that compounds 2f, 6c, and 2j could significantly suppress the growth of tested plant pathogens, namely Xanthomonas oryzae pv oryzae, X. axonopodis pv citri, and Pseudomonas syringae pv actinidiae, and provided the corresponding EC₅₀ values of 1.27, 0.993, and 0.603 μg/mL, which were significantly better than those of existing commercial drugs. In vivo studies confirmed their prospective applications for controlling plant bacterial diseases. Label-free quantitative proteomics analysis indicated that compound 2f could dramatically induce the up- and down-regulation of a total of 247 differentially expressed proteins, which was further validated by the parallel reaction monitoring technique. Moreover, fluorescence spectra and SEM images were obtained to further explore the antibacterial mechanism.

Full text of DOI:10.1021/acs.jafc.9b02036

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Functional Structure/Activity Relationships
Identi#cation of Racemic and Chiral Carbazole Derivatives
Containing an Isopropanolamine Linker as Prospective
Surrogates Against Plant Pathogenic Bacteria: In
Vitro and In Vivo Assays, and Quantitative Proteomics
Yong-Liang Zhao, Xing Huang, Li-Wei Liu, Peiyi Wang,
Qing-Su Long, Qing-Qing Tao, Zhong Li, and Song Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02036 • Publication Date (Web): 10 Jun 2019
Downloaded from http://pubs.acs.org on June 11, 2019
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Identification of Racemic and Chiral Carbazole Derivatives Containing an
Isopropanolamine Linker as Prospective Surrogates Against Plant Pathogenic
Bacteria: In Vitro and In Vivo Assays, and Quantitative Proteomics
a
a
a
a
a
Yong-Liang Zhao , Xing Huang , Li-Wei Liu , Pei-Yi Wang* , Qing-Su Long ,
a
b
a,b
Qing-Qing Tao , Zhong Li , Song Yang*
a
State Key Laboratory Breeding Base of Green Pesticide and Agricultural
Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering,
Ministry of Education, Center for R&D of Fine Chemicals of Guizhou University,
Guiyang, 550025, China.
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College of Pharmacy, East China University of Science & Technology, Shanghai,
China 200237.
* Corresponding authors.
E-mail: jhzx.msm@gmail.com (S. Yang), pywang888@126.com (P.-Y. Wang)
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Abstract
Recent observations on the emergence of drug-resistant plant pathogenic bacteria
have highlighted and elicited an acute campaign to develop novel, highly efficient
antibiotic surrogates for managing bacterial diseases in agriculture. Thus, a type of
racemic and chiral carbazole derivative containing an isopropanolamine pattern was
systematically synthesized to discover low-cost and efficient antibacterial candidates.
Screening results showed that compounds 2f, 6c, and 2j could significantly suppress
the growth of tested plant pathogens, namely Xanthomonas oryzae pv. oryzae, X.
axonopodis pv. citri, and Pseudomonas syringae pv. actinidiae, and provided the
corresponding EC values of 1.27, 0.993, and 0.603 µg/mL, which were extremely
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better than those of existing commercial drugs. In vivo study confirmed their
prospective applications for controlling plant bacterial diseases. Label-free
quantitative proteomics analysis indicated that compound 2f could dramatically
induce the up- and down-regulation of a total of 247 differentially expressed proteins,
which was further validated by the parallel reaction monitoring technique. Moreover,
fluorescence spectra and SEM images were performed to further explore the
antibacterial mechanism.
Keywords
carbazole, isopropanolamine, antibacterial, proteomics, action mechanism
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1. Introduction
Despite the persistent threat posed by plant bacterial diseases and the identification of
a variety of widespread, highly phytopathogenic bacteria, the shortage of effective
antibacterial agents in agriculture causes considerable concern.1-3 Currently, a limited
number of marketed bactericides, such as bismerthiazol (BT), thiodiazole copper
(TC), streptomycin (has been disabled for the risk potentials), triazoles, kocide, and
amobam, were exploited to manage these destructive bacterial diseases.4,5 However,
these agents have performed reduced field control efficiency, not only due to their
long-term usage and abuse, but also ascribing to the single mode of action
performed.6,7 Particularly, some of these pathogenic bacterial strains have
progressively experienced an elusive and prompt variation to acquire the flexible
defense mechanisms. Such complicated resistant pathogens can provide a quick and
adequate response against various existing external stimuli, putting agriculture in
considerable difficulty. For example, the application of marketed bactericide BT has
already resulted in the emergence of BT-resistant strains of Xanthomonas oryzae pv.
oryzae (Xoo) in Anhui Province, China.2,4 Therefore, exploring and developing new
chemotherapeutic agents normally bearing different modes of action to combat
wild-type and drug-resistant pathogens is urgently required.
