Synthesis and antimicrobial activities of novel sorbic and benzoic acid amide derivatives

Article abstract of DOI:10.1016/j.foodchem.2018.06.071

A series of sorbic and benzoic acid amide derivatives were synthesized by conjugating sorbic acid (SAAD, a₁–a₇) or benzoic acid (BAAD b₁–b₆) with amino acid esters and their antimicrobial activities were investigated against Escherichia coli, Bacillus subtilis and Staphylococcus aureus, mixed bacteria from rancid milk, Saccharomyces cerevisiae, and Aspergillus niger. The antimicrobial activity of sorbic acid amides was better than that of benzoic acid amides. The minimum inhibitory concentrations (MIC) of compound isopropyl N-[1-oxo-2, 4-hexadien-1-yl]-L-phenylalaninate (a₇) were 0.17 mM against B. subtilis, and 0.50 mM against S. aureus, while the MIC values of sorbic acid were more than 2 mM respectively. Also, compound a₇ displayed pH-independent antimicrobial activity in the range of pH 5.0–9.0 and was effective at pH 9.0. These results demonstrated that the conjugation of sorbic acid with amino acid esters led to significant improvement of in vitro antimicrobial attributes, but little effect was observed for benzoic acid amide derivatives.

Full text of DOI:10.1016/j.foodchem.2018.06.071

Accepted Manuscript
Synthesis and antimicrobial activities of novel sorbic and benzoic acid amide
derivatives
Qingyi Wei, Xiaomei Wang, Jun-Hu Cheng, Guangxiang Zeng, Da-Wen Sun
PII:
S0308-8146(18)31044-6
DOI:
https://doi.org/10.1016/j.foodchem.2018.06.071
FOCH 23032
Reference:
Food Chemistry
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Revised Date:
Accepted Date:
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30 May 2018
14 June 2018
Please cite this article as: Wei, Q., Wang, X., Cheng, J-H., Zeng, G., Sun, D-W., Synthesis and antimicrobial activities
of novel sorbic and benzoic acid amide derivatives, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.
2018.06.071
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Synthesis and antimicrobial activities of novel sorbic
and benzoic acid amide derivatives
Running title: Sorbic and Benzoic Acid Amide
Derivatives
Qingyi Weia, b, c, Xiaomei Wang a, b, c, Jun-Hu Cheng a, b, c, Guangxiang Zeng a, b, c, Da-Wen
Sun a, b, c, d, 
a School of Food Science and Engineering, South China University of Technology, Guangzhou
510641, PR China
b Academy of Contemporary Food Engineering, South China University of Technology,
Guangzhou Higher Education Mega Center, Guangzhou 510006, PR China
c Engineering and Technological Research Centre of Guangdong Province on Intelligent
Sensing and Process Control of Cold Chain Foods, Guangzhou Higher Education Mega Centre,
Guangzhou 510006, China
d Food Refrigeration and Computerized Food Technology (FRCFT), Agriculture and Food
Science Centre, University College Dublin, National University of Ireland, Belfield, Dublin 4,
Ireland
ABSTRACT
 Corresponding author: Tel: +353-1-7167342, Fax: +353-1-7167493, E-mail: dawen.sun@ucd.ie,
Website: www.ucd.ie/refrig; www.ucd.ie/sun.
1

A series of sorbic and benzoic acid amide derivatives were synthesized by
conjugating sorbic acid (SAAD, a1-a7) or benzoic acid (BAAD b1-b6) with amino acid
esters and their antimicrobial activities were investigated against Escherichia coli,
Bacillus subtilis and Staphylococcus aureus, mixed bacteria from rancid milk,
Saccharomyces cerevisiae, and Aspergillus niger. The antimicrobial activity of sorbic
acid amides was better than that of benzoic acid amides. The minimum inhibitory
concentrations
(MIC)
of
compound
isopropyl
N-[1-oxo-2,
4-hexadien-1-yl]-L-phenylalaninate (a7) were 0.17 mM against B. subtilis, and 0.50
mM against S. aureus, while the MIC values of sorbic acid were more than 2 mM
respectively. Also, compound a7 displayed pH-independent antimicrobial activity in
the range of pH 5.0-9.0 and was effective at pH 9.0. These results demonstrated that
the conjugation of sorbic acid with amino acid esters led to significant improvement
of in vitro antimicrobial attributes, but little effect was observed for benzoic acid
amide derivatives.
Keywords: Antimicrobial activity; Synthesis; Sorbic acid derivatives; Benzoic acid
derivatives; Amino acid esters
1. Introduction
Food safety is an important issue for both the food industry and consumers.
Chemical synthetic food preservatives are used for controlling microbial growth and
reducing incidences of food poisoning and spoilage (Eklund, 1980). Examples include
sorbic acids, benzoic acids and their respective potassium, sodium, calcium salts,
which belong to weak-acid preservatives. They are widely used to preserve various
foods against yeasts and molds, and many bacteria in acid media (Davidson et al.,
2

