Fine-tuning of the hydrophobicity of caffeic acid: studies on the antimicrobial activity against Staphylococcus aureus and Escherichia coli

Article abstract of DOI:10.1039/c5ra05840f

The increased bacterial multidrug resistance caused by inappropriate use and overuse of antimicrobials is a global concern. To circumvent this issue, a quest for the development of new active agents has been widely recognized. Some phytochemical products,

Full text of DOI:10.1039/c5ra05840f

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PAPER
Fine-tuning of the hydrophobicity of caffeic acid: studies on the
antimicrobial activity against Staphylococcus aureus and Escherichia
coli
Received 00th April 20xx,
Accepted 00th April 20xx
Mafalda Andradea¥, Sofia Benfeitob¥, Pedro Soaresb, Diogo Magalhães e Silvab, Joana Loureiroa,
Anabela Borgesa,b,c, Fernanda Borgesb* and Manuel Simõesa*
DOI: 10.1039/x0xx00000x
www.rsc.org/
The increased bacterial multidrug resistance caused by inappropriate use and overuse of antimicrobials is a global concern.
To circumvent this issue, a quest for the development of new active agents has been widely recognized. Some
phytochemical products, produced by plants as part of their chemical defense strategies, are regarded as new stimulus to
develop novel antimicrobials that are not as vulnerable as current drugs to bacterial resistance mechanisms. In this study,
the antimicrobial activity and mode of action of caffeic acid (CAF) and a series of CAF alkyl esters was assessed against
Escherichia coli and Staphylococcus aureus, with the aim of analyzing the influence of the alkyl ester side chain length on
the activity. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), changes in
physicochemical surface properties and intracellular potassium leakage were used as physiological indices for the
antimicrobial mode of action. CAF alkyl esters were found to be effective antimicrobial agents against both bacteria. Their
activity was directly dependent on their lipophilicity, which affected bacterial susceptibility, the physicochemical
properties of the bacteria and the integrity of the membranes. E. coli was less susceptible than S. aureus to the action of
the compounds. Longer alkyl side chains were more effective against the Gram-positive bacterium, while medium length
alkyl side chain compounds were more effective against the Gram-negative bacterium. Caffeic acid derivatives are
proposed to act as cell permeabilizers, inducing membrane alterations, causing rupture with potassium leakage,
particularly on the Gram positive bacterium, and consequent cell death.
agents have been widely recognized as being used inappropriately,
and constantly subjected to overuse, underuse and general misuse
over the years. On bacteria, these type of mishandlings create a
1 Introduction
Control of microbial growth is required in many microbiologically
sensitive environments, especially when the conditions for their
proliferation are favorable.1 For this purpose, antibacterial agents
selective pressure for the development of bacterial resistance
and/or multidrug resistance to these compounds, with resistance
being transmitted within and between individuals.2,5–8 The
emergence of resistant microorganisms is a global concern and has
led to a quest for the search and development of new alternative
antimicrobial products not so vulnerable as current drugs to
bacterial resistance mechanisms.7–10
2,3
such as biocides and antibiotics are usually employed
and their
selection differ by the extent of their pharmacological specificity
and of their degree of mammalian toxicity. Antibiotics usually have
a single biochemical target (i.e., a selective toxicity) and are used
against bacterial infections in human beings and animals. On the
other hand, biocides generally possess several distinct targets, with
diverse susceptibilities (i.e., a broad spectrum of usage) and are
often regarded as antiseptics (used externally on human skin),
disinfectants (for surface sanitizing), and/or preservatives
(incorporated in pharmaceutical, cosmetic or other types of
products to prevent microbial contamination).1,2,4 However, these
Plants produce
a
vast array of secondary metabolites
(phytochemicals), a number of which are commonly believed to be
involved in chemical strategies to protect themselves against
pathogen microbial attack of fungi, yeasts and bacteria.11,12 The use
of phytochemicals as antimicrobial agents is at present considered a
strategic approach to overpass the mentioned drawbacks because
they not only have multiple and different modes of action from
current antibiotics (posing a low risk for the development of
resistance), but are also derived from natural sources presenting a
green and safe status.13,14 Bacterial susceptibility to phytochemicals,
especially phenolic compounds, have been studied by several
authors and, in general, they present a promising antimicrobial
profile.8,9,15–22 Polyphenols are the most important and abundant
group of phytochemicals and can be found in diverse dietary
products like vegetables, fruits, chocolates and beverages (as
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coffee, tea or wine).23–25 Phenolic compounds can be divided into
two main groups: flavonoids and non-flavonoids that include
several chemical classes, such as flavonols, isoflavones,
anthocyanins, phenolic acids.24,26 Phenolic acids are the most
common non-flavonoid naturally occurring phenolics which contain
two distinguishing constitutive carbon frameworks: the
hydroxycinnamic (C6C3) and hydroxybenzoic (C6C1) structure. Only a
minor fraction of phenolic acids exists in the free form. Instead, the
majority are linked through ester, ether or acetal bonds either to
structural components of the plant, larger polyphenols or smaller
organic molecules (e.g., glucose, quinic acid). Particular attention is
at present given to hydroxycinnamic acids due to their remarkable
biological properties, including antimicrobial activity of broad
spectrum.24,26,27 The antimicrobial mode of action of phenolic acids
can be due to their ability to destabilize and permeabilize the
cytoplasmatic membrane, inhibition of enzymes involved in radical
generation and also the inhibition of the synthesis of nucleic acids
of both Gram-negative and -positive bacteria.8 Caffeic acid (Fig. 1) is
generally the most abundant hydroxycinnamic acid, representing
between 75% and 100% of the total hydroxycinnamic acid content
of diverse fruits and beverages.24,28,29
charcoal, when necessary. Solvents were evaporated in a Buchi
Rotavapor.
2.1.2 Apparatus. 1H and 13C NMR spectra were acquired, at room
temperature, on
a Brüker Biospin GmbH 400 spectrometer
operating at 400 and 100 MHz, respectively. Chemical shifts were
expressed in δ (ppm) values relative to tetramethylsilane (TMS) as
internal reference; coupling constants (J) were given in Hz.
Assignments were also made from DEPT (Distortionless
Enhancement by Polarization Transfer) (underlined values). Electron
impact mass spectra (EI-MS) were acquired on a VG AutoSpec
instrument; data were reported as m/z (% of relative intensity of
the most important fragments). Microwave-assisted synthesis was
performed in Biotage® Initiator Microwave Synthesizer.
