Discotic material hexakis(4-carboxyphenylethynyl)benzene inhibits Escherichia coli growth via the glycolysis pathway

Article abstract of DOI:10.1002/jccs.202000508

Discotic materials and nanoparticles are potential carriers of synthetic chemicals to increase the bioavailability. Several planar discotic compounds were prepared with C–C bond formation by the Sonagoshira reaction. Their toxicity was based on their inhi

Full text of DOI:10.1002/jccs.202000508

Received: 10 November 2020
DOI: 10.1002/jccs.202000508
Revised: 24 December 2020
Accepted: 28 December 2020
C O M M U N I C A T I O N
Discotic material hexakis(4-carboxyphenylethynyl)benzene
inhibits Escherichia coli growth via the glycolysis pathway
Hsiu-Hui Wu1 | Ho-Lun Chen1 | Chih Ying Hsu1 | Chih-Ling Yeh1
Hsiu-Fu Hsu2 | Chien-Chung Cheng1
|
1Department of Applied Chemistry,
Abstract
National Chia-Yi University, Chia-Yi City,
Taiwan, ROC
2Department of Chemistry, Tamkang
Discotic materials and nanoparticles are potential carriers of synthetic
chemicals to increase the bioavailability. Several planar discotic compounds
were prepared with C–C bond formation by the Sonagoshira reaction. Their
toxicity was based on their inhibition of Escherichia coli growth as well as a
simple proteomic analysis. The partial analogues of hexakis(4-car-
boxyphenylethynyl)benzene structures did not inhibit E. coli growth signifi-
cantly. The possible metabolic pathway of hexakis(4-carboxyphenylethynyl)
benzene (1) may involve the downregulation of glycolytic proteins detected
with a proteomic analysis. Hexakis(4-carboxyphenylethynyl)benzene is found
not only useful in liquid crystals but may also have utility in the development
of novel antibiotics.
University, New Taipei City, Taiwan, ROC
Correspondence
Chien-Chung Cheng, Department of Applied
Chemistry, National Chia-Yi University,
Chia-Yi City, Taiwan 60004, ROC.
Email: cccheng@mail.ncyu.edu.tw
Hsiu-Fu Hsu, Department of Chemistry,
Tamkang University, Taipei, Taiwan 251,
Republic of China.
Email: hhsu@mail.tku.edu.tw
Funding information
Ministry of Science and Technology
(MOST) in Taiwan
K E Y W O R D S
drug carrier, multiynylbenzenes, protemoic
The use of drug delivery to increase the bioavailability of
drugs in biological systems is gaining more attention.1–3
Large numbers of synthetic chemicals have been pre-
pared for the development of pharmaceutical agents
directed against a variety of diseases4–6; however, there
are usually no appropriate channels in biological systems
through which synthetic chemicals can traverse the lipid
bilayer.7,8 Most pathways for cell entry are mediated by
diffusion, osmotic pressure, or endocytosis.8 Even when
cell entry is permitted, synthetic chemicals normally have
a small volume, and therefore a short retention period in
the body after their uptake.9 These small chemicals have
recently been embedded in polymers,10 liposomes,2 or
nanoparticles11 to increase the efficiency of their trans-
portation and to improve their retention times.12 Discotic
materials with planar and nanoparticle functions13,14 are
potential carriers of these chemicals and should increase
their bioavailability; therefore, we investigated their tox-
icity based on their inhibition of Escherichia coli growth
as well as a simple proteomic analysis.
Discotic materials have long been investigated in the
development of liquid crystals,15 and discotic nematic liq-
uid crystals have been utilized as materials for improving
the viewing angle in liquid crystal display devices.16,17
A
discotic core structure makes a material easy to stack due
to the strong inter-planar interactions. The planar struc-
tures and multiple functional groups of discotic materials
may confer utility in the generation of micelle-like
nanoparticles that can act as drug carriers. With the capa-
bility of self-assembling into large entities, discotic mate-
rials can be a suitable candidate for increasing the
retention time in biological systems; therefore, we evalu-
ated the toxicity of some planar materials in a biological
system. (Supplementary Information S-1).