2005). These compounds have a positive effect at low pH levels but are ineffective in
foods at neutral pH levels (Tfouni & Toledo, 2002; Stratford et al., 2009). Some
derivatives of sorbic acid, such as sorbohydroxamic acid and sorbic aldehyde, were
synthesized by Troller & Olsen (1967), and they found that these derivatives were
more effective than the parent acid (Troller & Olsen, 1967). However, the commercial
applications of such derivatives are hindered by their disadvantages, such as strong
off-flavours and odours (Davidson et al., 2005). Up to now, the derivatives of sorbic
acid and benzoic acid as antimicrobial agents have not yet been fully exploited.
The emergence of new pathogens and the resistance of known pathogens
remains challenging to the food safety industry (Doyle et al., 2006). Currently, the use
and added levels of some preservatives have been restricted by legislations in
different foods. At the same time, the susceptibility of some microorganisms to the
most currently used preservatives is being reduced. Reports concerning the
possibility of acquired resistance to organic acid preservatives by microorganisms and
the resistance of a wide variety of yeasts to benzoic acid has been summarized
elsewhere (Davidson et al., 2005). For example, the resistance of the foodborne
pathogen Escherichia coli O157:H7 against benzoic acid (Lin et al. 1996) and acetic
acid (Bae & Lee, 2017) were observed and several acid-resistance systems were also
found, e.g., pathogenic E. coli could survive in extreme acidic pH. The inhibition of
microorganisms by sorbic acid and sorbate, depends on the differences in microbial
types, species and strains, etc. (Davidson et al., 2005). Neves et al. (1994) studied the
resistance of food spoilage yeasts to sorbic acid and found that most strains were
able to grow at 150 ppm, while 40% could grow at 500 ppm, and remarkably two
strains of Zygosaccharomyces bailii could tolerate sorbic acid at 800 ppm. Mollapour
3

et al. (2004) studied the increased sensitivity and resistance of sorbic acid to
Saccharomyces cerevisiae gene deletion mutants using robotic methods, and showed
that 34 mutants exhibited even higher sorbate resistance than the wild-type parental
strain. Recently, Nygård et al. (2014) exposed yeast strains with altered expression of
PDR12 to seven weak organic acids, including sorbic acid, and found that
overexpression of PDR12 increased the tolerance to acids with long chain lengths,
such as sorbic, propionic and levulinic acids. In addition, Holyoak et al. (1996) studied
the resistance mechanism of spoilage yeasts to weak organic acid preservatives and
indicated that the resistance involved ejection of protons via the plasma membrane
H+-ATPase proton pump. Similarly, resistance of fungi to sorbic acid was also found
(Kubo & Lee, 1998, Singh et al., 2016).
Therefore, new antimicrobial agents with a broader spectrum of activities and
low toxicity are needed. Recent attempts at preparing amino acid conjugates of
active substances have led to the development of a number of high activity
derivatives (Manjunatha et al., 2013). Noh & Lee (2011) and Noh et al. (2009)
synthesized Kojic acid and hydroxyphenolic acid derivatives, which were modified
with amino acids, and studied their tyrosinase inhibitory activities. They found that
the compounds with the phenylalanine moiety showed the strongest inhibitory
activities. On the other hand, in our previous works, cinnamoyl amides with an amino
acid ester moiety were synthesized through the conjugation of the corresponding
cinnamic acids with amino acid esters, which showed excellent tyrosinase inhibitory
activity (Fan et al., 2012) and antimicrobial activity against Bacillus subtilis,
Escherichia coli and Saccharomyces cerevisiae (Wei et al., 2012), confirming that the
activity of cinnamic acid derivatives could be enhanced after modification with
4

