Benzenetriol-derived compounds against citrus canker

Article abstract of DOI:10.3390/molecules26051436

In order to replace the huge amounts of copper salts used in citrus orchards, alternatives have been sought in the form of organic compounds of natural origin with activity against the causative agent of citrus canker, the phytopathogen Xanthomonas citri subsp. Citri. We synthesized a series of 4-alkoxy-1,2-benzene diols (alkyl-BDOs) using 1,2,4-benzenetriol (BTO) as a starting material through a three-step synthesis route and evaluated their suitability as antibacterial compounds. Our results show that alkyl ethers derived from 1,2,4-benzenetriol have bactericidal activity against X. citri, disrupting the bacterial cell membrane within 15 min. Alkyl-BDOs were also shown to remain active against the bacteria while in solution, and presented low toxicity to (human) MRC-5 cells. Therefore, we have demonstrated that 1,2,4-benzenetriol-a molecule that can be obtained from agricultural residues-is an adequate precursor for the synthesis of new compounds with activity against X. citri.

Full text of DOI:10.3390/molecules26051436

molecules
Article
Benzenetriol-Derived Compounds against Citrus Canker
Lúcia Bonci Cavalca 1,2, Ciaran W. Lahive 2, Fleur Gijsbers 1, Fernando Rogério Pavan 3, Dirk-Jan Scheffers 1,
*
and Peter J. Deuss 2,
*
1
Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute,
University of Groningen, 9747 AG Groningen, The Netherlands; l.bonci.cavalca@rug.nl (L.B.C.);
fleurgijsbers@gmail.com (F.G.)
Department of Chemical Engineering (ENTEG), University of Groningen, Nijenborgh 4, 9747 AG Groningen,
The Netherlands; c.w.lahive@rug.nl
2
3
School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara 14800-903, Brazil;
fernando.pavan@unesp.br
*
Correspondence: d.j.scheffers@rug.nl (D.-J.S.); p.j.deuss@rug.nl (P.J.D.)
Abstract: In order to replace the huge amounts of copper salts used in citrus orchards, alternatives
have been sought in the form of organic compounds of natural origin with activity against the
causative agent of citrus canker, the phytopathogen Xanthomonas citri subsp. Citri. We synthesized a
series of 4-alkoxy-1,2-benzene diols (alkyl-BDOs) using 1,2,4-benzenetriol (BTO) as a starting material
through a three-step synthesis route and evaluated their suitability as antibacterial compounds. Our
results show that alkyl ethers derived from 1,2,4-benzenetriol have bactericidal activity against X. citri,
disrupting the bacterial cell membrane within 15 min. Alkyl-BDOs were also shown to remain active
against the bacteria while in solution, and presented low toxicity to (human) MRC-5 cells. Therefore,
we have demonstrated that 1,2,4-benzenetriol—a molecule that can be obtained from agricultural
residues—is an adequate precursor for the synthesis of new compounds with activity against X. citri.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Keywords: Xanthomonas citri; antimicrobials; lignocellulosic biomass; bio-based chemicals; pheno-
lic compounds
Citation: Cavalca, L.B.; Lahive, C.W.;
Gijsbers, F.; Pavan, F.R.; Scheffers,
D.-J.; Deuss, P.J. Benzenetriol-Derived
Compounds against Citrus Canker.
Molecules 2021, 26, 1436. https://
doi.org/10.3390/molecules26051436
1. Introduction
Citrus canker is a bacterial plant disease caused by the pathogen Xanthomonas citri
subsp. citri that affects all cultivated citrus species, for example, oranges or lemons. This
disease causes lesions in the aerial tissues of citrus plants, generating spots and, in more
severe cases, the loss of leaves and premature fruit drop, being responsible for economic
Academic Editor: Paola Di Donato
Received: 2 February 2021
Accepted: 2 March 2021
Published: 6 March 2021
losses in agriculture and restrictions to international trade [1]. Citrus canker is believed
to have originated in Asia and is nowadays also present in Africa, Oceania, and in the
Americas, where it is already considered endemic to some of the main citrus-producing
areas, such as São Paulo state (Brazil) and Florida (USA).
Huge amounts of copper salts are sprayed annually in citrus orchards as the main
strategy for controlling the disease. This process threatens the environment and human
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iations.
health due to its toxic and cumulative effects [
compounds from natural sources have been investigated, and have so far shown promising
activity against the plant pathogen [ ]. In recent years, it has been demonstrated that sev-
eral alkylated compounds derived from hydroxybenzoic acids such as gallic, -resorcylic,
2]. As an alternative to copper salts, organic
3–5
β
Copyright:
© 2021 by the authors.
gentisic, and protocatechuic acids are capable of completely inhibiting X. citri (Figure 1a).