2.2 Synthesis of alkyl caffeates
2.2.1 Synthesis of alkyl caffeates with short chain length. The
synthetic procedure was adapted from Garrido et al.34 The cinnamic
acid (1.0 g), the ethanol or butanol (30 mmol) and 2 drops of
concentrated sulphuric acid were added to a glass vial (2-5 mL) that
The efficacy of phytochemicals as antimicrobials as well as their
diversity provides a renewed interest towards the discovery of new
antibacterial drugs, being a new hope to overcome the bacterial was then sealed. The system was heated 20 0C above the boiling
resistance problem8,16. They can constitute new scaffolds for drug
point of the alcohol for 10 min in microwave reactor cavity under
discovery and development programs as they can be tailored for
fine-tuning their drug-like properties and so replenish the antibiotic
mechanical stirring. After cooling to room temperature, the crude
products were extracted with ethyl acetate (3 × 10 mL) and washed
with water (3 × 10 mL). The combined organic layers were dried
with Na2SO4, filtered and solvent was evaporated. The resulting
residues were purified by flash chromatography using silica gel as
stationary phase and petroleum ether/ethyl acetate as eluent.
pipeline.30-33 In this context,
a structure-antimicrobial activity
relationship (SAR) study was carry out focused on caffeic acid
(trans-3,4-dihydroxycinnamic acid, CAF) (Fig. 1), a phytochemical
that has been shown to display interesting antibacterial activity,9
and an homologous series (C2 to C10) of alkyl esters derivatives
(Schemes 1 and 2). In addition, the study of the effects of the
increment of CAF lipophilicity on the planktonic bacterial growth of
Staphylococcus aureus and Escherichia coli was performed. Their
effects on bacterial physicochemical properties and membrane
integrity was also evaluated.
trans-Ethyl 3-(3,4-dihydroxyphenyl)propenoate (CAFC2):
Yield: 78%, 1H NMR (DMSO-d6): 1.23 (3H, t, J = 8.0 H(2’)), 4.14
(2H, m, H(1’)), 6.25 (1H, d, J = 16.0 H(β)), 6.76 (1H, d, J = 8.0
H(5)), 6.99 (1H, dd, J = 8.0; 2.0 H(6)), 7.04 (1H, d, J = 2.0 H(2)),
7.46 (1H, d, J = 16.0 H(α)). 13C NMR (DMSO): 14.3 (C(2’)), 59.7
(C(1’)), 114.1 (C(2)), 114.8 (C(α)), 115.7 (C(5)), 121.4 (C(6)),
125.5 (C(1)), 144.9 (C(β)), 145.6 (C(3)), 148.4 (C(4)), 166.5 (CO).
2 Materials and methods
+
•
MS/EI m/z: 209 (M , 30), 208 (100), 180 (33), 164 (23), 163
(96), 136 (55), 135 (40), 134 (45), 117 (21), 89 (47), 77 (25), 63
(24), 51 (23).
2.1 Chemistry
2.1.1 Reagents. Caffeic acid and all the other reagents were
purchased from Sigma–Aldrich Química S.A. (Sintra, Portugal) and
used without additional purification. The solvents were pro analysis
grade and were acquired from Merck (Lisbon, Portugal). Thin-layer
chromatography (TLC) was carried out on precoated silica gel 60
F254 (Merck) with layer thickness of 0.2 mm. For analytical control,
the following elution systems were used: dichloromethane, ethyl
acetate, dichloromethane/methanol and petroleum ether/ethyl
acetate in several proportions. The spots were visualized under UV
detection (254 and 366 nm). Flash column chromatography was
performed using silica gel 60 (0.040–0.063 mm) (Merck). Following
the workup and after extraction, the organic phases were always
dried over Na2SO4. Solutions were decolorized with activated
trans-Butyl 3-(3,4-dihydroxyphenyl)propenoate (CAFC4):
Yield: 80%, 1H NMR (CDCl3): 0.96 (3H, t, J = 7.4, H(4’)), 1.43
(2H, m, H(3’)), 1.69 (2H, m, H(2’)), 4.21 (2H, t, J = 6.6, H(1’)),
6.26 (1H, d, J = 16.0 H(β)), 6.88 (1H, d, J = 8.4 H(5)), 7.00 (1H,
dd, J = 2.0; 8.4 H(6)), 7.11 (1H, d, J = 2.0 H(2)), 7.58 (1H, d,
J=16.0 H(α)). 13C NMR (CDCl3): 13.8 (C(4’)), 19.2 (C(3’)), 30.8
(C(2’)), 64.8 (C(1’)), 114.4 (C(2)), 115.4 (C(α)), 115.5 (C(5)),
122.4 (C(6)), 127.4 (C(1)), 144.0 (C(3)), 145.3 (C(β)), 146.6
+
•
(C(4)), 168.4 (CO). MS/EI m/z: 236 (M , 78), 180 (98), 163
(100), 136 (43), 135 (26), 134 (30), 89 (27).
2.2.2 Synthesis of alkyl caffeates with long chain length
2 | J. Name., 2015, 00, 1-8
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2.2.2.1 Synthesis of malonic acid half esters. The synthetic
procedure was adapted from Menezes et al.35 Equimolar quantities
(10 mmol) of Meldrum’s acid and the appropriate long chain
alcohols were refluxed in toluene (5 mL) for 4 h. After cooling the
reaction to room temperature 10 mL of a saturated NaHCO3
solution was added. The formation of white foam is instantly
observed. The compounds were extracted with diethyl ether (3 × 10
mL) acidified with HCl, washed with water (3 × 10 mL). The
combined organic layers were dried with Na2SO4, filtered and
solvent was evaporated. The malonic acid half esters were
characterized and used in the next reaction without further
purification.