Discotic hexa-carboxylic-acid18 as well as tri- and
di-carboxylic-acid derivatives were synthesized, as shown
in Figure 1, to compare their structure–reactivity rela-
tionships, and to examine their toxicity during the inhibi-
tion of E. coli growth. The possible metabolic pathway
involved was determined using a proteomic analysis with
J Chin Chem Soc. 2021;68:239–244.
http://www.jccs.wiley-vch.de
© 2021 The Chemical Society Located in Taipei & Wiley-VCH GmbH
239

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WU ET AL.
HO2C
HO2C
HO2C
CO2H
HO2C
CO2H
CO2H
HO2C
CO2H
CO2H
CO2H
CO2H
HO2C
HO2C
1
CO2H
2
3
4
5
FIGURE 1 Structures of multiynylbenzenes
two-dimensional (2D) sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE). The
strategy used to synthesize target compound 1 (shown in
Figure 1) involved a C–C bond coupling reaction, medi-
ated by a palladium (Pd) catalyst, to construct the basic
framework.
Target compound 1 was characterized by nuclear mag-
netic resonance (NMR) and high-resolution mass spec-
troscopy (MS). In the proton NMR spectrum,
(Supplementary Information S-2) compound 1 showed
two peaks at 7.33 and 7.68 ppm, with a doublet splitting
pattern, and these were assigned to the neighboring pro-
tons in benzene, which is highly symmetric. The identity
and molecular weight (942.173) of compound 1 were con-
firmed using high-resolution fast atom bombardment
MS. The preparation and characterization of the di- and
tri-arm compounds were achieved using a similar proce-
dure with di- and tri-bromobenzene, respectively, instead
of hexabromobenzene, producing the corresponding
compounds with reasonable yields.
The toxicity of the multiynylbenzenes in a biological
system was examined by observing their inhibition of
E. coli (BL21) growth, as shown in Figure 2. Water-solu-
ble multiynylbenzenes 1–5 were prepared by neutralizing
the acid form in MeOH with an aqueous KOH solution
and evaporating it to dryness. The multiynylbenzene salts
were redissolved in water and examined further. Various
concentrations of the multiynylbenzenes were added
directly to the E. coli culture medium under optimized
A typical reaction scheme for the procedure is illus-
trated in Scheme 1. Bromobenzoic acid was converted to
an ester to reduce interference by the acidic proton. Con-
sequently, the ester product gave a better yield in the sub-
sequent C–C bond coupling reaction. An extended arm
with an alkyne group was introduced using the
Sonogashira reaction, in the presence of a Pd catalyst and
cupric iodide (CuI), to generate acetylene-extended com-
pound 6. After the removal of the trimethylsilyl (TMS)
group, compound 7 was reacted with hexabromobenzene
under the conditions of the Sonogashira reaction for
48 hr. It was then subjected to column purification, pro-
ducing the yellow ester compound 8, with six arms, with
a yield of 70%. Target compound 1 was obtained by fur-
ther hydrolysis of compound 8 with KOH at 70ꢀC for
48 hr, and subsequent acidification with an aqueous HCl
solution to produce the acidic form, with a yield of 80%.
O
O
(i)
O
O
(iii)
(ii)
91%
Br
TMS
Br
H
OR
97%
90%
OR
OH
OR
6
7
RO2C
CO2R
HO2C
CO2H
Br
Br
Br
Br
Br
Br
(v)
80%
RO2C
CO2R
HO2C
CO2H
(iv)
70%
R= - Et
RO2C
CO2R
HO2C
CO2H
8
1
TBAF/THF, r.t. ; (iv) Pd(PPh3)2Cl2/PPh3/TMSA, TEA, 80 o
oC
(i)
; (iii)
; (v) KOH/THF/H O, reflux
C
2
; (ii)Pd(PPh ) Cl /PPh /TMSA, TEA, 80
DCC/DMAP/CH2Cl2, rt
3 2
2
3
SCHEME 1 Synthesis of hexakis(4-carboxyphenylethynyl)benzene

WU ET AL.