Elaborate observation found that agrochemicals, which can be commercialized
preferentially, possess the following features: simple structural architecture, short
synthetic procedures, facile operations for drug production, low manufacturing cost,
and superior biological activity. Concurrently, modern agricultural science has
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proposed strict or even higher requirements, such as good biocompatibility and
degradability, low residues, negligible phytotoxicity, permissible environmental
friendliness, high selectivity for the target species, and low cytotoxicity toward
non-target organisms, for the development of pesticide products. Therefore, the
reasonable capture of a building block as the original template for exploring highly
active antibacterial leads has elicited considerable concern. One of the effective
sources for the selection is from the natural products in which the biological effects of
various key skeletons have been carefully explored and highlighted. Carbazole
skeleton with outstanding electronic and charge-transport properties has been
frequently discovered and reported from many naturally occurring products.8,9
Moreover, this privileged building block can be easily decorated with various
functional groups, which endows carbazole-based derivatives and analogs with
extensive and distinct biopharmaceutical activities, including antimicrobial,10-14
anti-cancer,15-17
insecticidal,18,19
anti-viral,20
anti-α-glucosidase,21,22
anti-inflammatory,23,24 anti-malarial,25,26 anti-diabetic,27,28 neuroprotective,29,30 and
anti-Alzheimer activities.31-33 Given that the carbazole motif plays a crucial and
versatile role in determining the final biological action of a target molecule, a large
batch of natural and non-natural carbazole-tailored substrates acquiring prospective
and practical applications in many fields, especially in the antimicrobial aspect, has
also been presented.8,34 As shown in Figure 1a, these kinds of typical natural
carbazole derivatives were discovered to possess good antimicrobial effects against a
wide range of medicinal bacteria and fungi including Escherichia coli, Salmonella
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typhi, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus,
Micrococcus luteus, Proteus vulgaris, Candida albicans, and Trichophyton
rubrum.35,36
Considering the powerfully broad-spectrum bioactivity of carbazole-based
compounds, the relevant action mechanisms for their pharmaceutical effects have also
been extensively studied. Consequently, a variety of target species, such as diverse
enzymes or kinases (Pim-1 kinase, anaplastic lymphoma kinase, topoisomerase I, and
topoisomerase II), DNA, and G-quadruplexes (G-tetrads or G4-DNA), were
reported.37-39 Inspired by the aforementioned studies, the rational design of title
compounds based on natural carbazole skeleton may probably lead to the discovery of
highly active antibacterial surrogates intended for the future battle against plant
bacterial diseases. Moreover, this kind of carbazole-tailored molecules may be
endowed with flexibly privileged functions for interacting with multiple targets of
pathogenic microorganisms, which probably reduces the risk of developing
bacteria-resistance. Few studies were performed to describe the general antibacterial
effects of carbazole-tailored compounds toward plant pathogens. Herein, a type of
simple racemic and chiral N-substituted carbazole derivative bridging by
isopropanolamine patterns (Figure 1b) was systematically synthesized and their
antibacterial effects were screened. In this study, three seriously invasive
phytopathogenic bacterial strains, including Xoo, Xanthomonas axonopodis pv. citri
(Xac), and Pseudomonas syringae pv. actinidiae (Psa), will be evaluated in vitro.
These pathogenic strains are known to cause destructive and widespread bacterial leaf
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blight, citrus bacterial canker, and kiwifruit bacterial canker in agriculture, which
makes a mass of economic losses every year.3,40,41 Subsequently, in vivo study will be
performed to verify the practical application for controlling bacterial diseases. In
addition, label-free quantitative proteomics and parallel reaction monitoring (PRM)
techniques, fluorescence spectra, and scanning electron microscopy (SEM) will be
utilized to study the possible antibacterial mechanism.
2. Materials and methods
2.1 Instruments and Chemicals
NMR spectra were obtained by using a Bruker Biospin AG-400 apparatus. Chemical
shifts were reported in parts per million (ppm) down field from TMS with the solvent
resonance as the internal standard. Coupling constants (J) were reported in Hz and
referred to apparent peak multiplications. SEM images were visualized and obtained
using Nova Nano SEM 450. Fluorescence spectra measurements were performed on a
Fluoromax-4 spectrofluorimeter (Horiba Trading CO., LTD).
2.2 In vitro antibacterial bioassay (turbidimeter test)
In our study, all the synthesized target compounds were evaluated for their
antibacterial activities against Xoo, Xac, and Psa by the turbidimeter test in vitro.6
Dimethylsulfoxide in sterile distilled water served as a blank control, Bismerthiazol
and Thiodiazole Copper served as positive controls. Approximately 40 μL of solvent
NB (1.5 g beef extract, 2.5 g peptone, 0.5 g yeast powder, 5.0 g glucose, and 500 mL
distilled water; pH = 7.0-7.2) containing Xoo (or Xac, or Psa), incubated on the phase
of logarithmic growth, was added to 5 mL of solvent NB containing different
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concentrations of the test compounds and positive control, such as 20, 10, 5, 2.5, 1.25
μg/mL or 10, 5, 2.5, 1.25, 0.625 μg/mL (depending on the bioactivity of different
compounds, the concentrations were chosen in two times decline trend to make sure
the EC values are inside the concentration ranges tested). The inoculated test tubes
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were incubated at 28 ± 1 °C and continuously shaken at 180 rpm for 24-48 h until the
bacteria were incubated on the logarithmic growth phase. The growth of the cultures
was monitored on a microplate reader by measuring the optical density at 595 nm
(OD ) given by turbidity corrected values = OD − ODno bacterial wilt, and the
bacterial wilt
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inhibition rate I was calculated by I = (C − T)/C × 100%. C is the corrected turbidity
values of bacterial growth on untreated NB (blank control), and T is the corrected
turbidity values of bacterial growth on treated NB. By using the SPSS 17.0 software
and the obtained inhibition rates at different concentrations, a regression equation was
provided. The results of antibacterial activities (expressed by EC ) against Xoo, Xac,
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and Psa were calculated from the equation and the value was within the concentration
ranges. The experiment was repeated three times.