121.8 (C(6)), 126.0 (C(1)), 145.5 (C(β)), 146.0 (C(3)), 148.8
+
(C(4)), 167.1 (CO). MS/EI m/z (%): 264 (M , 80), 181 (30), 180
•
(100), 163 (90), 136 (49), 135 (34), 89 (38).
trans-Octyl 3-(3,4-dihydroxyphenyl)propenoate (CAFC8):
Yield 53%. 1H NMR (DMSO-d6): 0.86 (3H, t, J = 7.0 H(8’)), 1.30
(10H, m, H(3’)-H(7’)), 1.62 (2H, m, H(2’)), 4.10 (2H, t, J = 6.6
H(1’)), 6.26 (1H, d, J = 15.6 H(β)), 6.76 (1H, d, J = 8.4 H(5)), 6.94
(1H, dd, J = 2.0; 8.4 H(6)), 7.04 (1H, d, J = 2.0 H(2)), 7.47 (1H,
d, J = 15.6 H(α)), 9.40 (2H, 2x s, 2xOH). 13C NMR (DMSO-d6):
15.3 (C(8’)), 23.5 (C(7’)), 26.8 (C(6’)), 29.7 (C(5’)), 30.0 (C(4’),
C(3’)), 32.6 (C(2’)), 65.1 (C(1’)), 115.4 (C(2)), 116.2 (C(α)), 117.1
(C(5)), 122.7 (C(6)), 126.7 (C(1)), 146.4 (C(β)), 146.9 (C(3)),
+
•
149.7 (C(4)), 168.0 (CO). MS/EI m/z: 292 (M , 50), 181 (25),
Monohexyl malonate (1): Yield 61%. 1H NMR(CDCl3): 0.89 (3H,
t, J = 7.0 H(6’)), 1.33 (6H, m, H(3’)-H(5’)), 1.65 (2H, m, H(2’)),
3.43 (2H, s, H(2)), 4.16 (2H, t, J = 6.6 H(1’)). 13C NMR(CDCl3):
14.2 (C(6’)), 22.4 (C(5’)), 25.8 (C(4’)), 29.1 (C(3’)), 31.6 (C(2’)),
41.5 (C(2)), 64.8 (C(1’)), 166.9 (C(3)), 170.5 (C(1)).
180 (100), 163 (70), 136 (30).
trans-Decyl 3-(3,4-dihydroxyphenyl)propenoate (CAFC10):
Yield 42%. 1H NMR (CDCl3): 0.88 (3H, t, J = 6.8 H(10’)), 1.32
(14H, m, H(3’)-H(9’)), 1.70 (2H, m, H(2’)), 4.20 (2H, t, J = 6.6
H(1’)), 6.27 (1H, d, J = 15.6 H(β)), 6.88 (1H, d, J = 8.4 H(5)), 7.01
(1H, dd, J = 8.4; 2.0 H(6)), 7.12 (1H, d, J = 2.0 H(2)), 7.59 (1H, d,
J = 15.6 H(α)). 13C NMR (CDCl3): 14.1 (C(10’)), 22.7 (C(9’)), 26.0
(C(8’)), 28.7 (C(7’)), 29.3 (C(6’), C(5’)), 29.6 (C(4’), C(3’)), 31.9
(C(2’)), 65.0 (C(1’)), 114.5 (C(2)), 115.5 (C(5), 115.6 (C(α)),
122.4 (C(6)), 127.5 (C(1)), 143.9 (C(3)), 145.0 (C(β)), 146.4
Monooctyl malonate (2): Yield 48%. 1H NMR(CDCl3): 0.89 (3H,
t, J = 7.0 H(8’)), 1.30 (10H, m, H(3’)-H(7’)), 1.65 (2H, m, H(2’)),
3.43 (2H, s, H(2)), 4.16 (2H, t, J = 6.6 H(1’)). 13C NMR(CDCl3):
14.3 (C(8’)), 22.4 (C(7’)), 29.3 (C(5’)), 29.4 (C(4’)), 26.9 (C(3’)),
29.2 (C(2’)), 32.3 (C(6’)), 41. 2 (C(2)), 64.7 (C(1’)), 166.8 (C(3)),
169.9 (C(1)).
+
•
(C(4)), 168.1 (CO). MS/EI m/z (%): 320 (M , 28), 180 (100), 163
(47), 134 (22), 98 (24), 84 (24), 57 (40), 55 (34).
Monodecyl malonate (3): Yield 55%. 1H NMR(CDCl3): 0.88 (3H,
t, J = 6.8 H(10’)), 1.30 (14H, m, H(3’)-H(9’)), 1.65 (2H, m, H(2’)),
3.43 (2H, s, H(2)), 4.16 (2H, t, J = 6.6 H(1’)). 13C NMR(CDCl3):
14.0 (C(10’)), 22.7 (C(9’)), 25.7 (C(3’)), 28.8 (C(2’)), 29.2 (C(7’)),
29.5 (C(4’)), 29.7 (C(5’)), 29.9 (C(6’)), 32.1 (C(8’)), 40.8 (C(2)),
64.6 (C(1’)), 166.6 (C(3)), 168.4 (C(1)).
2.3 Microbiological studies
2.3.1 Microorganisms and chemical solutions. S. aureus CECT 976
and E. coli CECT 434, obtained from the Spanish Type Culture
Collection, were selected for this study. The bacteria were
cryopreserved at -80 ⁰C, in a mixture of Mueller-Hinton broth
(MHB, Merck) and 30% (v/v) glycerol and subcultured in Mueller-
Hinton agar (MHA), at 30 ⁰C for 24 h, before testing. Stock solutions
of all tested compounds were prepared in dimethyl sulfoxide
(DMSO, Fisher), under sterile conditions, and kept in the dark, at
room temperature, for a maximum of two weeks. Serial dilutions of
the stock solutions were prepared in DMSO, when needed.
2.2.2.2 Condensation of malonic acid half esters with 3,4-
dihydroxybenzaldehyde. The synthetic procedure was adapted
from Menezes et al.35 Equimolar quantities (1.0 mmol) of the
malonic acid half esters and the 3,4-dihydroxybenzaldehyde were
added to cyclohexane (3-5 mL). Then, anhydrous pyridine (1 mmol)
and β-alanine (1.6 mmol) were added and the mixture was refluxed
for 6-10 h. After cooling the mixture in an ice bath, concentrated
HCl was added dropwise. After pH neutralization the mixture was
extracted with diethyl ether (3 × 10 mL) and water (3 × 10 mL). The
2.3.2 Evaluation of minimum inhibitory concentration (MIC) and
organic layer was dried with Na2SO4, filtered and solvent was minimum bactericidal concentration (MBC). Bacterial cells were
evaporated under vacuum. The residues were purified by flash
chromatography using gradient elution (petroleum ether/ethyl
acetate).