phenylalanine. Later, Manjunatha et al., (2013) synthesized amino acid conjugates of
tetrahydrocurcumin and evaluated their antibacterial and anti-mutagenic properties,
demonstrating that the conjugation of tetrahydrocurcumin at the phenolic position
with amino acids contributed to significant improvement in its in vitro biological
activities. In addition, Kaushik et al. (2014) synthesized a series of N-protected
L-amino acid propargyl esters with amino acid-linked 1, 4-disubstituted 1, 2,
3-bistriazole conjugates, which evaluated their antimicrobial activity against
gram-positive and gram-negative bacteria, and fungi, and found that most of the
compounds had good antibacterial activity compared to the reference drug
ciprofloxacin.
Recently,
Panda
et
al.
(2016)
synthesized
novel
fluoroquinolone–pyrazine derivatives modified with amino acids and exhibited that
the synthesized conjugates had antimicrobial properties comparable to the parent
drugs. Moreover, Baltina et al. (2017) synthesized several conjugates of glycyrrhizic
acid (GA) with amino acids and dipeptides and showed the GA conjugate with
tyrosine exhibited weak but consistent antiviral activity.
Inspired by the above findings, in the current study, two series of sorbic acid
amide derivatives (SAAD, a1-a7, see Fig. 1 for structures) and benzoic acid amide
derivatives (BAAD b1-b6, see Fig. 1 for structures) were synthesized by conjugating
sorbic acid or benzoic acid with amino acid esters and their antimicrobial activities
were studied. It is hoped that the current study would encourage further research in
searching for new antimicrobial agents.
2. Materials and methods
2.1. Chemical reagents and instruments
5

Sorbic acid, benzoic acid, glycine, and l-phenylalanine were purchased from
Sigma-Aldrich Co. (St. Louis, MO, USA). Carbinol, ethanol, n- propyl alcohol and
isopropyl alcohol were obtained from Shanghai Medpep Co., Ltd. (Shanghai, China).
The water used was re-distilled and ion-free. All the other reagents were of analytical
grade.
The melting points of the synthesized compounds were determined on a RY-2
melting point tester (Tianjin Guoming Medicinal Equipment Co., Ltd., Tianjin, China).
1H NMR data for the structures of the synthesized compounds were acquired using a
nuclear magnetic resonance (NMR) spectrometer (Bruker 400 MHz NMR, Bruker Co.,
WI, USA), operating at 400 MHz and room temperature, with CDCl3 or DMSO-d6 used
as solvent, and the chemical shifts were expressed in δ (parts per million) values
relative to tetramethylsilane (TMS) as an internal reference. The masses of the
synthesized compounds were measured using a mass spectrometer (Saturn 2000,
Varian B.V., Middelburg, The Netherlands). A Spectronic Genesys 8 UV/VIS
spectrophotometer (Thermo Fisher Scientific Inc., MA, USA) was used for
antibacterial assays.
2.2. Synthesis
Seven SAAD derivatives and six BAAD derivatives were synthesized from sorbic
acids or benzoic acids and the corresponding amino acid esters, according to the
literature, with some modifications (Orlandi et al., 2001; Qu, 2007). The synthesis
method was exemplified by the preparation of compound a1 as shown in Fig. 2, with
the procedure given below.
A solution of thionyl chloride (4.5 ml, 0.06 mol) in anhydrous ethanol (30 ml,
6

0.52 mol) was added to glycine (3.3 g, 0.02 mol) with stirring at 0oC for 1 h and then
at room temperature for another 3 h. L-ethyl glycinate hydrochloride was
synthesized after removal of the solvent.
Sorbic acid (2.24 g, 0.02 mol) was added to thionyl chloride (4.5 ml, 0.06 mol)
with stirring at room temperature for 1 h. Sorbic chloride was synthesized after
removal of the redundant thionyl chloride. A solution of L-ethyl glycinate
hydrochloride and sorbic chloride in chloroform (15 ml) was added to triethylamine
o
(2 ml, 0.014 mol) with stirring at 0 C for 3 h and then at 60 oC for 4 h. The reaction
mixture was poured into water (25 ml), extracted with ethyl acetate (3×25 ml),
washed with NaOH solution (0.5 mol/l, 2×25 ml) and then dried over anhydrous
MgSO4. After removal of the solvent under reduced pressure, the residual paste was
purified by column chromatography (silica gel, petroleum ether / ethyl acetate, 2:1)
to give Etheyl N-[1-oxo-2, 4-hexadien-1-yl] glycinate (a1).
Other compounds (a2-a7 and b1-b7) were synthesized using procedures similar
to that described above for compound a1. All the reactions proceeded in 10-12 h to
provide products in good yields (53%-73%). The structures of the synthesized
compounds were established by 1H NMR. In addition, propyl N-[1-oxo-2,
4-hexadien-1-yl]-L-phenylalaninate
(a6)
and
isopropyl
N-[1-oxo-2,
4-hexadien-1-yl]-L-phenylalaninate (a7) were characterized by high resolution mass
spectrometry (HRMS) (Agilent 1290/maxis Impact, Bruker Daltonik GmbH, Bremen,
Germany).
2.3. Antimicrobial activity assay
Thirteen newly synthesized SAAD (a1-a7) and BAAD (b1-b6) were evaluated for their in
7