2.3 In vivo bioassay against rice bacterial leaf blight
The curative and protection activities of compound 2f against rice bacterial leaf blight
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were determined by Schaad’s method with some slight modifications. Bismerthiazol
(20% wettable powder) and thiodiazole copper (20% suspending agent), the
bactericides registered for rice bacterial leaf blight and purchased from the market,
served as the positive controls. The curative activity in potted plants for reducing rice
bacterial leaf blight of compound 2f was determined under controlled conditions in a
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growth chamber. After sowing the rice seeds of variety “Fengyouxiangzhan”
approximately 8 weeks, rice leaves were inoculated with Xoo, which was incubated at
logarithmic growth using sterilized scissors. One day after inoculation, 2f solution at
200 μg/mL was uniformally sprayed onto the rice leaves until dripping down, whereas
distilled water was uniformally sprayed onto the negative control plants. Then, all
inoculated rice plants were placed in a plant growth chamber (28 °C and 90% RH). At
14 days after spraying, the disease index of the inoculated rice leaves was measured.
Similarly, the protection activity for reducing rice bacterial leaf blight of compound 2f
was also conducted under controlled conditions. After sowing the rice seeds of variety
“Fengyouxiangzhan” approximately 8 weeks, compound 2f solution at 200 μg/mL
was uniformally sprayed onto the rice leaves until dripping down, whereas distilled
water was uniformally sprayed onto the negative control plants. One day after
spraying, Xoo, which was incubated at logarithmic growth, was inoculated on the rice
leaves using sterilized scissors. All inoculated rice plants were placed in a growth
chamber (28 °C and 90% RH). At 14 days after inoculation, the disease index (C or T)
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of the inoculated rice leaves was measured. Firstly, the spot area of each leaf and the
whole leaf area were measured, and then the percentage of the spot area in the whole
leaf area was calculated. Secondly, these leaves were classed according to the
following grading standards. Grade 1: the area of disease spot accounts for less than
5% of the whole leaf area; Grade 3: the area of disease spot accounts for 6-10% of the
whole leaf area; Grade 5: the area of disease spot accounts for 11-20% of the whole
leaf area; Grade 7: the area of disease spot accounts for 21-50% of the whole leaf
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area; Grade 9: the area of disease spot accounts for more than 50% of the whole leaf
area. Finally, the disease index (C or T) was calculated using the following formula:
Disease index (C or T) = ∑(the number of leaves at each Grade × the corresponding
Grade) / (the total number of leaves × the superlative Grade).
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The control efficiencies I for the curative and protection activities are calculated
by the following equation: Control efficiency I (%) = (C - T) / C ×100. In the
equation, C is the disease index of the negative control and T is the disease index of
the treatment group.
2.4 Label-free quantitative proteomics analysis
2.4.1 Bacterial strains and growth conditions
The bacterial strain used in this study was Xoo, which is the causative pathogen of
rice bacterial leaf blight. The Xoo was cultivated in liquid nutrient broth medium (1.5
g beef extract, 2.5 g peptone, 0.5 g yeast powder, 7.5 g sucrose, added into 500 mL
distilled water, pH 7.0–7.2) at 28–30°С for 12 h. Cells were harvested when OD600
(optical density at 600 nm) reached approximately 0.1. Then compound 2f with the
concentration of 6.35 μg/mL (5×EC ) was added into the mixture and cultured at
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28-30 °C. When the OD600 of the control group reached to 0.6-0.8, all the samples
o
were collected and centrifuged at 8000 rpm at 4 C, respectively. Three biological
replicates were prepared for each condition.
2.4.2 Protein extraction and trypsin digest
The extraction of all Xoo proteins were performed based on the methods with a slight
modification.43 Briefly, the sample was sonicated three times on ice using a high
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intensity ultrasonic processor (Scientz) in lysis buffer (8 M urea, 1% Protease
Inhibitor Cocktail). The remaining debris was removed by centrifugation at 12,000 g
at 4 °C for 10 min. Finally, the supernatant was collected and the protein
concentration was determined with BCA kit according to the manufacturer’s
instructions. For digestion, the protein solution was reduced with 5 mM dithiothreitol
for 30 min at 56 °C and alkylated with 11 mM iodoacetamide for 15 min at room
temperature in darkness. The protein sample was then diluted to a urea concentration
(less than 2 M). Finally, the trypsin was added into the protein sample by 1:50
trypsin-to-protein mass ratio for the first digestion overnight, and 1:100
trypsin-to-protein mass ratio for a second 4 h-digestion.