grown overnight in MHB at 30 ⁰C and 120 rpm in a Sartorius
Certomat® BS-1 (Portugal) incubator. Afterwards, an inoculum was
taken and adjusted to an optical density (OD) of 0.1 ± 0.02 (OD600
nm). MIC values were determined in sterile 96-well flat-bottomed
polystyrene tissue culture microtiter plates (Orange Scientific,
Belgium). In each well, a volume of 20 μL of compound’s solution
was added to 180 μL of cell culture. All compounds were tested in a
range of different concentrations to reach the MIC. Cell suspensions
with DMSO and cell suspensions without caffeic acid and
derivatives were used as negative controls. Ciprofloxacin (Sigma–
Aldrich Química S.A.) was used as positive control for MIC and MBC
determination. The OD600 was measured at t = 0 h and at t = 24 h,
trans-Hexyl 3-(3,4-dihydroxyphenyl)propenoate (CAFC6):
Yield 69%. 1H NMR (DMSO-d6): 0.87 (3H, t, J = 7.0, H(6’)), 1.31
(6H, m, H(3’)-H(5’)), 1.62 (2H, m, H(2’)), 4.10 (2H, t, J = 6.6
H(1’)), 6.26 (1H, d, J = 15.6 H(β)), 6.76 (1H, d, J = 8.4 H(5)), 6.94
(1H, dd, J = 2.0; 8.4 H(6)), 7.04 (1H, d, J = 2.0 H(2)), 7.47 (1H,
d, J = 15.6 H(α)), 9.12 (1H, s, OH), 9.57 (1H, s, OH). 13C NMR
(DMSO-d6): 14.4 (C(6’)), 22.5 (C(5’)), 25.6 (C(4’)), 28.7 (C(3’)),
31.4 (C(2’)), 64.2 (C(1’)), 114.5 (C(2)), 115.3 (C(α)), 116.2 (C(5)),
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using a microtiter plate absorbance reader (Biotek Synergy HT),
after incubation at 30 ⁰C and 120 rpm. The MIC was defined as the
lowest concentration of tested compound at which no bacterial
growth was detected.20,37 After MIC determination, a volume of 10
µL of each concentration tested for MIC assessment was plated out
on MHA. Plates were incubated at 30 ⁰C for 24 h and growth was
visually inspected. The MBC was determined as the lowest
concentration of compound in which total inhibition of growth on
solid medium was observed (no colony forming units - CFU - were
detected).1
be determined by simultaneous resolution of three equations
(accounting for the three different liquids used for measuring the
contact angles) of the form:
ꢑꢒꢑ
ꢊ
ꢋ ꢊ
ꢎ
ꢐ
ꢄ
1 + cos ꢏ ꢅꢁ
= 2 ꢃ ꢅꢁꢆꢇꢅꢂꢆꢇ + ꢄꢅꢁ ꢅꢂꢋ + ꢄꢅꢁ ꢅꢂ ꢈ
(2)
where θ is the contact angle and γTOT=γLWγAB.
2.3.4 Evaluation of zeta potential. S. aureus and E. coli were
incubated overnight in MHB at 30 ⁰C and under 120 rpm agitation.
The cells were centrifuged twice at 3202 g for 10 min (at 25 ⁰C) and
washed with sterile distilled water. The cell suspensions were
adjusted to OD640 nm = 0.2 ± 0.02. A volume of 1.8 mL of this culture
was added to 200 μL of test compound (to a final concentration of
0.1 mM) and incubated for 1 h at 30 ⁰C and 120 rpm. A negative
control was prepared with DMSO. The zeta potential of the
bacterial suspensions was determined, according to the procedure
described by Borges et al.8, using a Nano Zetasizer (Malvern
Instruments) equipment, in carefully filled zeta potential cells
(DTS1060, Malvern), at room temperature.
2.3.3 Characterization of bacterial surface hydrophobicity.
Overnight cultures grown in MHB were centrifuged at 3202 g for 10
min and washed twice with sterile saline solution (0.85% (w/v)
NaCl, BDH Prolabo). Subsequently, the OD640
of the cell
nm
suspension was set to 0.4 ± 0.04. A volume of 45 mL of the cell
suspension was added to 5 mL of each test compound (to a final
concentration of 0.1 mM) and incubated for 1 h at 30 ⁰C and 120
rpm. All compounds were tested at the same concentration,
regardless their MIC and MBC. A negative control was prepared
with DMSO. Bacterial lawns (i.e. homogeneous layers of cells) were
then prepared according to Busscher et al.38 After exposure to the
compounds, cell cultures were filtered into nitrocellulose sterile
filters (47 mm of diameter and pore size of 0.45 μm; Advantec) and
contact angle measurements were carried out according to Simões
et al.39, by using the sessile drop contact angle method to
determine the surface tension of the bacterial surfaces (at least 20
determinations for each liquid and for each microorganism). The
measurements were performed at room temperature using three
different liquids of well-known surface tension components (two
polar - ultrapure water and formamide - and one apolar - α-
bromonaphtalene; Sigma). Contact angles were automatically
determined using an OCA 15 Plus (Dataphysics) video-based optical
measurement instrument, which allowed image acquisition and
data analysis. The values of the liquids surface tension components
were taken from the literature.40 Hydrophobicity was assessed after
2.3.5 Evaluation of potassium leakage. Bacterial suspensions of S.
aureus and E. coli were prepared in MHB and incubated at 30 ⁰C
under 120 rpm agitation. After overnight grow, the cells were
centrifuged at 3202 g for 15 min and washed twice with sterile
distilled water. The cell suspensions were adjusted to OD640 nm = 0.2
± 0.02 and incubated for 1 h at 30 ⁰C and 120 rpm, in contact with
each test compound (at a final concentration of 0.1 mM). A
negative control was prepared with DMSO. A positive control was
performed with benzyldimethyldodecylammonium chloride (Sigma–
Aldrich Química S.A.) according to Ferreira et al.1 The bacterial
suspensions were then filtered in sterile cellulose nitrate membrane
filters (pore size 0.2 μm) (Whatman, UK). Atomic absorption
spectroscopy (conditions: flame air-acetylene; wavelength 769.9
nm) were used for K+ titration in the filtered bacterial solutions,
using a GBC ASS 932plus device with the GBC Avante 1.33
software.1
contact angle measurements using the approach of van Oss et al.41
,
where the degree of hydrophobicity of a given surface (s) is
expressed as the free energy of interaction between two entities of
that surface, when immersed in water (w): ΔGsws. If the interaction
between two entities is stronger than the interaction of each one of
the entities with water, then ΔGsws>0 mJ m-2 and the surface of
bacterial cells are considered hydrophobic. Contrariwise, if ΔGsws<0
mJ m-2 the bacterial cell surfaces are hydrophilic. ΔGsws can be
calculated from the surface tension components of the interacting
entities:
2.4 Statistical analysis.
All experiments were carried out in triplicate with at least three
repeats. The data was analyzed using the GraphPad Prism 5
software. The mean and standard deviation within samples was
calculated for all cases. To assess the statistical significance of the
data, an unpaired Student’s t-test was used (confidence level ≥
95%); p<0.05 was considered statistically significant.