vitro antibacterial activities against two gram-positive bacteria (Bacillus subtilis MTCC
441 and Staphylococcus aureus ATCC 25923), one gram negative bacteria (Escherichia
coli ATCC 25922), and one mixed bacteria from rancid milk. One yeast
(Saccharomyces cerevisiae ATCC 2365) and one mold (Aspergillus niger ATCC 16404)
were also employed for antifungal activity evaluation (Bhardwaj et al., 2016). These
microorganisms were available from the College of Food Science and Engineering,
South China University of Technology, Guangdong, China. Commercial sorbic acid and
benzoic acid were used as positive controls in all the following antimicrobial assays.
The synthesized compounds were dissolved in anhydrous ethanol at proper
concentrations (Smith et al., 2008; Yesilel et al., 2010). Respectively, serial dilutions
of the synthesized compounds were prepared in sterile nutrient broth medium agar
plates for the bacteria, in sterile potato extract glucose medium agar plates for yeast
and in sterile potato sucrose medium agar plates for mold. For the evaluation of
antimicrobial activities, all microorganisms were introduced and evenly distributed
on the surface of agar plates with the aid of a sterile glass spreader. The plates were
inoculated with 1.0×107 spores/ml for mold and 1.0×107 cfu/ml for both bacteria and
o
o
yeast. The incubation period was 24 h at 37 C for bacterial strains, 48 h at 28 C for
o
yeast strains and 72 h at 28 C for fungal strains. The minimal inhibitory
concentration (MIC), at which no growth was observed, was taken as the MIC value
(mM). In order to verify the antimicrobial activities of the synthesized compounds,
the highest aliquot with pure ethanol was used as control, and it was verified that the
ethanol did not inhibit microorganism growth. Three independent experiments were
performed. MIC was determined as the lowest concentration of the compound
inhibiting the complete growth of the microbe being tested (Guarda et al., 2011).
8

2.4. Effect of pH on antimicrobial activity assay
The concentration of compounds (compound a2, a5, a7, sorbic acid) was 2.5
mmol/l. The growth of the mixed bacteria from rancid milk was evaluated every 6 h
by determining the absorbance at 560 nm in the Spectronic Genesys 8 UV/VIS
spectrophotometer (Wei et al., 2011; Bevilacqua et al., 2008). Microorganisms were
cultivated in liquid nutrient broth media supplemented with test compounds at
various pH levels. The pH of cultures was adjusted to 5.0, 7.0 and 9.0 by the addition
of HCl solution and NaOH solution. Cultures were diluted to achieve 1.0×107 cfu/ml.
0.1ml of the diluted microorganism was added to a conical flask with 100 ml of liquid
media at each pH with 2.5 mmol/l of test compounds, which were dissolved in 0.6 ml
tween-20 sterile medium with microbial inoculums and tween-20 was used as a
control. Liquid media without any addition served as a blank. All analyses were
performed in triplicate. Data reported from each assay were the average of three
independent experiments. The inhibitory index was calculated as follows:
t,k  t,i
I (%) =
×100
t,k
(1)
where t,k represents the absorbance of the control subtracting that of the blank
sample; t,i is the absorbance of the treated samples subtracting that of the blank
sample.
2.5. Toxicity study on cells
The breast cancer cell line (MCF-7) was used as a cell model, which was
9

provided by Sun Yat-sen University, Guangzhou, China. The cell cultures were grown
at 37 ◦C in an incubator with a humidified atmosphere of 5% CO2 / 95% air, and
cultured with RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum,
penicillin (100 kU/l), streptomycin (100 mg/l) and 2 mM of glutamine. The MTT
colorimetric assay (Hussain et al., 1993) was applied to evaluate cell viability. Briefly,
100 l of exponentially growing MCF-7 cells (5×104 cells/ml) were plated in each well
of a 96-well plate and incubated with various concentrations (10, 50, 100, and 200
M) of the tested conjugates dissolved in culture medium with 0.1%
dimethylsulfoxide (DMSO) for 48 h. After that, 10 l MTT solution (5 mg/ml) in PBS
o
was added to each well and incubated for an additional 4 h at 37 C in a 5% CO2
incubator. Then 110 l lysis buffer (10% SDS – 0.1 M HCl) were added to each well
o
and incubated over 8 h at 37 C. The optical density (OD) of formazan was recorded
by an ELISA microplate reader (Model 550, Bio-Rad Laboratories Inc., CA, USA) at 570
nm. Four replicate wells were applied for each point.
2.6. Statistical analysis
The results were exhibited as mean±standard deviations (SD) with three parallel
tests. All data in Figures 3 and 4 were calculated with the Origin statistical and
analysis software (Version 8.0, OriginLab Co., MA, USA).
3. Results
3.1. Characterization of the synthesized compounds
1
The structures of the synthesized compounds as established by H NMR with
propyl a6 and a7 characterized by high resolution mass spectrometry are shown in Fig.
10