241
FIGURE 2 Inhibition of the growth of E. coli BL21 in the presence or absence of various concentrations of multiynylbenzenes,
monitored using an ELISA reader. After treatment with (a) compound 1, (b) compound 2, and (c) compound 3. The x axis represents time in
hours, and the y axis represents the variation in intensity at an absorption wavelength of 600 nm
FIGURE 3 Selected spots on a 2D gel containing proteins downregulated by compound 1 at various concentrations. (a) Control
reaction; (b) 40 μM compound 1; (c) 80 μM compound 1; (d) 120 μM compound 1. Selected spots varied in intensity by >2 or < 0.5
bacterial growth conditions, and the cells were grown to
show absorbance at the wavelength of 600 nm (A600) of
0.1. (Supplementary Information S-3) Compound 1
showed the greatest inhibition of bacterial growth after
incubation for 5 hr, evident as the changes in A600
monitored with an enzyme-linked immunosorbent
assay (ELISA) reader. Di-acid compounds 4 and 5, with
linear geometries, and compounds 2 and 3, with planar
geometries, were examined under similar conditions
for comparison. The capacities of these compounds to
inhibit bacterial growth decreased in the order:
1 ꢁ 2 ~ 3 ~ 4 ~ 5, as shown in Figure 2. Negatively
charged species are known to be disadvantaged in
transporting cargo molecules through the lipid bilayers of
cells. Compound 1 has more negative charges than the
other compounds, which may generate more charge
repulsion with biomolecules. Remarkably, compound 1
most strongly inhibited E. coli growth, with approxi-
mately 64% inhibition of cell growth at a concentration of
100 μM. Compound 2 has a triple-arm form and a triskel-
ion-like structure. In biological system, it is known that
triskelia are capable of binding to clathrin,19,20 allowing
their intracellular trafficking in the cytoplasm. It may
indicate that compound 2 to possess a similar binding
affinity to induce the toxicity. As a result, even com-
pound 2, which had fewer negatively charged groups
than 1, inhibited E. coli growth less effectively than com-
pound 1. This suggests that the density of the negative
charge is not necessarily a factor that affects the toxicity
of the compound to E. coli.
Compound 3 has two phenylethynyl groups at the
ortho positions of benzene. It is known that an enediyne
moiety is capable of inducing Bergman-type
cyclization,21,22 causing irreversible double-stranded
DNA scission, when the distance between two triple
bonds is smaller than 3.4 Å. However, compound 3, with
a diyne structure, did not significantly inhibit E. coli
growth; neither did compound 4, with two phenylethynyl
groups at the para positions, nor compound 5, with no
central benzene ring, significantly inhibited E. coli
growth at all. Clearly, the number of carboxylate groups
with negative charges and partial structural frameworks

242
WU ET AL.
of the multiynylbenzenes are not factors that predomi-
nantly affect the toxicity of the discotic material, hexakis
(4-carboxyphenylethynyl)benzene. This suggests that the
hexa-arm structure has a unique property that allows the
discotic material to associate with certain proteins to
inhibit E. coli growth.
To understand the possible inhibitory pathway of
compound 1, the total proteins of E. coli were reacted
with compound 1 salt and analyzed with SDS-PAGE
using a proteomic technique. The total proteins of E. coli
(BL21) were separated by the sequential procedures of
cell harvesting, centrifugation, collection of the precipi-
tate, sonication to liberate the proteins, precipitation with
acetone, quantification with the Bradford assay using
Coomassie Brilliant Blue G-250 and an ELISA reader,
and visualization with SDS-PAGE. Several bands on SDS-
PAGE differed when the cells were treated (or not) with
compound 1, as shown in Figure 3. The target proteins
were identified with the typical procedures of band exci-
sion from the gel, in-gel digestion with trypsin, analysis
TABLE 1 Protein functions of selected spots in E. coli affected by compound 1, hexakis(4-carboxyphenylethynyl)benzene
Conc.