2.5. Calculation of drug-likeness properties
ꢉ
ꢆꢇ
ꢆꢇ
ꢊ
ꢋ ꢊ
ꢅꢂ ꢈ + 4 ꢃꢄꢅꢁ ꢅꢂꢋ + ꢄꢅꢁ ꢅꢂ
−
ꢄ
ꢄ
∆ꢀꢁꢂꢁ = −2 ꢃ ꢅꢁ
−
The parameters for drug-likeness were evaluated according to the
Lipinski’s ‘rule-of-five’, using the MolinspirationWebME Editor
[http://www.molinspiration.com].
ꢄꢅꢁꢊꢅꢁꢋ − ꢄꢅꢂꢊꢅꢂꢋꢈ
(1)
where γLW is the Lifshitz-van der Waals component of the surface
free energy and γ+ and γ- are the electron acceptor and electron
donor parameters, respectively of the Lewis acid-base component
(given by ꢅꢌꢍ = 2 × ꢄꢅꢊꢅꢋ ). The surface tension components can
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that lipophilicity is an important property for data interpretation
the theoretical partition coefficients (clogP) were also calculated
(Table 2). In general, for each bacterial strain, a clear tendency was
observed: MIC or MBC values decreased with increasing length of
the alkyl ester chain. A simple modification of the lipophilicity of
CAF (see CAF vs CAFC2) caused a relevant MIC decrease. It must be
stressed that the esterification process also preclude the ionization
process of CAF to a carboxylate ion (-COOH pka1= 4.36) at the
3 Results and discussion
3.1 Chemistry
The alkyl caffeates with short chain length (Scheme 1) were
synthesized by a nucleophilic acyl substitution reaction often called
Fischer esterification. Esterification of carboxylic acids by primary or
secondary alcohols is conventionally carried out under catalytic
conditions, namely with sulfuric acid, tosylic acid and Lewis acids.
Despite being a reaction widely used in (bio)organic synthesis it has
some disadvantages, including long reaction times, safety and working pH. The data is in accordance with the results obtained by
Merkl et al.20 using similar systems.
hazardous environmental problems, tedious purification processes
and average to low reaction yields.34,42 The use of microwave-
In S. aureus, octyl (CAFC8) and decyl caffeates (CAFC10) presented
assisted organic synthesis over conventional reactions may offer a
the lowest MIC values. As observed in Fig. 2 (A and B) these alkyl
convenient solution to overpass some of the mentioned problems,
lengths seem to represent the maximum threshold of the parabolic
allowing a reduction of reaction time and/or the amount of
curve of antimicrobial activity vs lipophilicity described by Kubo et
reagent/solvent. In our case, yields around 80% (20 min of reaction
al.17,18 for gallic acid esters. The same tendency was observed from
time) have been obtained for CAFC2 and CAFC4 using caffeic acid
(CAF) as starting material and ethanol and butanol as reagent,
respectively (Scheme 1). Compared with the classic Fisher
MBC data, as the maximum of antibacterial activity is observed for
CAFC8 (Fig. 3A and 3B).
The antibacterial activity of the compounds towards E. coli followed
the same tendency as observed for S. aureus. MIC values decreased
esterification, microwave conditions significantly reduced the
reaction time (10 min versus 5 hours to 5 days, if conducted at
room temperature), increased the yields and simplified the with the increasing alkyl length of the compounds. The maximum
purification process.43,44 The same reaction was used to obtain the
threshold of antimicrobial activity was also observed for CAFC8 and
alkyl caffeates with long chain length (Scheme 2). However, due to
purification problems, mainly related to the higher boiling points of
the primary alcohols and low yields, a different synthetic strategy
was envisaged. Alkyl caffeates (CAFC6, CAFC8 and CAFC10) have
been synthesized in moderate yields by synthetic strategy
encompassing two steps: a) the first one consists in the formation
of monomalonate compounds (1-3) by heating Meldrum’s acid with
the appropriate alcohols in toluene; b) in the second the
CAFC10 (Fig. 2A and 2B). MBC differed in several orders of
magnitude from the MIC and showed a more prominent parabolic
behavior than the one observed for S. aureus, with maximum
antibacterial activity being achieved for CAFC4 (Fig. 3A and 3B).
These data propose that E. coli viability (bactericidal effects) is more
affected by the length of the alkyl side chain than growth inhibition
(bacteriostatic effects). In general, E. coli was less susceptible than
S. aureus to the action of caffeate alkyl esters, which is in
accordance with the observations made by several authors for
condensation
of
malonate
half
esters
to
3,4-
dihydroxybenzaldehyde, in the presence of dry pyridine and β-
alanine, was performed by
Knoevenagel reaction.45
a
Verley-Doebner modification of similar systems.7,11,16 This behavior is most likely explained by the
fact that Gram-negative bacteria possess an outer membrane with
a hydrophilic coating of lipopolysaccharides (LPS)48, creating a
greater barrier to antimicrobial agents. Contrarily, Gram-positive
3.2 Antibacterial activity of caffeic acid and its alkyl esters
bacteria do not have an outer membrane and the cell wall consists
almost entirely of peptidoglycan.49 The activity seems to be also
reliant on the presence of a catechol moiety as the analogues based
on ferulic (trans-3-methoxy-4-hydroxycinnamic acid) or sinapic
(trans-3,5-dimethoxy-4-hydroxycinnamic acid) acids did not display
remarkable activity (data not shown). This particular behavior can
be related with the antioxidant/prooxidant and iron chelating
properties displayed by catechol cinnamic systems.50
CAF and its alkyl esters (C2 to C10) were screened towards S. aureus
CECT 976 and E. coli CECT 434 strains. Although esters with a higher
chain length (C12, C14 and C16) have been synthesized, water
solubility problems preclude their use. So, the study was performed
only with the compounds that present drug-like properties (Tables
1 and 2). According to the Lipinski’s ‘rule-of-five’ most "drug-like"
molecules must have logP ≤ 5, molecular weight ≤ 500, number of
hydrogen bond acceptors ≤10, and number of hydrogen bond
donors ≤ 5. Molecules violating more than one of these parameters
may have problems with bioavailability, namely poor oral
absorption or membrane permeability.46,47
The MIC values obtained in this study are in the range of those
described in other works with CAF and related compounds as well
as reviewed by Guzman et al.31 These compounds showed a broad
spectrum of antimicrobial activity, with MIC values between 0.49-8
mM and 0.12-6 mM against E. coli and S. aureus, respectivly. For
instance, in a work performed by Parkar et al.51 the MIC of CAF
against S. aureus and E. coli was 0.69 mM and 2.78 mM,
respectively. In other studies, MIC values in the range of 0.16 to
˃5.5 mM against E. coli were found.52-55 In the same way, other
natural cinnamic acids, such as ferulic and sinapic, showed
significant activity against S. aureus with MIC between 0.64 and
0.55 mM, respectively. Similar values were found against E. coli
with these compounds.56 Interesting antimicrobial activity was
found in a study performed with caffeic acid phenethyl ester and
In general cafeic alkyl esters present drug-like properties, only
CAF10 has its partion coefficient on boundary, possessing an
adequate number of proton acceptor and proton donor groups to
ensure efficient interaction with the hydrogen bonding groups of
the receptors. The predictive topological polar surface area (TPSA)
data allow concluding that they could have a good capacity for
penetrating cell membranes (Tables 1 and 2). From the data one
can notice that caffeic acid has not satisfactory properties to cross
membranes effectively.