All of these new compounds with anti-X. citri activity have similar structures, being formed
by esterification between benzoic acid derivatives with two or three hydroxyls (phenols)
and a linear aliphatic chain of up to 11 carbons.
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Attribution (CC BY) license (https://
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Molecules 2021, 26, 1436. https://doi.org/10.3390/molecules26051436
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Molecules 2021, 26, 1436
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Figure 1.
(a) Reported organic compounds with activity against Xanthomonas citri [3–5]; (b) Retrosynthetic analysis (design
strategy) for novel BTO (bio)-based agents against citrus canker. BTO: 1,2,4-benzenetriol; HMF: hydroxymethylfurfural.
Taking into account that the antibacterial activity of these compounds seems to be
directly related to the presence of the phenols and the linear aliphatic carbon chain, we
designed a new series of compounds to be synthesized using 1,2,4-benzenetriol (BTO,
Figure 1b). BTO was demonstrated to be attainable from biomass by hydrothermal reaction
of hydroxymethylfurfural (HMF) in the presence of catalysts [
as a key bio-based platform chemical and is projected to be available at low price once
produced at large scale from agricultural residues [ ]. BTO is composed of an aromatic
6,7]. HMF has been reported
8,9
ring with three phenolic hydroxyls, of which the one in the 4 position can be selectively
modified to obtain ethers (Figure 1b) [10]. Lateral carbon chains from 4 to 14 carbons
were chosen. These compounds were tested for X. citri in order for us to provide a range
which could allow us to determine optimal lateral chain length and possible correlations
between antibacterial activity and carbon chain size. Moreover, in the designed molecules
the aromatic group was linked to the alkyl chain by an ether bond instead of an ester
bond, allowing comparisons between both bonds and the identification of possible roles
for these bonds in the antibacterial activity of these compounds. Furthermore, the potential
activity against other bacteria was investigated as well as their mode of action and toxicity
to mammalian cells.
2. Results and Discussion
2.1. A Three-Step Synthesis Route from BTO to 4-Alkoxy-1,2-Benzenediols (Alkyl-BDOs)
To obtain the new compounds based on 1,2,4-benzenetriol, we were first interested in
a one-pot synthesis catalyzed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as this could be
considered to provide an easy route to the proposed set of compounds [10]. However, this
route proved to be inconvenient as, in contrast to the earlier report [10], the etherification
of BTO showed no clear preference for substituting the position 4 selectively, resulting
in a mixture of products etherified in any one, two, or all three positions. Only very
low yields (<1%) of individual compounds were achieved after separation of the product
mixture by chromatography. Furthermore, the correct identification of each product as to
the position(s) to which the carbon chain was added proved to be a challenge, leading to
us not pursuing this route further.
As an alternative, a three-step synthetic route consisting of BTO protection [11],
etherification, and deprotection was designed (Scheme 1) consisting of: (a) protection of
BTO; (b) purified protected BTO (pBTO) etherification; and (c) crude protected alkylated
BTO deprotection and purification. This route achieved target compound synthesis with a
reasonable overall yield of up to 9% for the alkyl-BDOs (Table S1, in SI); the main challenge
of this approach was to obtain protected BTO, which was achieved with a final yield of 30%
(after purification). Our attempts showed that the presence of oxygen or a high substrate
concentration in the reaction favored the formation of byproducts that were related to the
tendency of BTO to dimerize as we previously reported [7,12]. Moreover, BTO protection

Molecules 2021, 26, 1436
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only occurred in the presence of a large excess of protecting agent (2,2-dimethoxypropane),
and ethyl acetate proved to be a more suitable solvent than toluene for this reaction. The
steps of etherification and deprotection were straightforward and etherification was also
shown to be suitable for other phenolic substrates, such as sesamol. Although not ideal
in terms of green chemistry, this route allowed us to obtain a sufficient quantity of these
compounds from BTO for an initial evaluation, keeping open the possibility of returning to
optimization of this synthetic route in case the compounds are deemed of interest for further
studies. Synthesis routes using other starting materials have been previously described to
obtain some of the target compounds reported here. The synthesis of 4-alkoxy catechols
and other catechol derivatives from 4-butoxyphenol and 3,4-dihydroxybenzaldehyde was
described respectively by [13–15].