2.4.3 LC-MS/MS analysis
The tryptic peptides were dissolved in 0.1% formic acid, directly loaded onto a
home-made reversed-phase analytical column (15 cm length, 75 μm i.d.). The
gradient was comprised of an increase from 6% to 23% (0.1% formic acid in 98%
acetonitrile) over 26 min, 23% to 35% in 8 min and climbing to 80% in 3 min, then
holding at 80% for the last 3 min, all at a constant flow rate of 400 nL/min on an
EASY-nLC 1000 UPLC system.
The peptides were subjected to NSI source followed by tandem mass
spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC.
The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for
full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000.
Peptides were then selected for MS/MS using NCE setting as 28 and the fragments
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were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure
that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s
dynamic exclusion. Automatic gain control (AGC) was set at 50000. Fixed first mass
was set as 100 m/z.
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2.4.4 Protein identification
The resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8).
Tandem mass spectra were searched against Xoo database concatenated with reverse
decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 2 missing
cleavages. The mass tolerance for precursor ions was set as 20 ppm in First search and
5 ppm in Main search, and the mass tolerance for fragment ions was set as 0.02 Da.
Carbamidomethyl on Cys was specified as fixed modification and oxidation on Met
was specified as variable modifications. The false discovery rate (FDR) for peptide
and protein identifications was set to 0.01 and minimum score for peptides was
set >40. It should be combined and reported as one protein group to identify peptides
which all appear between two proteins. We used label-free quantification with a
minimum of two ratio counts to determine the normalized protein intensity to
compare between samples. We used the iBAQ algorithm to rank the absolute
abundance of different proteins within a single sample. Protein tables were filtered to
eliminate the interference from common contaminants and reverse database. A
two-sample unpaired t-test was used to identify the differentially accumulated
proteins between control and treatment groups. The iBAQ data were used for the
t-test, and proteins with p value ≤0.05 were considered differentially expressed.
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2.4.5 Bioinformatics analysis
Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA
database (www. http://www.ebi.ac.uk/GOA/). Firstly, Converting identified protein
ID to UniProt ID and then mapping to GO IDs by protein ID. If some identified
proteins were not annotated by UniProt-GOA database, the InterProScan soft would
be used to annotated protein’s GO functional based on protein sequence alignment
method. GO items can be divided into three categories, namely, biological process
(BP), cellular components (CC), and molecular functions (MF). In this study, we
mapped the differentially displayed proteins (fold changes >1.5, P<0.05) into the GO
database (http://www.geneontology.org/). It was computable for the amount of
proteins at each GO term, and the target list used for result which came from
label-free. The list was constructed by downloading the data on the GO database.
Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to annotate
protein pathway. Firstly, using KEGG online service tools KAAS to annotated
protein’s KEGG database description. Then mapping the annotation result on the
KEGG pathway database used KEGG online service tools KEGG mapper.
2.5 Fluorescence analysis
The total DNA was isolated from Xoo by TIANamp bacterial DNA kit. The
concentration of DNA was determined by UV absorption at 260 nm using a molar
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absorption coefficient ξ260 = 6600 L mol cm (expressed as molarity of phosphate
groups) by Bouguer-Lambert-Beer law. The purity of DNA was checked by
monitoring the ratio of the absorbance at 260 nm to that at 280 nm. The solution gave
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a ratio of >1.8 for A /A , which indicated that DNA was sufficiently free from
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protein. Then compound 2f was added into a TRIS-HCl buffer solution (50 nM, pH =
7.4) containing different amounts of DNA. The final volume was fixed to 10 mL and
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the final concentration of compound 2f was 1×10 mol L . After incubating the
solution for 20 min at room temperature, fluorescence spectra were recorded on
F-7000 Spectrofluorimeter (Hitachi, Tokyo, Japan) equipped with 1.0 cm quartz cells,
the widths of both the excitation and emission slit were fixed as 5 nm, and the
excitation wavelength was 363 nm.
2.6 Scanning electron microscopy (SEM)
In this assay, 1.5 mL Xoo (or Xac or Psa) cells incubated at the logarithmic phase
were centrifuged and washed with PBS (pH = 7.2), and re-suspended in 1.5 mL of
PBS buffer (pH = 7.2). After that, bacteria Xoo (or Xac or Psa) were incubated with
compound 2f at concentrations of 12.5 μg/mL, 25.0 μg/mL, and an equivalent volume
of DMSO (solvent control) for 6 h at room temperature. After incubation, these
samples were washed 3 times with PBS (pH = 7.2). Subsequently, the bacterial cells
were fixed for 8 h at 4 ℃ with 2.5% glutaraldehyde, and then dehydrated with graded
ethanol series and pure tert-butanol (2 times with 10 min/time). Following
dehydration, samples were freezing dried and coated with gold, and visualized using
Nova Nano SEM 450.