The MIC and MBC data of CAF and its alkyl ester derivatives (C2-
C10) gathered along the study are shown in Table 2. Considering
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derivatives, where it was obtained MIC values ranging from 41 to 86 obtained from zeta potential measurements with S. aureus and E.
µM against S. aureus, including strains of MRSA.57
coli in the absence and presence of CAF and its alkyl esters are
The MIC and MBC values of CAF and its alkyl esters were shown in Table 4. Bacterial cells normally present a negative surface
significantly higher than those of the positive control, ciprofloxacin charge, due to the presence of anionic groups in their membranes,
(MIC = 7.5 × 10-4 mM and MBC = 1.8 × 10-2 mM for E. coli; MIC = 1.5 such as carboxylate and phosphate groups.61,62 In the present study,
× 10-3 mM and MBC = 1.8 × 10-2 mM for S. aureus) and comparable zeta potential values of -30.9 mV and -22.6 mV were obtained for S.
to the susceptibilities previous reported for E. coli and S. aureus.58,59 aureus and E. coli, respectively, without exposure to the
Neverthless, CAF and its alkyl esters can be considered compounds. Changes in the surface charge of S. aureus to less
antimicrobials. In fact, plant-derived compounds are routinely negative values were obtained after exposure to CAF and CAFC2.
classified as antimicrobias on the basis of susceptibility tests that The highest change on surface charge, for E. coli, was caused by
produce MIC in the range of 100 to 1000 μg/mL, orders of CAF. This effect was probably due to its ionization to a carboxylate
magnitude weaker than those of typical antibiotics produced by ion (-COOH pka1= 4.36) at the working pH.63 In general, the other
bacteria and fungi (MIC in the range of 0.01 to 10 μg/mL).9,11
CAF alkyl esters did not change significantly the surface charge
(p>0.05) of the selected bacteria.
3.3 Effects of caffeic acid and alkyl esters on the physicochemical
surface properties of bacterial cells
3.4 Effects of caffeic acid and its alkyl esters on the cell membrane
integrity
The results on the hydrophobicity and other surface tension
parameters (polar, apolar, electron acceptor and donor
components) of S. aureus and E. coli strains in the absence and
presence of CAF and its alkyl esters are depicted in Table 3. Both
bacterial surfaces presented hydrophilic properties (ΔGTOT>0 mJ/m2)
and this parameter was slightly changed due to the exposure to CAF
and its alkyl esters. S. aureus surface hydrophilicity decreased in the
presence of alkyl caffeates with exception of CAFC2 (p<0.05), where
an increase was found. No significant variation of hydrophilicity
values was observed for CAFC4 and CAFC6, when comparing with
untreated cells (p>0.05). This reduction in the hydrophilic character
was significant (p<0.05) for S. aureus exposed to CAFC8 and
CAFC10.
Potassium leakage is considered to be a good indicator of microbial
cytoplasmatic membrane damage.64 In fact, the internal ionic
environment of prokaryotic and eukaryotic cells is rich in potassium,
which means that membranolytic events in bacteria (i.e., any
impairment to the barrier between cytoplasm and the extracellular
medium) generally cause release of this ion.8,64 Table 5 shows the
effects of CAF and its alkyl esters on K+ release by E. coli and S.
aureus. A significant increase in K+ release was detected for S.
aureus exposed to CAFC6, CAFC8 and CAFC10. This indicates an
alteration in the cytoplasmic membrane permeability. However, no
significant K+ release was found due to exposure to CAF and alkyl
ester derivatives (CAFC2 and CAFC4). K+ release by E. coli was found
for cells treated with CAF, CAFC4, CAFC6 and CAFC10, even if at
lower extents than for S. aureus, when comparing to the control.
The application of benzyldimethyldodecylammonium chloride
(positive control) at 0.2 mM caused K+ release of 1.6 ± 0.28 µg/mL
and 2.8 ± 0.18 µg/mL for E. coli and S. aureus, respectively. This
biocide is known to cause cellular disruption and loss of membrane
integrity with consequent leakage of essential intracellular
constituents, including K+.1 Therefore, comparing with the positive
control, the overall data propose that alkyl caffeates inflicted
damage on the bacterial membranes of both bacteria, with
remarkable effects on S. aureus. Therefore, one can propose that
these compounds may act as membrane permeabilizers, i.e. as
antimicrobial agents that weaken the outer membrane of the cells,
inducing changes on its permeability and, consequently, in the
bacterial physicochemical characteristics.63,64 It is possible that the
antimicrobial activity of alkyl caffeates is associated with a fine
balance between affinity for the lipid bilayers of cell membranes
and the ability to cause disruption of the membrane, which visibly
differs from Gram-positive to Gram-negative bacteria (due to their
different cell wall structure and composition) and is dependent on
the length of the alkyl ester side chain. Therefore, it is proposed
that in Gram-negative bacteria, smaller length alkyl ester chains
might have a better lipophilicity balance, and thus, be more
effective in crossing the LPS layer of the outer membrane.48 This in
accordance with the results obtained for MBC and K+ leakage,
where medium length CAF alkyl esters, such as CAFC4 and CAFC6,
The surface of E. coli cells was changed with the exposure to the
compounds tested. Their hydrophilic character decreased with the
ester caffeates, with the minimum values attained for cells treated
with CAFC6 and CAFC8. Exposure to CAFC10 did not show a
significant effect on the cell surface hydrophobicity, when
compared with the control.