(Vari)a
Accession
name
Mascot
score
No
18
Name
Mass
Protein functions
40-(0.48)
120-(0.45)
80-(0.38)
120-(0.31)
Soluble cytochrome b562 precursor
P0ABE8
14,054
229
Electron transport; transport; iron
binding
26
29
DNA-binding protein H-NS (histone-
like protein
P0ACF8
15,399
784
Transcription regulation; repressor
HLP-II) (protein H1) (protein B1)
Bacterioferritin
40-(0.1)
120-(0.08)
40-(0.16)
P0ABD3
18,484 1,566
Iron storage; ferric binding;
oxidoreductase activity
34
38
RNA polymerase-binding
transcription factor DksA
P0ABS1
P0AFH8
17,517
21,061
353
507
Zinc ion binding
40-(0.21)
80-(0.26)
120-(0.11)
80-(0.37)
120-(0.45)
Osmotically-inducible protein Y
Stress-regulated protein
48
Inorganic pyrophosphatase
Q8FAG0
P0AE08
19,588
664
Hydrolase
Alkyl hydroperoxide reductase
subunit C
20,617
25,646
460
491
Antioxidant; Oxidoreductase
52
60
80 + (2.21)
40 + (2.83)
80 + (3.76)
120 + (2.92)
80-(0.39)
Chloramphenicol acetyltransferase
P62577
P09551
Acyltransferase; Transferase
Transport
Lysine-arginine-ornithine-binding
periplasmic protein
28,089 4,196
101
Glycerophosphoryl diester
phosphodiesterase
P09394
40,817
681
Hydrolase; glycerol metabolism;
calcium-binding
108
117
40-(0.35)
Phosphoglycerate kinase
P0A799
P0A6F7
40,962 1,363
57,162 920
Transferase
40 + (2.17)
60KDa chaperonin (protein Cpn60)
(groEL protein)
Chaperone; ATP binding
123
40-(0.19)
80-(0.12)
120-(0.03)
Enolase
P0A6P9
45,495 1,829
Transferase, the degradation of
carbohydrates
Elongation factor Tu (EF-Tu)
Dihydrolipoyl dehydrogenase
P0A6N1
P0A9P0
43,155 1,039
50,942 2,827
Elongation factor
143
40-(0.23)
Hydrolase; oxidoreductase;
glycolysis
Note: The variation number was calculated by comparing the gel containing proteins from E. coli treated with compound 1 and a control gel of proteins from E.
coli not treated with compound 1. An intensity ratio (with/without compound 1) > 2 or < 0.5 was considered to indicate effective variation.
aconc (var): “40+” indicates increasing intensity after reaction with compound 1 (40 μM); “40– “indicates decreasing intensity after reaction with compound
1 (40 μM); the number in parentheses (x.x) indicates the ratio of the spot intensity compared with that in the control gel.

WU ET AL.
243
with
matrix-assisted
laser
desorption/ionization
(four proteins), which corresponded to ~30% of the 13
downregulated proteins. Analysis of the biological pro-
cesses showed that these downregulated proteins are
largely related to carbohydrate metabolism, particularly
glycolysis, and are involved in energy pathways that are
important for bacterial growth.
(MALDI), and data mining in specific databases. Each
band in a gel usually represents more than one protein,
and membrane proteins are usually not detected in 2D
gel analyses. Treatments of different compound 1 concen-
trations led to intensity variations in some of the bands
on SDS-PAGE as compared to the control in which the
proteins of untreated E. coli were subjected to 2D gel elec-
trophoresis. (Supplementary Information S-4).