5. The details are as follows:
1
Etheyl N-[1-oxo-2, 4-hexadien-1-yl] glycinate (a1): yield = 53.5%, yellow liquid; H
NMR (400 MHz, CDCl3): δ = 7.28-7.18 (m, 1H, CH=C-C=O), 6.20 (br s, 1H, NH),
6.16-6.12 (m, 1H, C-C=CH-C), 6.11-6.08 (m, 1H, C-CH=C-C), 5.87 (d, 1H, J=14.8Hz,
C=CH-C=O), 4.22-4.11 (m, 4H, CH2-C=O and OCH2), 1.82 (d, 3H, J=5.2Hz, CH3-C=C),
1.25 (t, 3H, J=6.8Hz, O-C-CH3).
Propyl N-[1-oxo-2, 4-hexadien-1-yl] glycinate (a2): yield = 58.8%, yellow solid;
1
melting point (mp) = 74.9-77.7 oC; H NMR (400 MHz, CDCl3): δ = 7.28-7.20 (m, 1H,
CH=C-C=O), 6.19 (br s, 1H, NH), 6.16-6.13 (m, 1H, C-C=CH-C), 6.11-6.07 (m, 1H,
C-CH=C-C), 5.86 (d, 1H, J=15.2Hz, C=CH-C=O), 4.16-4.13 (m, 4H, CH2-C=O and OCH2),
1.87 (d, 3H, J=6.0Hz, CH3-C=C), 1.69 (m, 2H, C-CH2-C), 0.95 (t, 3H, J=7.4Hz,
O-C-C-CH3).
Isopropyl N-[1-oxo-2, 4-hexadien-1-yl] glycinate (a3): yield = 55.2%, yellow solid;
mp = 69.2-72.1 oC; 1H NMR (400 MHz, CDCl3): δ = 7.28-7.19 (m, 1H, CH=C-C=O), 6.18
(br s, 1H, NH), 6.15-6.12 (m, 1H, C-C=CH-C), 6.10-6.06 (m, 1H, C-CH=C-C), 5.88 (d, 1H,
J=14.8 Hz, C=CH-C=O), 5.08 (m, 1H, OCH), 4.09 (d, 2H, J=5.2 Hz, CH2-C=O), 1.78 (d, 3H,
J=6.0 Hz, CH3-C=C), 1.27 (d, 6H, J=6.0 Hz, -O-C- (CH3) 2).
Methyl N-[1-oxo-2, 4-hexadien-1-yl]-L-phenylalaninate (a4): yield = 71.2%, white
o
1
solid; mp = 108.6-111.5 C; H NMR (400 MHz, CDCl3): δ = 7.30-7.28 (m, 1H,
CH=C-C=O), 7.25-7.10 (m, 5H, C6H5-C), 6.18 (br s, 1H, NH), 6.18-6.05 (m, 1H,
C-C=CH-C), 5.95 (d, 1H, J=7.2 Hz, C-CH=C-C), 5.74 (d, 1H, J=15.2 Hz, C=CH-C=O), 4.98
(q, 1H, J=6.4 Hz, Ph-C-CH), 3.73 (s, 3H, OCH3), 3.16 (m, 2H, CH2-Ph), 1.83 (d, 3H, J=5.6
Hz, CH3-C=C).
Ethyl N-[1-oxo-2, 4-hexadien-1-yl]-L-phenylalaninate (a5): yield = 61.8%, yellow
11