Scheme 1.
Three-step synthesis of 4-alkoxy-1,2-benzenediols (alkyl-BDOs) from 1,2,4-benzenetriol (BTO).
pBTO: protected BTO (2,2-dimethylbenzo[d][1,3]dioxol-5-ol); p alkyl-BDO: protected alkyl-BDO (2,2-dimethyl-5-
(alkyloxy)benzo[d][1,3]dioxole).
2.2. Alkyl-BDOs Inhibit Bacterial Cell Growth
The ability of alkyl-BDOs with varying alkyl chain lengths to inhibit bacterial cell
growth was initially assessed by monitoring the growth of Xanthomonas citri subsp. citri
and Bacillus subtilis liquid cultures treated with the compounds at different concentrations.
B. subtilis is used as a model to test for activity against Gram-positive bacteria. Alkyl-
BDOs with lateral chains ranging from five to eight carbons completely inhibited both
Xanthomonas citri subsp. citri and Bacillus subtilis cell growth for 24 h at the concentration
of 100
concentration of 50
µ
g
·
mL−1 (see Supporting Information S2). The inhibitory effect was strong at the
mL−1 and there was a mild effect at 12.5 mL−1, showing a clear
µg
·
µg
·
dose–response correlation. The presence of alkyl-BDOs caused a reduction in the maximum
optical density (OD) of bacterial populations and a delay in the initiation of exponential
growth phase.
Although the method does not allow a precise quantification of this inhibitory effect,
we can observe that (a) at 100
effect, X. citri is able to start bacterial growth after ~12 h of treatment; (b) at 50
the growth curve of X. citri treated with 9-BDO already shows a slope similar to the control
curve (delayed by about 9 h), where X. citri treated with 8-BDO starts growth after ~12 h
and only the compounds with 4 to 7 carbons in their lateral chain are capable of maintaining
µg
mL−1, although 9-BDO initially shows an inhibitory
·
µg ,
mL−1
·
total inhibition over 24 h; (c) at 25
4- and 5-BDO also resemble the control curve (delayed by about 6 h) and X. citri treated
µg
mL−1, the growth curves of X. citri treated with
·
with 6-, 7- and 8-BDO start growth after ~9 h, without reaching exponential phase; and
(d) at 12.5
µg
mL−1, all growth curves eventually reach maximum OD, with treatment
·
with 7-BDO notably causing the longest delay in the onset of bacterial growth. It is notable
that, while 7-BDO is one of the most active compounds, its protected intermediate p7-BDO
does not exert any inhibitory activity against the tested bacteria, confirming the essential
role of the two phenolic groups in the activity of these molecules, as also observed for
dihydroxybenzoates and dihydroxyphenyl alkanoate [4,16].

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2.3. Alkyl-BDOs Have Bactericidal Activity
Alkyl-BDO activity against Gram-negative (Xanthomonas citri subsp. citri and Es-
cherichia coli) and Gram-positive bacteria (Bacillus subtilis and Lactococcus lactis) was further
assessed and the minimum inhibitory concentrations (MICs) determined by broth microdi-
lution method with concentrations ranging from 100 to 12.5
µg
mL−1. X. citri, B. subtilis,
·
and L. lactis showed susceptibility (MIC up to 100
µ
g
·
mL−1) to all alkyl-BDOs with lateral
chains ranging from 5 to 8 carbons (Table 1). Therefore, we found no evidence of specificity
for Gram-positive or -negative bacteria by these compounds. The minimum inhibitory
concentration of alkyl-BDOs against X. citri is within the same range as the MIC found for
commercially available copper oxychloride (43.1 µg·mL−1) [4].
The minimum bacteriostatic/bactericidal concentration (MBC) assay results confirmed
the bactericidal nature of alkyl-BDO
showing complete absence of bacterial growth after treatment with the active compounds.
As expected, the positive control (kanamycin, 20
mL−1) showed a total absence of
´s inhibitory activity (see Supporting Information S2),
µg
·
bacterial growth, while there was no growth inhibition in the negative control with dimethyl
sulfoxide (DMSO 1% v/v). Therefore, the bactericidal activity of alkyl-BDOs was shown to
be similar to that of di and trihydroxybenzoates, with only 8- and 9-BDOs being slightly
less active than the corresponding esters (Figure 1a). Despite this small difference, there is
no evidence that the ester or ether groups could play a significant role in the activity of
these molecules, since the MIC/MBC values determined for both series were very similar,
especially when compared to the variations observed between compounds with carbon
chains of different sizes.