3. Results and Discussion
An isopropanolamine linker was elaborately investigated and will be integrated into
the title compounds to efficiently fabricate a type of simple carbazole derivative,
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which probably possesses adequate competence to inhibit the growth of plant
pathogens; this linker has been extensively exploited as a versatile tool or bridge to
construct pharmacological substrates, which displayed impressive biological activities
in many fields.14,34 Therefore, a series of carbazole compounds bridging by this
isopropanolamine linker was synthesized following Figure 2. Briefly, carbazole was
treated with racemic epichlorohydrin under strong alkali conditions of KOH to give
the key intermediate 1 bearing an epoxy tail, which was subsequently ring-opened by
various substituted benzylamines to afford title molecules 2a–2k. All these racemic
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molecular structures were confirmed by H NMR, C NMR, and HRMS. For their
antibacterial evaluation, the typical turbidimeter test was conducted, while the mainly
used agricultural agents BT and TC were co-assayed as reference drugs. As indicated
in Table 1, most of the designed compounds displayed significant antibacterial powers
against the tested three bacterial strains. For anti-Xoo and anti-Xac activity, numerous
compounds provided the corresponding EC values ranging from 1.39 to 4.30 µg/mL
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and 1.80 to 2.54 µg/mL except compounds 2d and 2g, which afforded relatively high
EC values of 10.2, 35.4 µg/mL and 8.01, 30.5 µg/mL, respectively. For anti-Psa
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assays, these compounds also showed outstanding performances in suppressing the
pathogen growth with EC50 values within 0.603 and 29.7 µg/mL apart from
compounds 2d and 2i, which presented the negligible antibacterial ability with EC50
values exceeding 100 µg/mL. The above findings indicated that the design of
carbazole-based compound bridging by the isopropanolamine linker could promote
the discovery of highly efficient molecules probably applied in agriculture. The
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structure–activity relationship (SAR) was elucidated according to the obtained EC50
values and the effect of different substituents on the benzene ring. The electronic
property, the type of halogens, the position, and steric hindrance of substituents
clearly have a significant influence toward the bioactivity. Compared with
unsubstituted compound 2c (EC values of 2.76, 2.46, 3.94 µg/mL against Xoo, Xac,
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and Psa, respectively), introducing a weak electron-donating group 4–CH (2b, 1.51,
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3.83 µg/mL) or 4-F (2j, 2.04, 2.42, and 0.603 µg/mL) would improve the bioactivity.
Notably, the antibacterial effects on Psa were increased by approximately two folds
after introducing a strong electron-donating group (4-OCH , 2a, 1.79 µg/mL) and
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were reduced by approximately two folds after placing a strong electron-withdrawing
group (4-CF , 2k, 9.24 µg/mL) on the benzene ring. Appreciably, the position of the
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substituents provided different tendencies toward the three strains. For anti-Xoo
activity, the order of activities followed para (R = 4-Cl or 4-F, 2f and 2j, 1.27 and
2.04 µg/mL) > ortho (R = 2-Cl or 2-F, 2d and 2h, 10.2 and 4.30 µg/mL). By contrast,
different patterns were observed for the anti-Xac activity, providing the results of para
(R = 4-Cl, 2f, 1.96 µg/mL) > ortho (R = 2-Cl, 2d, 8.01 µg/mL). Additionally, the
substituent located at the para-position afforded the most powerful potency against
Psa with the EC value of 0.603 µg/mL (4-F, 2j). In another aspect, a remarkable
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observation indicated that the antibiotic action was dramatically decreased after
introducing an additional steric group on the benzene ring, which was illustrated by
comparing the bioactivity of compounds 2f (4-Cl, 1.27, 1.96, 3.83 µg/mL) and 2g
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(2,4-diCl, 35.4, 30.5, 29.7 µg/mL). From the above results, admirable antibacterial
effects with the minimal EC values of 1.27, 1.80, and 0.603 µg/mL against the
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corresponding strains of Xoo, Xac, and Psa were achieved.