solid; mp = 69.8-72.2 oC; 1H NMR (400 MHz, CDCl3): δ = 7.29-7.27 (m, 1H, CH=C-C=O),
7.26-7.08 (m, 5H, C6H5-C), 6.15 (br s, 1H, NH), 6.18-6.06 (m, 1H, C-C=CH-C), 5.97 (d,
1H, J=7.6 Hz, C-CH=C-C), 5.77 (d, 1H, J=14.8 Hz, C=CH-C=O), 4.95 (q, 1H, J=6.4 Hz,
Ph-C-CH), 4.16 (q, 2H, J=7.1 Hz, OCH2), 3.16 (m, 2H, CH2- Ph), 1.85 (d, 3H, J=6.4 Hz,
CH3-C=C), 1.24 (t, 3H, J=7.2 Hz, O-C-CH3).
Propyl N-[1-oxo-2, 4-hexadien-1-yl]-L-phenylalaninate (a6): yield = 60.3%, white
o
1
solid; mp = 107.4-109.7 C; H NMR (400 MHz, CDCl3): δ = 7.31-7.30 (m, 1H,
CH=C-C=O), 7.28-7.12 (m, 5H, C6H5-C), 6.19 (br s, 1H, NH), 6.21-6.07 (m, 1H,
C-C=CH-C), 5.96 (d, 1H, J=7.2 Hz, C-CH=C-C), 5.76 (d, 1H, J=14.8 Hz, C=CH-C=O), 4.99
(q, 1H, J=6.4 Hz, Ph-C-CH), 4.09 (m, 2H, OCH2), 3.19 (m, 2H, CH2- Ph), 1.88 (d, 3H,
J=5.6 Hz, CH3-C=C), 1.66 (m, 2H, C-CH2-C), 0.92 (t, 3H, J=7.4 Hz, O-C-C-CH3) ; MS (ESI)
m/z 302 [M+H]+; high resolution MS (ESI) CALCD for C18H24NO3[M+H]+: 302.1751,
found: 302.1750.
Isopropyl N-[1-oxo-2, 4-hexadien-1-yl]-L-phenylalaninate (a7): yield = 67.6%,
1
bronzing solid; mp = 86.4-92.8 oC; H NMR (400 MHz, CDCl3): δ = 7.29-7.27 (m, 1H,
CH=C-C=O), 7.25-7.11 (m, 5H, C6H5-C), 6.15 (br s, 1H, NH), 6.19-6.08 (m, 1H,
C-C=CH-C), 6.02 (d, 1H, J=7.6 Hz, C-CH=C-C), 5.73 (d, 1H, J=14.8 Hz, C=CH-C=O), 4.92
(q, 1H, J=6.4 Hz, Ph-C-CH), 4.13 (m, 1H, OCH), 3.07 (m, 2H, CH2- Ph), 1.85 (d, 3H,
J=5.6 Hz, CH3-C=C), 1.24 (d, 6H, J=6.4 Hz, -O-C- (CH3) ) ; MS (ESI) m/z 302 [M+H]+;
2
high resolution MS (ESI) CALCD for C18H24NO3[M+H]+: 302.1751, found: 302.1745.
1
Ethyl N-benzoyl-glycinate (b1): yield = 63.9%, yellow liquid; H NMR (400 MHz,
CDCl3): δ = 7.84-7.44 (m, 5H, C6H5-C=O), 6.76 (br s, 1H, NH), 4.30-4.25 (m, 4H,
CH2-C=O and OCH2), 1.32 (t, 3H, J=7.0 Hz, -O-C-CH3).
Propyl N-benzoyl-glycinate (b2): yield = 61.7%, yellow liquid; 1H NMR (400 MHz,
12