Table 1. MIC/MBC (in
µ
g
·
mL−1) of alkyl-BDOs and two intermediate compounds. MIC: minimum inhibitory concentration;
MBC: minimum bacteriostatic/bactericidal concentration; MW: Molecular Weigth; B. subtilis: Bacillus subtilis; E. coli:
Escherichia coli; L. lactis: Lactococcus lactis.
Compound
R
MW
X. citri
B. subtilis
E. coli
L. lactis
4-BDO
5-BDO
6-BDO
7-BDO
8-BDO
9-BDO
12-BDO
14-BDO
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)6CH3
(CH2)7CH3
(CH2)8CH3
(CH2)11CH3
(CH2)13CH3
182.22
196.25
210.27
224.30
238.33
252.35
294.44
322.49
50
50
50
>100
100
50
50
50
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
100
50
25
25
100
100
>100
alkyl-BDOs
50
100
>100
>100
>100
Intermediates
pBTO
p7-BDO
H
166.18
264.37
>100
>100
>100
>100
>100
>100
>100
>100
(CH2)6CH3
As observed for dihydroxybenzoates [4], alkyl-BDOs with side chains longer than
7 carbons exhibited little or no activity, as phenolic compounds with strongly hydrophobic
tails tend to have their solubility reduced in the culture medium, preventing them from
crossing the bacterial cell membrane. In addition, although compounds 4- to 7-BDO have
an identical MIC value, those with less than seven carbons in their side chain did not
have as strong and prolonged an effect as 7-BDO at lower concentrations (see item 2.2),
indicating that the ideal length of the side chain for optimal alkyl-BDOs activity against X.
citri is seven carbons.
2.4. Alkyl-BDOs Permeabilize the Bacterial Cell Membrane
To investigate the mode of action of the active alkyl-BDOs, the membrane integrity
of X. citri and B. subtilis cells was evaluated by staining with nucleic acid dyes (Syto9
and propidium iodide) and fluorescence microscopy. Syto9 crosses intact membranes
and stains all cells, while propidium iodide can only penetrate cells with a permeabilized

Molecules 2021, 26, 1436
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membrane and quenches Syto9, allowing the identification of both intact and permeabilized
cells. Imaging of X. citri and B. subtilis treated with the intermediate compound p7-BDO
(
Figure 2A,E) and compound 7-BDO (Figure 2B,F) showed that while the intermediate
does not disturb the membrane in the majority of cells, compound 7-BDO was able to
permeabilize cells of both Gram-negative and Gram-positive bacteria within 15 min of
treatment (Table 2). Panels C and G in Figure 2 correspond to the negative control cells
with intact membrane and panels D and H to positive control cells with permeabilized
membrane. Membrane disruption has also been highlighted as the main mode of action of
phenolic molecules with antimicrobial activity by several other studies [17
–21], including
anti-X. citri compounds previously reported by our group [3,4,22,23].
−1
Figure 2. Fluorescence microscopy images of X. citri (
A
–
D
) and B. subtilis (
E
D
–
,
H
H
) treated with p7-BDO at 100
µg
·
mL
(A,E),
−1
7-BDO at 50
µg
·
mL
(
B,F
), negative control (
C,G
), and positive control (
). Panels show, respectively, phase contrast,
FITC, and TRITC images.
Table 2. Percentage of X. citri and B. subtilis cells with intact or permeabilized membrane after
treatment with intermediate p7-BDO and compound 7-BDO observed by fluorescence microscopy
imaging.
Treatment
X. citri
Permeabilized
B. subtilis
Permeabilized
Intact
Intact
p7-BDO
7-BDO
negative
positive
69
0
93
0
31
100
7
94
0
86
0
6
100
15
100
100
n > 225 cells per treatment.
2.5. Alkyl-BDOs Remain Active against X. citri While in Solution
Taking into account that active compounds against citrus canker are sprayed in
aqueous solution [24] combined with our earlier observation of decomposition pathways
for BTO [7], the ability of alkyl-BDOs to keep their antibacterial activity while stored in a

Molecules 2021, 26, 1436
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solution was assessed. This was done by comparing the MICs initially determined to MICs
observed in broth microdilution assays performed with compounds pre-incubated for 24 h
at 30 ◦C prior to inoculation. After pre-incubation, all alkyl-BDOs kept the same level of
activity against X. citri but were no longer able to inhibit B. subtilis growth (Table 3). In
addition to possible discrepancies caused by the metabolism of each species, the standard
growth media used to cultivate and determine the MIC of X. citri and B. subtilis also differ
in composition. As a standard, X. citri is grown in nitrogen–yeast–glycerol medium (NYG)
medium and B. subtilis in Lysogeny broth (LB)–Lennox, which could be the cause for the
total loss of activity of alkyl-BDOs against B. subtilis in contrast to the maintained effect
against X. citri.