Further modification from various aspects was conducted based on the preceding
study to explore even superior bioactive molecules. First, influence of replacing the
benzene ring into the heterocyclic scaffolds on the antibacterial effect was examined,
and compounds 2l, 2m, and 2n bearing furan, thiofuran, or pyridine moieties were
prepared. Although these compounds still performed strong inhibition powers against
the three pathogens with EC values ranging from 2.06 to 7.23 µg/mL, except for
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compound 2m against Psa (EC value > 100 µg/mL), they could not afford a better
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antibacterial action. Second, substituted arylamines were employed to ring-open the
epoxy motif instead of substituted benzylamines and resulted in the fabrication of title
compounds 2o–2r. Even though compounds 2p and 2r provided a tolerable inhibition
action toward Psa with the corresponding EC values of 12.2 and 32.0 µg/mL, the
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antibacterial effects of these compounds were significantly blocked after removing the
methylene group. This finding suggested that ring-opening the epoxy motif with a
relatively flexible and relaxed amine would be beneficial to the bioactivity. A series
of title compounds (2s–2x) bearing the saturated alkyl amine motifs were synthesized
to verify this hypothesis and their bioactivities were tested. Notably, the EC values
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of these compounds experienced three steps of variation tendencies: first, increasing
to extend the alkyl chain length, suggesting that slightly raising the hydrophobicity of
title compounds was disadvantageous to the activity; then being decreased after
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introducing a long alkyl chain (for example, n-butyl, 2w, providing the minimal EC50
values of 2.09, 1.90, and 4.84 µg/mL against Xoo, Xac, and Psa, respectively),
indicating that the title molecule bearing a providential and adjustable lipophilic tail
would elevate the antibacterial potency; finally, elevation along with the introduction
of a sterically hindered group (tert-butyl, 2x, providing EC values of 7.34, 12.5, and
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5.55 µg/mL against Xoo, Xac, and Psa, respectively), indicating that a bulk group
located at the molecular tail would block the additional interactions with target
species. Based on the preceding modification and obtained bioassay results, even
slight changes in the molecular patterns would significantly affect the final
bioactivity. Therefore, molecular framework maps must be carefully and
systematically optimized.
Given that compounds 2f, 2b, and 2j bearing the corresponding 4-chlorobenzyl,
4-methylbenzyl, and 4-fluorobenzyl groups exerted the best antibacterial potency
against Xoo (EC = 1.27 µg/mL), Xac (EC = 1.80 µg/mL), and Psa (EC = 0.603
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µg/mL), respectively, the substituents on the carbazole ring toward bioactivity were
investigated. Therefore, compounds 4a–4h bearing 3,6-diBr, 3,6-ditert-butyl, or
3,6-diacetyl motifs were prepared (Figure 3) and biologically evaluated. As shown in
Table 2, the anti-Psa activity was significantly decreased after introducing the
substituents on the carbazole ring and resulted in the EC50 values exceeding 100
µg/mL, suggesting that the additional substituted groups could seriously block the
interactions with Psa target species. For anti-Xoo and anti-Xac activity, an increased
pharmacological effect was observed via the introduction of the 3,6-diBr moiety (4c)
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and provided the corresponding EC values of 1.10 and 1.62 µg/mL. By contrast, an
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opposite pattern was observed from EC values of compounds 4a (17.9 and >100
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µg/mL) and 4b (17.2 and >100 µg/mL), indicating that an electron-donating group
(-CH , 4c) on the benzene ring was beneficial to bioactivity in comparison to those
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weak electron withdrawing groups (4-Cl, 4a; 4-F, 4b). Notably, introducing a
hydrophobic and bulk group (3,6-ditert-butyl, 4d-4f) led to a comparative activity
toward Xoo and slightly improved efficiency toward Xac, respectively. By contrast,
the introduction of 3,6-diacetyl motif (4g and 4h) significantly reduced the
antibacterial potency, demonstrating that an electron-withdrawing group was
unfavorable to the bioactivity. Given the preceding results, the antibacterial
competence could be affected by various substituents on the carbazole ring, which
provides a new template and guidance for the careful optimization of the molecular
framework.
After evaluating the antibacterial effect of substituents on the carbazole ring, the
absolutely chiral carbazole derivatives toward activity must be clarified. Therefore, 6a
(R), 6b (S), 6c (R), and 6d (S) were synthesized by replacing the racemic
epichlorohydrin into the corresponding chiral material (Figure 4). Bioassay results
indicated that the fixed configuration had certain effects toward the antibacterial
efficiency (Table 3). For the anti-Xoo activity, their EC50 values presented the
following order, R-forms (1.38 µg/mL, 6a; 0.902 µg/mL, 6c) < R/S-forms (1.47
µg/mL, 4d; 1.32 µg/mL, 4f) < S-forms (1.51 µg/mL, 6b; 1.43 µg/mL, 6d), suggesting
that the R-forms of target compounds were beneficial to bioactivity. For anti-Xac
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bioassay, a distinct pattern was provided as R = 4-chlorobenzyl, and their EC
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values followed the order of R/S-form (2.42 µg/mL, 4d) < S-form (3.11 µg/mL, 6b) <
R-form (5.49 µg/mL, 6a). This finding indicates that the combination of S- and
R-form can improve the antibacterial power. Based on the preceding investigation, the
final bioactivity could be affected by the absolute configuration of the chiral center in
the designed molecular structure.