CDCl3): δ = 7.84-7.40 (m, 5H, C6H5-C=O), 6.75 (br s, 1H, NH), 4.27 (d, 2H, J=4.8 Hz,
CH2-C=O), 4.15 (t, 2H, J=4.8 Hz, OCH2), 1.73 (m, 2H, C-CH2-C), 0.97 (t, 3H, J=7.4 Hz,
-O-C-C-CH3).
Isopropyl N-benzoyl-glycinate (b3): yield = 67.5%, white solid; mp = 85.8-87.9 oC;
1H NMR (400 MHz, CDCl3): δ = 7.77-7.18 (m, 5H, C6H5-C=O), 6.66 (br s, 1H, NH), 5.05
(m, 1H, OCH), 3.28 (d, 2H, J=4.8 Hz, CH2-C=O), 1.26 (d, 6H, J=3.7Hz, -O-C- (CH3) 2).
Methyl N-benzoyl-L-phenylalaninate (b4): yield = 69.4%, yellow solid; mp =
o
1
117.8-119.8 C; H NMR (400 MHz, CDCl3): δ = 7.73-7.40 (m, 5H, C6H5-C=O),
7.31-7.13 (m, 5H, C6H5-C), 6.59 (br s, 1H, NH), 5.10 (q, 1H, J=6.4 Hz, CH-C=O), 3.75 (s,
3H, OCH3), 3.26 (m, 2H, CH2- Ph).
Ethyl N-benzoyl-L-phenylalaninate (b5): yield = 66.1%, yellow solid; mp =
103.5-105.6 oC; 1H NMR (400 MHz, CDCl3): δ = 7.76-7.42 (m, 5H, C6H5-C=O), 7.33-7.17
(m, 5H, C6H5-C), 6.62 (br s, 1H, NH), 5.09 (q, 1H, J=6.4 Hz, CH-C=O), 4.16 (q, 2H, J=7.2
Hz, OCH2), 3.29 (m, 2H, CH2-Ph), 1.29 (t, 3H, J=7.2 Hz, -O-C-CH3).
Isopropyl N-benzoyl-L-phenylalaninate (b6): yield =60.8%, white solid; mp =
1
78.9-80.8 oC; H NMR (400 MHz, CDCl3): δ = 7.77-7.43 (m, 5H, C6H5-C=O), 7.31-7.18
(m, 5H, C6H5-C), 6.66 (br s, 1H, NH), 5.09-5.05 (m, 2H, CH-C=O and 0-CH), 3.28 (m,
2H, CH2-Ph), 1.29 (d, 3H, J=3.5Hz, -O-C- (CH3) 2).
3.2. Antimicrobial activities of a1-a7 and b1-b6
The minimum inhibitory concentrations of the 13 new synthesized SAAD and
BAAD derivatives are summarized in Table 1, indicating that a1-a7 possessed strong
antibacterial activities against gram positive and negative bacteria when compared to
the standard parent sorbic and benzoic acids. The MIC values of a2-a7 against B.
13

subtili were between 0.17 to 2.20 mM, while MIC values of sorbic acid and benzoic
acid were 2.23 and 3.28 mM, respectively. For the antibacterial activity against the
mixed bacteria from rancid milk, a2, a5 and a7 exhibited good activity with MIC values
of less than 1.66 mM. All the compounds, except a7, tested against the yeast and
mold were less effective, with a7 having the highest inhibitory activity against
microorganisms (B. subtilis, S. aureus, E. coli, and the mixed bacteria from rancid
milk), yeast (S. cerevisiae) and mold (A. niger). At the same time, the inhibitory effect
of a7 was better than those of both sorbic and benzoic acids. In addition, a1-a7 had
greater antibacterial activities against gram positive bacteria than gram negative
bacteria. On the other hand, b1-b6 was less effective against the six microorganisms
tested, with only b6 exhibiting antibacterial activity against B. subtilis having a MIC of
1.45 mM.
3.3. Effect of pH on antimicrobial activity
Compounds a2, a5 and a7 and sorbic acid were selected for examining the effect
of pH on their antimicrobial activities. The mixed bacteria from rancid milk were
cultivated in liquid nutrient broth media supplemented with the compounds at
various pH levels. As shown in Fig. 3 (a), a7 and sorbic acid could inhibit the growth
of the mixed bacteria for more than 72 h at pH 5, while the inhibitory times, at which
the inhibitory rate was more than 90%, of a2 and a5 were only 24 h and 36 h,
respectively. At pH 5.0, the inhibition strength of a2, a5 and a7 and sorbic acid
followed the following order: a7 ≈ sorbic acid > a5 > a2. At pH 7.0, the antimicrobial
activity of sorbic acid decreased quickly, and its inhibitory time was 24 h, and
compounds a2, a5 showed the same inhibitory activities. However the inhibitory
14

effect of a7 was continuously at more than 80% within 72 h, as illustrated in Fig. 3 (b).
Moreover, Fig. 3 (c) shows that at pH 9.0, the antimicrobial activity of sorbic acid
nearly disappeared, while a7 could still inhibit the growth of the mixed bacteria for 72
h. The inhibition strength of compounds a2, a5 and a7 and sorbic acid followed the
following order: a7 > sorbic acid > a5 > a2, and a7 maintained the highest antimicrobial
activity at pH 5.0, pH 7.0 and pH 9.0.
3.4. Toxicity study on cells
The in vitro cytotoxicity of compounds a2, a5 and a7 and sorbic acid in MCF-7 cells
was studied. The cell viability was evaluated by the MTT colorimetric assay and the
compounds in these experiments did not inhibit the proliferation of MCF-7 cells (data
shown in Fig. 4). The cell viability was not reduced after treatment with 10, 50 and
100, and 200 M, indicating that a2, a5 and a7 had low cytotoxicity to MCF-7 cells in
vitro.
4. Discussion
A large number of amino acid conjugates have been synthesized and their
biological activities were evaluated. Certain curcumin conjugates modified with
esters and peptides were reported and these compounds showed higher antifungal
and antibacterial activities than curcumin, because of better cellular uptake, higher
cellular amounts and increased receptor binding (Kapoor et al., 2007). Kojic acid and
hydroxyphenolic acid derivatives conjugated with amino acids exhibited higher
tyrosinase inhibitory activities compared to parent compounds (Noh et al., 2011; Noh
et al., 2009). Recently, some amino acid conjugates of tetrahydrocurcumin (THC)
15