Table 3. Comparison of MIC (in
compounds’ pre-incubation.
µg
mL−1) of alkyl-BDOs and two intermediates with and without
·
Compound
X. citri
B. subtilis
Pre-Incubation
No
24 h
No
24 h
4-BDO
5-BDO
6-BDO
7-BDO
8-BDO
9-BDO
pBTO
50
50
50
50
50
50
>100
100
50
50
50
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
50
50
100
>100
>100
>100
100
>100
>100
>100
p7-BDO
Since the results observed in the broth microdilution assays performed with com-
pounds’ pre-incubation suggested that medium composition could be a factor affecting
the ability of alkyl-BDOs to retain their antibacterial activity in solution, the test was then
applied for the same bacteria (X. citri) in different media, allowing for a comparison of
possible medium composition effects. As observed with B. subtilis, all alkyl-BDOs had
their activity reduced against X. citri when pre-incubated for 24 h in LB–Lennox medium
(Table 4), while the same does not happen in NYG medium, suggesting that the differences
in composition between LB–Lennox and NYG media should have an influence over the
stability of the compounds. The main differences between both media are that NYG is com-
posed of 2% glycerol and LB–Lennox has a NaCl concentration more than 60 times higher
than that of the NYG medium; the relatively high salt concentration in LB–Lennox may
promote the aggregation of the compounds and reduce their availability in the medium
over time.
Table 4. Comparison of MIC (in
growth media with different composition, with and without pre-incubation of the compounds. NYG:
nitrogen–yeast–glycerol medium; LB: Lysogeny broth.
µg
mL−1) of alkyl-BDOs and two intermediates against X. citri in
·
X. citri MIC/MBC
Compound
NYG
No
NYG
24 h
LB–Lennox
No
LB–Lennox
24 h
Pre-Incubation
4-BDO
5-BDO
6-BDO
7-BDO
8-BDO
9-BDO
pBTO
50
50
50
50
50
50
50
50
50
100
100
100
>100
>100
>100
>100
>100
50
50
50
100
100
>100
>100
100
100
>100
>100
100
100
>100
>100
p7-BDO

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2.6. Alkyl-BDOs Cytotoxicity
The cytotoxicity of compounds 6-BDO, 7-BDO, and 8-BDO against two mammalian
cell lines was determined by the resazurin reduction method. While none of the tested
compounds were toxic against the rat ascite macrophage cell line J774A.1, they all showed
low toxicity to human lung fibroblast cells MRC-5 (Table 5) when compared to doxorubicin,
a widely used chemotherapy medication [25]. The toxicity levels shown by alkyl-BDOs
against MRC-5 cells are similar to those reported for other polyphenolic molecules with
promising activity against Xanthomonas citri subsp. citri, namely alkyl gallates [26] and
dihydroxybenzoates [4]. Some gallates (propyl, octyl, and dodecyl) and other similar com-
pounds (alkyl hydroxybenzoates) are regulated by the U.S. Food and Drug Administration
(FDA) as food additives for human consumption, being used by the food industry as
antioxidants, flavorings/adjuvants or antimicrobials; therefore, alkyl-BDOs should also be
expected to be safe for application in the field or even for contact with food.
Table 5. Cytotoxicity index (IC50) of compounds 6-BDO, 7-BDO, and 8-BDO against cell lines MRC-5
and J774A.1.
IC50
*
Compound
MRC-5
J774A.1
6-BDO
7-BDO
8-BDO
38.00 ± 4
34.41 ± 4
39.33 ± 5
8.7
>100
>100
>100
-
doxorubicin
* values expressed in µg·mL−1.