In vivo studies were conducted to fight against rice bacterial leaf blight and
estimate the prospective applications of this kind of compounds against plant bacterial
diseases. In this study, compound 2f was selected due to its superior anti-Xoo activity
(1.27 μg/mL) and accessibility for synthesis. As shown in Table 4 and Figure 5,
compound 2f exhibited good in vivo curative and protection activities toward bacterial
leaf blight diseases with the corresponding control efficiency of 50.77% and 48.71%
at 200 μg/mL, respectively, which were superior to those of BT (43.47% and 40.91%)
and TC (42.60% and 39.17%). This outcome suggests that this type of molecular
structure can be considered as a template for the future exploration and development
of highly efficient alternatives.
A label-free quantitative proteomic analysis was conducted in response to the
compound 2f stimulation to examine the underlying antibacterial mechanism of this
type of designed compound toward Xoo. In this study, three biological replicates were
conducted for each group (2f and CK) to ensure the reliability of the results (detected
by relative standard deviation (RSD) analysis, Figure 6a) and the total proteins of
samples were identified through label-free LC-MS/MS. The result showed that a total
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of 2337 proteins were preliminary monitored for CK and 2f samples, among which
2095 proteins with quantitative information were identified (Table S1). Comparing
the treatment (2f) and control (CK) groups, 1848 proteins (88.2%) presented common
expression levels, while 247 proteins (100 up-regulated, 147 down-regulated; fold
changes > 1.5, p < 0.05) were confirmed differentially expressed (Table S2, Figure
6b). A volcano plot map was also utilized to filter the significant differentially
expressed proteins (Figure 6c). This outcome indicates that compound 2f could
significantly provoke the pathogen to provide a physiological response against the
external stimulus, and consequently produced a series of differentially expressed
proteins.
These differentially expressed proteins were analyzed for GO categories in
biological process (BP), molecular function (MF), and cellular component (CC) to
preliminarily clarify their biological functions.43-45 According to their BP (Figure 7A),
a large proportion of these proteins were clearly involved in the metabolic process,
localization, cellular process, and single-organism process. Figure 7B showed that a
large quantity of these differentially expressed proteins was involved in catalytic
activity, binding transporter activity on the basis of their MF. Meanwhile, CC analysis
showed that most of these proteins were found in membrane and cell (Figure 7C).
Moreover, the subcellular localization chart revealed that most of these differentially
expressed proteins were mainly found in the cytoplasmic (Figure Sa). Clusters of
Orthologous Groups (COG) chart was exploited to further expound the functional
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classification of these unique proteins. As indicated in Figure Sb, 21 representative
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functions, such as “Energy production and conversion,” “Amino acid transport and
metabolism,” “Carbohydrate transport and metabolism,” “Lipid transport and
metabolism,” “Cell wall/membrane/envelope biogenesis,” and “General function
prediction only”, were predicted. This observation suggests that a variety of
physiological processes could be dramatically influenced and triggered by the
stimulation from compound 2f.
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The KEGG pathway enrichment bubble plot chart (Figure Sc) was presented to
determine the receivable action pathways containing multiple differentially expressed
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proteins induced by compound 2f. Two main metabolic pathways containing biotin
metabolism pathway and starch and sucrose metabolism pathway involving additional
differentially expressed proteins were locked. The two pathways are known to play
one of the key approaches in the biosynthesis of biotin and D-glucose, which are
normally participants in a variety of physiological processes including more than the
metabolisms of fat, protein, and energy. The disturbance or blockage of the access
will result in serious problems, such as delayed growth, energy deficiency, and
biochemical disorder.48,49 In the biotin metabolism pathway (Figure 8), the
harmonious cooperation of these proteins is essential for the normal operation.
However, an array of down-regulated proteins was observed in this pathway. For
example, beta-ketoacyl-synthase I (Fab B) and 3-oxoacyl-(Acyl-carrier-protein)
reductase (Fab G) are the main enzymes performing significant roles in the
biosynthesis of fatty acid; by contrast, the expression quantity of biotin synthase
(2.8.1.6), ATP-dependent dethiobiotin synthetase BioD (6.3.3.3), and
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8-amino-7-oxononanoate synthase (2.3.1.47) would further influence the synthesis of
biotin. Simultaneously, in the starch and sucrose metabolism pathway, the evidently
decreased expression of beta-glucosidase (3.2.1.21) might directly lead to the reduced
conversion of GDP-glucose into D-glucose, which would disrupt the normal energy
metabolism of bacteria and consequently resulted in the failure of normal life
activities of bacteria. By contrast, the up-regulated proteins, including endoglucanase
(3.2.1.4), trehalose-6-phosphate synthase (2.4.1.15), and trehalose 6-phosphate
phosphatase (3.1.3.12), would cause the imbalance of these key metabolic
intermediates, further breaking the normal pathway metabolism. This outcome
suggested that compound 2f might have the privileged competence to induce the
expression of differential proteins affecting the crucial metabolic pathways, and
consequently resulted in the disorder of normal physiological processes and final
bacteria death.