The apolar character of both bacteria (γLW) was reduced with the
increase of the lipophilicity of the compounds. The polar
component (γAB) showed a parabolic behavior for E. coli, increasing
with the lipophilicity of the compounds tested, being the maximum
obtained for cells treated with CAFC6. However, no significant
changes in this parameter was found for S. aureus (p>0.05). The
electron acceptor properties were also maximized for E. coli
exposed to CAFC6 and minimized for S. aureus exposed to CAFC2,
while CAFC8 and CAFC10 caused a reduction of the electron donor
properties of both bacteria (p<0.05). The overall data on
hydrophobicity and its components clearly demonstrate that the
CAF and its alkyl esters derivatives interact with the bacterial
surface. A similar mode of action was found by Borges et al.8 for
ferulic and gallic acids.
The surface charge of cells is frequently determined based on their
zeta potential, which is calculated from their electrophoretic
motility in the presence of an electric field, under defined pH and
salt concentrations.60 When applying an electric field across a
bacterial suspension, bacteria with non-zero zeta potential migrate
towards the electrode of the opposite charge, with a velocity
proportional to the magnitude of their zeta potential.1 The results
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were more effective as bactericidal agents and proven to cause The authors declare that there is no conflict of interests regarding
more damage to the membrane integrity. It is also noteworthy that the publication of this paper. The authors alone are responsible for
lower susceptibilities observed for Gram-negative bacteria, might the content and writing of the article.
also be credited to the fact that the outer membrane is an effective
barrier.17,49 On the Gram-positive bacterium longer alkyl side chain
Acknowledgements
compounds, such as CAFC8 and CAFC10, reveal to be the best
bacterial growth inhibitors and the compounds that have a greater
This work was supported by Operational Programme for
impact on destabilizing the membrane based on the results of
Competitiveness Factors–COMPETE and by FCT–Portuguese
MIC/MBC, hydrophobicity and K+ leakage.
Foundation for Science and Technology through Projects FCUP-CIQ-
The selected compounds possess a head-and-tail structure, similar
UP-NORTE-07-0124-FEDER-000065 and PTDC/DTP-SAP/1078/2012
to an amphiphile. Amphiphiles are molecules which present both
and the grants awarded to S. Benfeito (SFRH/BD/99189/2013) and
hydrophilic and hydrophobic parts, usually
a long-chain alkyl
A. Borges (SFRH/BPD/98684/2013). Further financial support was
obtained from Project UID/EQU/00511/2013-LEPABE, by the
FCT/MEC with national funds and co-funded by FEDER in the scope
of the P2020 Partnership Agreement; Project NORTE-07-0124-
FEDER-000025-RL2_ Environment&Health, by FEDER funds through
Programa Operacional Factores de Competitividade – COMPETE, by
the Programa Operacional do Norte (ON2) program and by national
funds through FCT - Fundação para a Ciência e a Tecnologia. M.
Andrade acknowledges the grant provided by the European
Research Project SusClean (Contract n_ FP7-KBBE-2011-5, project
number: 287514). The work was undertaken as part of the COST
Actions FA1202 and TD1305.
group.48 In this case, the amphiphile properties of caffeic acid esters
are associated with the presence of the phenolic groups
(hydrophilic moiety) and the alkyl side chain (lipophilic tail)15, which
contributes to the parabolic function attained when antimicrobial
activity is plotted against lipophilicity.15–18 This biological behavior
could be correlated with the cutoff phenomenon, very distinctive of
amphiphilic substances: antimicrobial properties of amphiphiles
tend to increase with increasing alkyl chain length till a limit (the
cutoff effect). However, it must be stressed that the length
increasing lead to a concomitant shrinkage of compounds solubility
in aqueous media a fact that can be also related with the decrease
of their antimicrobial activity.48
Notes and references
4 Conclusions
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a groundbreaking green
2
approach to the discovery of broad-spectrum antimicrobials. This
study shows that CAF is an interesting scaffold for the development
of antimicrobial agents. CAF alkyl esters were more effective than
CAF in controlling bacterial growth of S. aureus and E. coli. The
lipophilicity of the compounds influenced its activity. Longer alkyl
side chain compounds appeared to be more effective in inhibiting
bacterial growth of the Gram-positive bacterium, particularly CAFC8
and CAFC10. Medium length alkyl side chain compounds were more
effective against the Gram-negative bacterium, particularly CAFC4.
These molecules had broad spectrum activity causing significant
changes in cell surface hydrophobicity, charge and induced K+
leakage, an effect more significant for S. aureus. Moreover, as some
cinnamic derivatives, have demonstrated good efficacy in the
treatment of patients with tuberculosis65, the CAF alkyl esters are of
potential interest to be used against Mycobacterium tuberculosis.
Indeed, cinnamic derivatives already revelead high potency and
selectivity against this bacterium, with MIC only at micromolars.66,67
In summary, the study reinforce the idea that phytochemicals can
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This journal is © The Royal Society of Chemistry 20xx
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Figures, Tables and Schemes
Fig. 1 Chemical structure and IUPAC numbering of caffeic acid (CAF).

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A
4,0
3,5
3,0
2,5
2,0
1,5
1,0
Saureus
E Coli
0
2
4
6
8
10
Number of Carbon Atoms in Alkyl Chain
B
4,0
3,5
3,0
2,5
2,0
1,5
1,0
Saureus
E Coli
1
2
3
4
5
Log P
Fig. 2 (A) Comparison of antimicrobial activity of CAF and alkyl esters against S. aureus (○) and E. coli (●). The best
curve fit was obtained with the Boltzmann equation y =3.9 – 6811.03/(1+exp (x+12.28)/1.55), R2 = 0.9197) by S.
aureus and polynomial equation y = 2.011+0.359x-0.016 x2), R2= 0.6256) by E. coli. (B) Regression plots of
log(1/MIC) towards S. aureus (○) and E. coli (●) for CAF and alkyl esters derivatives tested.

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A
4,0
3,5
3,0
2,5
2,0
1,5
Sareus
E Coli
0
2
4
6
8
10
Number of Carbons
B
4,0
3,5
3,0
2,5
2,0
1,5
1,0
Saureus
E Coli
1
2
3
4
5
Log P
Figure 3 - (A) Comparison of antimicrobial activity of CAF and alkyl esters against S. aureus (○) and E. coli (●). The
best curve fit was obtained with the Boltzmann equation y = 4.13 – 2.88/(1+exp (x-4.16)/2.03), R2 = 0.9176) by S.
aureus and Gaussian equation y = 1.607+ 0.883 exp(-0.5(x-4.027)/1.305)2, R2 = 0.7591) by E. coli. Regression plots of
log(1/MBC) towards S. aureus (○) and E. coli (●) for CAF and alkyl esters derivatives tested.