3. Materials and Methods
3.1. Chemicals and Analytical Equipment
All reagents and solvents were acquired from commercial courses using the highest
available purity; BTO (95%, Fluorochem), ethyl acetate (
99.8%, ChromAR®), pyridinium
p-toluenesulfonate ( 99%, Sigma-Aldrich), silica for column chromatography (FlashPure
12 g, Büchi), acetone (
99.5%, ChromAR®), by cesium carbonate (99%, Sigma-Aldrich),
tetrabutylammonium iodide ( 99%, Sigma-Aldrich) High Resolution Mass Spectrometry
≥
≥
≥
≥
(HRMS) and Fourier Transform Infrared Spectroscopy (FTIR). NMR and FTIR spectra and
compounds’ characterization can be found in the SI. 1H and 13C NMR spectra were recorded
on an Agilent Technologies 400 MHz NMR Premium Shielded Magnet using DMSO-d6
as solvent at room temperature. HRMS were measured with an Thermo LTQ Orbitrap
XL with ESI ionization; samples were injected and eluted with 0.15 mL/min acetonitrile
+ 0.1% NH3. FTIR analyses were performed on Shimadzu IRTracer-100 equipment with
ATR Specac Golden Gate KRS5. Melting points were determined with Büchi Melting Point
M-560 equipment.
3.2. Synthesis, Purification, and Characterization of Alkyl-BDOs
The general protocol for the synthesis of a series of new phenolic compounds based
on 1,2,4-benzenetriol was established as a three-step route: (a) protection of BTO was
achieved by refluxing 7.9 mmol of BTO with 117.2 mmol of dimethoxypropane in 250 mL of
ethyl acetate, catalyzed by pyridinium p-toluenesulfonate (0.44 mmol) for 20 to 24 h ([11],
with modifications) under a nitrogen atmosphere, followed by extraction with ethyl ac-
etate/water and purification by silica column (heptane/3–5% gradient of EtOAc); (b) pro-
tected BTO was etherified under normal atmosphere by refluxing 2.4 mmol with 3.6 mmol
of alkyl bromide in 36 mL of acetone and catalyzed by cesium carbonate (0.36 mmol) and
tetrabutylammonium iodide (3.1 mmol) for 24 h, followed by the solvent evaporation and
extraction with diethyl ether/water; and (c) deprotection of crude BTO derived ethers
was achieved under normal atmosphere at acidic conditions by refluxing the product
for 12 to 14 h in 7.5 mL of acetic acid added of 2.5 mL of HCl 3 M, followed by solvent

Molecules 2021, 26, 1436
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evaporation, extraction with diethyl ether/water and purification by silica chromatography
(heptane/7–12% gradient of EtOAc). The progress of each reaction was verified by thin
layer chromatography (7:3 n-heptane:ethyl acetate) and the products were characterized
by 1H and 13C Nuclear Magnetic Resonance (NMR).,
3.3. Bacterial Strains and Standard Growth Conditions
The Gram-negative and Gram-positive bacteria tested and the standard growth condi-
tions used are summarized in Table 6. Xanthomonas citri subsp. citri 306 is the pathogenic
strain sequenced and described by the São Paulo State consortium [27] and was kindly
provided by Dr. H. Ferreira (UNESP, Rio Claro/SP, Brazil). The other strains are all from
our laboratory collection and are available on request.
Table 6. Bacterial strains and standard growth conditions.
Strain
Growth Conditions *
◦
Xanthomonas citri subsp. citri 306
Bacillus subtilis 168
NYGB (30 C)
◦
LB–Lennox (30 C)
LB–Lennox (30 C)
M17 + glucose 0.5% (30 C)
◦
Escherichia coli MG1655
Lactococcus lactis MG1363
◦
* NYGB: peptone 5 g
·
L
−1, yeast extract 3 g
·
·
L
−1, glycerol 20 g
·
L−1 (pH ~6.7). LB–Lennox: tryptone 10 g −1, yeast
L
·
extract 5 g
·
L
−1, NaCl 5 g
L−1 (pH ~7). M17: tryptone 5 g
·
L
−1, peptone 5 g
·
L
−1, yeast extract 5 g −1, beef extract
L
·
2.5 g·L−1, ascorbic acid 0.5 g·L−1, MgSO4 0.25 g·L−1, disodium β-glycerophosphate 19 g·L−1 (pH ~7).