Protein–protein interaction networks were presented using STRING database
version 10.5 to further study and assign the function and association of these
differentially expressed proteins. As indicated in Figure 9, 12 functions, including
“Fatty acid biosynthesis,” “Translation ribosomal structure and biogenesis,” “Lipid
transport and metabolism,” “RNA processing and modification,” “Ion transport and
metabolism,” “Amino acid transport and metabolism,” “Phosphotransferase system,”
“Nucleotide transport and metabolism,” “Cytochrome oxidoreductase,”
“Oxidoreductase,” “Biotin metabolism,” and “Starch and sucrose metabolism” were
enriched. This finding further suggested that various physiological processes could be
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significantly affected by compound 2f stimulation and consequently led to the
disturbance and final bacteria death.
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PRM technique was performed to further validate the label-free quantitative
proteomics result, because it is another widely used and efficient method which can
precisely quantify and verify an array of target proteins of interest.50-52 In this study,
twenty significant differentially produced proteins involved in these GO terms and
pathways were selected for PRM analysis (Table S3). Moreover, two unique peptides
with anticipated chemical stability were chosen for each protein, and the relative
protein abundance was expressed as the average of the two normalized peptide peak
areas (Table S4). The PRM result was shown in Figure 10 and afforded the same
trends with the label-free quantitative proteomics data.
The natural carbazole scaffold is known to normally possess the privileged
ability to interact with DNA, which results in changes in fluorescence intensity.13,34
Fluorescence spectrometry was used to monitor the interactions between the total
DNA extracted from pathogen Xoo and compound 2f. Primarily, time dependent
fluorescent intensity of compound 2f in the TRIS-buffer revealed that the fluorescence
was constant after 20 min (Figure 11a). After the addition of an equivalent DNA, the
fluorescent intensity was first decreased, and then became constant for 20 min
incubation (Figure 11b). This outcome might be attributed to the encapsulation of the
carbazole moiety into the grooves of DNA helical structures, and consequently
reduced the total amount of carbazoles to produce fluorescence. A gradually
decreased fluorescent intensity for compound 2f with different dosages of DNA
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further confirmed that the designed compounds bearing carbazole patterns had strong
interactions with the DNA of pathogens (Figure 11c), which was in accordance with
the observation of differentially expressed proteins.
The SEM images of Xoo, Xac, and Psa were presented to study the morphology
changes after being subjected to different dosages of compound 2f. As indicated in
Figure 12, the shapes of these pathogens were changed from well-proportioned
(Figures 12a, 12d, and 12g) to partially corrugated or broken (Figures 12b, 12e, and
12h) after treatment with compound 2f at the dosage of 12.5 µg/mL. Furthermore,
nearly all these pathogens were destroyed when the drug concentration was elevated
to 25 µg/mL and consequently afforded the observation of various debris and large
leakage holes on the bacteria surfaces (Figures 12c, 12f, and 12i). This result further
confirmed that the designed compounds performed strong interactions with the target
species of bacteria, which subsequently led to a series of physiological process
changes and final bacteria death.
In summary, a type of simple racemic and chiral carbazole derivative possessing
an isopropanolamine pattern was systematically synthesized and evaluated their
antibacterial activities. Bioassay results showed that compounds 2f, 6c, and 2j could
evidently inhibit the growth of plant pathogens Xoo, Xac and Psa with the
corresponding EC values of 1.27, 0.993, and 0.603 µg/mL, which were extremely
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superior to those of BT (92.6 µg/mL) and TC (121.8, 77.0, and 87.0 µg/mL).
Moreover, the SAR analysis indicated that the electronic property, the type of
halogens, the position, steric hindrance of substituents, and molecular absolute
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configuration had a dramatic influence toward the bioactivity. In vivo study confirmed
the prospective application for developing these compounds as agricultural agents
against plant bacterial diseases. Quantitative proteomics analysis indicated that
compound 2f could induce a total of 247 proteins (100 up-regulated, 147
down-regulated) differentially expressed comparing to the negative control, which
was further validated by the PRM result. This occurrence significantly affected
various physiological processes of pathogens and eventually resulted in their
morphological variations from SEM images. Moreover, fluorescence spectra
suggested that this kind of compounds had strong interactions with the DNA of
pathogens. Considering their privileged features and multi-interactions with the target
species of tested pathogens, these versatile carbazole derivatives could be considered
as a suitable template for exploring highly efficient antibacterial surrogates against
plant bacterial infections and disabling the bacterial resistance.
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Acknowledgment
We acknowledge the financial support of National Natural Science Foundation of
China (21877021, 21662009, 21702037, 31860516), the Research Project of Ministry
of Education of China (20135201110005, 213033A), and Guizhou Provincial S&T
Program ([2012]6012, LH [2017]7259, [2017]5788).
Supporting Information
Supplementary data including experimental procedures for PRM, partial racemic or
chiral intermediates and target compounds were analyzed by HPLC (Figures S1-S10),
Figures Sa-Sc, Tables S1-S5, synthetic procedures for the intermediates and target
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compounds and H NMR, 13C NMR, and HRMS spectra of target compounds
associated with this article can be found, in the online version, at
https://pubs.acs.org/journal/jafcau.
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Conflict of interest
The authors declare no competing financial interest.
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