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Table 1 Structural properties of caffeic acid and alkyl esters.
Molecular
weight
n-
TPSA
(Å2)
n-ON
n-ROTB
Volume
OHNH
acceptors
(Å3)
(g/mol)
donors
R
4
4
154.50
188.83
222.43
256.03
289.64
323.24
Caffeic acid
CAF
180.16
208.21
77.76
66.76
4
4
3
2
CAFC2
CAFC4
CAFC6
CAFC8
CAFC10
6
236.27
264.32
292.38
320.43
66.76
66.76
66.76
66.76
4
4
4
4
2
2
2
2
Caffeic
8
alkyl esters
10
12
a
n-ROTB, number of rotable bonds; TPSA, topological polar surface area; n-ON, number of hydrogen bond
acceptors; n-OHNH, number of hydrogen bond donors. The data was determined with Molinspiration calculation
software

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Table 2 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of caffeic acid
(CAF) and its alkyl ester derivatives (CAFC2-C10) against S. aureus and E. coli.
S. aureus
E. coli
cLoga
MIC
MBC
MIC
MBC
(mM)
(mM)
(mM)
(mM)
R
Caffeic acid
CAF
19.5
0.4
1.6
0.4
0.1
0.1
32.5
6.5
39
0.4
0.4
0.15
0.1
0.1
39
13
1.15
1.75
2.66
3.49
4.33
5.16
CAFC2
CAFC4
CAFC6
CAFC8
CAFC10
3.25
0.6
3.25
> 13
> 19.5
> 19.5
Caffeic
alkyl esters
0.1
0.15
aTheoretical estimated using ChemBioDraw Ultra 13.0 program.

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Table 3 Surface tension parameters and hydrophobicity of E. coli and S. aureus exposed to caffeic acid (CAF) and its
alkyl ester derivatives (CAFC2-C10) at 0.1 mM, for 1 h
Surface tension parameters (mJ/m2)
ΔGTOT (mJ/m2)
γLW
γAB
γ+
γ-
Control
CAF
34.3 ± 1.2
32.4 ± 2.2
34.8 ± 0.7
34.1 ± 1.6
31.7 ± 0.3
25.6 ± 4.4
24.8 ± 2.0
32.7 ± 1.4
31.1 ± 1.4
30.1 ± 0.8
29.9 ± 0.9
24.0 ± 1.6
22.6 ± 1.4
24.0 ± 2.3
21.5 ± 1.2
23.5 ± 2.8
18.0 ± 1.3
20.8 ± 2.3
22.0 ± 2.1
17.2 ± 4.0
18.9 ± 2.0
21.6 ± 2.6
25.5 ± 2.1
26.2 ± 1.5
26.9 ± 1.3
32.3 ± 1.3
22.4 ± 4.8
19.9 ± 6.0
2.4 ± 0.3
2.8 ± 0.6
1.6 ± 0.2
2.3 ± 0.5
2.7 ± 0.5
2.1 ± 0.9
2.3 ± 0.7
2,2 ± 0.5
3.1 ± 0.5
3.3 ± 0.3
3.4 ± 0.3
5.1 ± 0.5
3.2 ± 1.5
2.2 ± 1.5
47.6 ± 3.0
49.2 ± 1.4
50.0 ± 1.2
47.9 ± 1.1
46.2 ± 1.4
38.0 ± 4.4
39.7 ± 4.1
54.5 ± 1.8
53.8 ± 0.4
51.9 ± 1.0
52.8 ± 0.2
50.9 ± 1.0
43.3 ± 4.4
51.6 ± 4.0
23.0 ± 3.5
24.3 ± 0.7
27.5 ± 1.4
23.8 ± 1.8
22.1 ± 2.0
15.5 ± 4.9
17.6 ± 5.5
31.3 ± 2.7
28.6 ± 1.1
26.4 ± 0.4
27.1 ± 0.3
23.1 ± 1.5
16.8 ± 2.4
34.7 ± 4.3
CAFC2
CAFC4
CAFC6
CAFC8
CAFC10
Control
CAF
S. aureus
CAFC2
CAFC4
CAFC6
CAFC8
CAFC10
E. coli
ΔGTOT > 0 mJ/m2 – Hydrophilic; ΔGTOT < 0 mJ/m2 - Hydrophobic

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Table 4 Effects of caffeic acid (CAF) and its alkyl ester derivatives (CAFC2-C10) in the surface charge of S. aureus and
E. coli cells at 0.1 mM, for 1 h.
Zeta Potential (mV)
S. aureus
-30.9 ±3.3
-10.2±4.5
-16.4±2.8
-24.7±3.7
-28.3±1.3
-25.5±4.5
-27.0±5.6
E. coli
Control
CAF
-22.6±6.2
-11.8±3.0
-28.1±4.7
-17.4±3.2
-18.4±0.9
-20.5±0.8
-19.6±2.1
CAFC2
CAFC4
CAFC6
CAFC8
CAFC10

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Table 5. K+ concentration in the solution after exposure of E. coli and S. aureus cells to CAF and its alkyl ester
derivatives (CAFC2-10) at 0.1 mM, for 1 h.
Concentration of K+ in solution (μg/mL)
S. aureus
E. coli
Control
CAF
0.904 ± 0.272
1.177 ± 0.305
0.845 ± 0.344
0.927 ± 0.280
2.770 ± 0.095
2.745 ± 0.035
2.789 ± 0.004
0.263 ± 0.001
0.417 ± 0.010
0.356 ± 0.092
0.447 ± 0.021
0.566 ± 0.081
0.348 ± 0.071
0.502 ± 0.370
CAFC2
CAFC4
CAFC6
CAFC8
CAFC10

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Scheme 1 Synthesis of ethyl (CAFC2) and butyl (CAFC4) caffeate from caffeic acid (CAF). Reagents and conditions:
alkyl alcohol, H2SO4, microwave irradiation, 20min.
Scheme 2 Synthesis of long alkyl chain cinnamates (CAFC6, CAFC8, CAFC10) from monomalonates 1-3. (i) Meldrum's
acid, toluene, reflux, 4h; (ii) 3,4-dihydroxybenzaldehyde, pyridine, β-aniline, cyclohexane, reflux 6-10h.