3.4. Bacterial Growth Inhibition Assessment
X. citri, B. subtilis, E. coli, and L. lactis were cultivated overnight, diluted in fresh medium
(Table 6) to an optical density at 600 nm (OD600) of ~0.1, incubated until OD600 = 0.3–0.4
and then diluted again 100 times in fresh medium. Aliquots with 100 L of culture
dilutions treated with alkyl-BDOs at concentrations ranging from 100 to 12.5
mL−1
,
µ
µg
·
were distributed in a 96-well microplate and incubated at 30 ◦C during 24 h with constant
agitation. The minimum inhibitory concentrations (MICs) were determined by broth
microdilution method as the lowest concentration in which bacterial growth could not
be detected after 24 h incubation. To assess the nature of alkyl-BDOs inhibitory activity,
the minimum bacteriostatic/bactericidal concentration (MBC) method was applied by
transferring a few microliters of each well (after incubation with the compounds) to an
agar plate without compound followed by incubation under standard conditions to allow
bacterial growth. MIC and MBC values were established according to the results of three
biological replicates. The broth microdilution method provides a range for the MIC and
MBC (for example, between 25 to 50
µg
mL−1) and, as standard, the higher value of this
·
range is defined as the MIC/MBC. To follow growth inhibition in real time, X. citri and
B. subtilis were precultured as described, the 96-well microplate was incubated at 30 ◦C
during 24 h with constant agitation in a Synergy Power Wave reader (BioTek), and OD600
was measured every 30 min. For every condition, three cultures were monitored. Plots of
the growth curves (mean of three with SD indicated) were prepared using the software
Graphpad Prism 6. Stock solutions were prepared in DMSO and the growth conditions
were performed as indicated in Table 6. Kanamycin (20
were used as positive and negative controls, respectively.
µg
mL−1), and DMSO 1% (v/v)
·
3.5. Membrane Integrity Evaluation
Cell membrane integrity was assessed by fluorescence microscopy. Fluorescent dyes
SYTO9 and propidium iodide were applied prior to visualization following instructions for
Live/Dead BacLight bacterial viability kit (Molecular Probes L7007). X. citri and B. subtilis
cells were harvested at OD600 = 0.3–0.4 and treated with alkyl-BDOs for 15 min at 30 ◦C.
◦
DMSO 1% was used as negative control, heat shock (65 C for 15 min) was used as positive
control for X. citri and nisin (5
immobilized in agarose layer and observed with Nikon Ti microscope equipped with an
µ
g
·
mL−1) as positive control for B. subtilis. Cells were

Molecules 2021, 26, 1436
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ORCA-flash 4.0 camera (Hamamatsu) and filters FITC and TRITC. Images were acquired
with NIS Elements 4.10 software and processed using ImageJ 1.52p.
3.6. Alkyl-BDOs Stability
−1
Broth microdilution plates with alkyl-BDOs dilutions ranging from 100 to 12.5
µg mL
·
were prepared and pre-incubated for 24 h at 30 ◦C with constant agitation prior to X. citri
and B. subtilis inoculation. Broth microdilution assay was then performed as described in
Section 3.3, following the standard growth conditions for each bacteria. The results were
compared to MICs determined in the same medium without pre-incubation. To evaluate
the influence of medium composition on alkyl-BDOs stability, the same test was applied to
X. citri in both NYG and LB–Lennox media.
3.7. Determination of the Cytotoxicity Index (IC50)
The cytotoxicity index (IC50) for three alkyl-BDOs (6-BDO, 7-BDO, and 8-BDO) was
determined by metabolic reduction of resazurin. Mammalian cell lines MRC-5 (ATCC
CCL-171) and J774A.1 (ATCC TIB-67) were cultivated respectively in DMEM (GIBCO®)
and RPMI (GIBCO®) media supplemented with 10% fetal bovine serum and 1% antibiotic
and antimycotic (GIBCO®). In 96-well plates, 100
µL of cell suspension at a concentration
of 106 cells/mL were placed in each well. The plates were incubated in a humidified
incubator for 24 h at 37 ◦C under 5% CO2 atmosphere to allow cell adhesion to the plate.
The cultivation medium was then replaced by 100
DMSO were diluted in cultivation medium, with final DMSO concentration no higher than
1%) in concentrations ranging from 100 to 0.39
mL−1 and the plates were incubated
µL of compound solution (stocks in
µg
·
for 24 h under the same conditions. Resazurin solution was added to each well and
fluorescence readings (530 nm excitation, 590 nm emission) were performed after 2 h
incubation. Metabolic reduction of resazurin to resorufin was used as indicator of cell
viability and IC50 was defined as the highest compound concentration at which 50% of the
cells were viable relative to the negative control [28]. A doxorubicin (Fauldoxo®) control
was used for comparison; cell growth in cultivation medium and sterile medium were used
respectively as positive and negative controls.