Further detailed analysis was performed. In the con-
trol, a total of ~200 obvious spots were identified and
approximately 104 spots were detected when 80 μM com-
pound 1 was added to E. coli, and these spots were ana-
lyzed using a 2D imaging program after normalization.
Because the intensity of a band on a gel is affected by the
protein concentration, among other factors, the variation
in the numbers of spots was determined after normaliza-
tion and compared with the spots in the control gel. As
the intensity of a band on a gel is approximately propor-
tional to the protein concentration, the ratios of the spot
intensities with and without compound 1 can be compared
for the realization of effect of 1 on various E. coli proteins.
A more than two-fold intensity variation is considered to
be a significantly influenced band. Under such screening
condition, compound 1 at 80 μM led to 36 significantly
influenced bands, 13 of which showed increased intensity
and 23 of which showed reduced intensity.
In summary, several planar discotic compounds were
successfully prepared with C–C bond formation, medi-
ated by the Sonagoshira reaction, with reasonable yields.
Their reactivities in inhibiting E. coli growth decreased in
the order: 1 ꢁ 2 ~ 3 ~ 4 ~ 5. Among these discotic
multiynylbenzenes, compound 1, with six carboxylic acid
arms, showed the best reactivity. The di- and tri-arm ana-
logues with partial hexakis(4-carboxyphenylethynyl)ben-
zene structures did not inhibit E. coli growth
significantly. These findings suggest that the negative
charges and partial structural frameworks of discotic
compounds are not essential factors for their toxicity. The
possible metabolic pathway of compound 1, detected
with a proteomic analysis, involves the downregulation
of glycolytic proteins. Therefore, compound 1 may
severely inhibit energy generation, to futher induce cell
death. The discotic structure of hexakis(4-car-
boxyphenylethynyl)benzene is not only useful in liquid
crystals, but may also have utility in the development of
novel antibiotics.
Thirteen of the thirty-six significantly influenced pro-
tein bands by compound 1 were further characterized, as
listed in Table 1. The proteins upregulated by compound 1
included transport proteins (lysine–arginine–ornithine-
binding periplasmic protein, involved in amino acid trans-
port), chaperonins (60-kDa chaperonin of GroEL protein,
involved in protein folding), and antibiosis proteins (chlor-
amphenicol acetyltransferase, involved in bacterial resis-
tance to chloramphenicol). The downregulated proteins
included transferases (enolase, involved in the degradation
of carbohydrates via glycolysis; glycerophosphoryl diester
phosphodiesterase, involved in glycolysis, generating alco-
hol and sn-glycerol 3-phosphate; inorganic pyrophos-
phatase, involved in hydrolysis during lipid degradation;
dihydrolipoyl dehydrogenase, involved in glycolysis; phos-
phoglycerate kinase, involved in glycolysis), transcription
factors (DNA-binding protein H-NS, a regulatory repressor;
RNA polymerase-binding transcription factor DksA,
involved in zinc ion binding), storage proteins
(bacterioferritin, involved in iron storage), and transport
proteins (soluble cytochrome b562, an electron-transport
protein; osmotically inducible protein Y, involved in the
stress response).
ACKNOWLEDGMENTS
The authors thank Prof. Tung-Kung Wu at National
Chao-Tung University for kindly providing the mass
spectroscopy facilities. We gratefully acknowledge the
financial support of the Ministry of Science and Technol-
ogy (MOST) in Taiwan.
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SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
How to cite this article: Wu H-H, Chen H-L,
Hsu CY, Yeh C-L, Hsu H-F, Cheng C-C. Discotic
material hexakis(4-carboxyphenylethynyl)benzene
inhibits Escherichia coli growth via the glycolysis
pathway. J Chin Chem Soc. 2021;68:239–244.
https://doi.org/10.1002/jccs.202000508
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