4. Conclusions
We have shown that 1,2,4-benzenetriol (BTO) is a suitable precursor for the synthesis
of a series of new compounds that are active against citrus canker. This was done by
the synthesis of alkyl derivatives (alkyl-BDOs) via a three-step protocol which showed
bactericidal activity against X. citri. The main economic and environmental advantages
of using BTO as starting material for the synthesis of new compounds are that it can be
obtained from low-cost raw material while providing a use for agricultural waste. However,
the synthesis route by which it was possible to obtain the compounds described here does
not conform to the principles of green chemistry and needs to be improved by selective
alkylation or esterification procedures. Alkyl-BDOs showed bactericidal activity against
Gram-positive (B. subtilis and L. lactis) and negative (X. citri) bacteria, acting through the
permeabilization of their cell membranes. The obtained alkyl-BDOs had a performance
similar to that of gallates and dihydroxybenzoates (with greener synthesis route) previously
studied by our group, and therefore, it seems to us that the disadvantages caused by the
synthesis of alkyl-BDOs do not outweigh the possible advantages of using BTO as a
starting material. Nevertheless, we consider that 1,2,4-benzenetriol is certainly a molecule
of interest as a precursor for the synthesis of new compounds against citrus canker in the
future, especially if HMF, and thus BTO, become general cheap, readily available bio-based
building blocks.
Supplementary Materials: The following are available online. Section S1: NMR spectra and other-
compound characterization; Section S2: Bacterial growth inhibition assessment.

Molecules 2021, 26, 1436
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Author Contributions: Conceptualization, D.-J.S., P.J.D., C.W.L., F.R.P. and L.B.C.; methodology,
P.J.D., D.-J.S., C.W.L., F.R.P. and L.B.C.; validation, L.B.C., F.G. and F.R.P.; formal analysis, L.B.C., F.G.
and F.R.P.; investigation, L.B.C., F.G. and F.R.P.; writing—original draft preparation, L.B.C., P.J.D. and
D.-J.S.; writing—review and editing, P.J.D., D.-J.S. and L.B.C.; visualization, L.B.C., F.G. and F.R.P.;
supervision, D.-J.S., P.J.D. and C.W.L.; project administration, D.-J.S. and P.J.D.; funding acquisition,
D.-J.S., P.J.D. and F.R.P. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the collaborative grant “Using agricultural waste to combat
plant pathogens-environmental-friendly ways to combat Xanthomonas citri” from the Netherlands
Organization for Scientific Research (grant 729.004.026) and FAPESP (grant 2017/50216-0).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We would like to thank Henrique Ferreira (UNESP, BR) for kindly providing
the X. citri strain, ing. Renze Sneep (University of Groningen, NL) for the HRMS analyses and ing.
Léon Rohrbach (University of Groningen, NL) for the FT-IR analyses.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds described in this manuscript are available from the
authors.
Abbreviations
alkyl-BDOs 4-alkoxy-1,2-benzene diols
BTO
1,2,4-benzenetriol
HMF
hydroxymethylfurfural
DBU
pBTO
1,8-Diazabicyclo[5.4.0]undec-7-ene
Protected BTO; 2,2-dimethylbenzo[d][1,3]dioxol-5-ol
Protected intermediate; 2,2-dimethyl-5-(heptyloxy)benzo[d][1,3]dioxole
4-(butoxy)benzene-1,2-diol
4-(pentyloxy)benzene-1,2-diol
4-(hexyloxy)benzene-1,2-diol
4-(heptyloxy)benzene-1,2-diol
4-(octyloxy)benzene-1,2-diol
4-(nonyloxy)benzene-1,2-diol
4-(dodecyloxy)benzene-1,2-diol
4-(tetradecyloxy)benzene-1,2-diol
Xanthomonas citri subsp. citri
Bacillus subtilis
p7-BDO
4-BDO
5-BDO
6-BDO
7-BDO
8-BDO
9-BDO
12-BDO
14-BDO
X. citri
B. subtilis
E. coli
Escherichia coli
L. lactis
MICs
MBC
DMSO
NYG
Lactococcus lactis
Minimum inhibitory concentrations
Minimum bacteriostatic/Bactericidal concentration
Dimethyl sulfoxide
Nitrogen–yeast–glycerol medium
LB–Lennox Lysogeny broth–Lennox medium
IC50
Cytotoxicity index
EtOAc
NMR
HRMS
FTIR
Ethyl acetate
Nuclear magnetic resonance
High-resolution mass spectrometry
Fourier-transform infrared spectroscopy
Optical density at 600 nm
OD600

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