were synthesized and their antibacterial and anti-mutagenic activities were shown to
be enhanced compared to tetrahydrocurcumin (Manjunatha et al., 2013). In our early
work, cinnamoyl amides with an amino acid ester moiety were studied and excellent
tyrosinase inhibitory activity (Fan et al., 2012) and antimicrobial activities against B.
subtilis, E. coli, and S. cerevisiae (Wei et al., 2012) were shown. The activity of
cinnamic acid derivatives could be further enhanced after being modified with
phenylalanine (Fan et al., 2012). In the current study, a7 had higher antimicrobial
activities than that of sorbic acid, which was also modified with phenylalanine, but
the antimicrobial activities of b1-b6 were decreased after modification with amino
acid esters. As shown in Table 1, among the SAAD series, a1–a7 bear different amino
acids and different lengths of hydrocarbon chains at the amino acid esters, while a7
bears the more active isopropoxy- at the phenylalanine ester. However, in the BAAD
series, phenylalanine and isopropyl groups have little effect on the benzoic acid
derivatives for antimicrobial activity, thus showing lower antimicrobial activities than
benzoic acid.
The inhibition mechanism of acid preservatives is based on the “classical
weak-acid theory”, in which, weak acids can partially dissociate in water, achieving a
dynamic equilibrium between molecular acids and charged anions (Stratford et al.,
2013). In acid media, the equilibrium is on the side of molecular acids, which can
enter into the cell cytoplasm through cell membrane by simple diffusion, while in
neutral or alkaline circumstances, the equilibrium is in favour of charged anions,
which are hard to diffuse through cell membrane (Davidson et al., 2005; Brul & Coote,
1999). Sorbic acid can inhibit many microbes, such as yeasts and molds. Microbial
inhibition by sorbic acid and sorbate varies and depends on bacterial species and
16

strains, composition of substrate and its pH, additives and their concentrations,
temperature and packaging, etc. The inhibition mechanism of sorbate and sorbic acid
depends on the media pH. The inhibition activities increase with the decrease of
media pH (Davidson et al., 2005, Bae & Lee, 2017). In the current study, due to its pH
dependence, sorbic acid exerted high antimicrobial activity at acidic conditions but
low antimicrobial activity at pH 7.0 and no antimicrobial activity at pH 9.0 was
observed. The antimicrobial properties of all weak-acid preservatives are greatly
increased at acidic pH. On the other hand, by bearing isopropoxy- at the
phenylalanine ester, a7 displayed largely pH independent antimicrobial activity in pH
5.0-9.0 range. The increased antimicrobial activity of sorbic acid at lower pH values
was attributed to the increased amount of non-dissociated acid molecules (Davidson
et al., 2005). However a7 is an amide compound, which is not dissociated in neutral
and basic conditions, thus a7 had effective antimicrobial activity at pH 5.0, 7.0 and
9.0. Therefore compound a7 exhibited higher antimicrobial properties, which could
be more applicable for the industry compared to sorbic acid.
5. Conclusions
In the current study, a series of sorbic and benzoic acid amide derivatives were
designed and synthesized, and their antimicrobial activities were investigated. Results
indicated that when sorbic acid was conjugated with amino acid esters, higher
antimicrobial activities were achieved when compared to their parent sorbic acid.
However, for benzoic acid amide derivatives, little antimicrobial effectiveness was
observed. Among the SAAD series, compound a7 presented the best antimicrobial
activities, depending on the pH levels ranging from pH 5.0 to pH 9.0. For the use of
17

these novel antimicrobial agents as food preservatives, further studies about their
effectiveness and antimicrobial mechanisms on real foods should be conducted.
Acknowledgments
The authors are grateful to the support provided by the Fundamental Research Funds
for the Central Universities (2017MS075). This research was also supported by the
National Key R&D Program of China (2017YFD0400404), Collaborative Innovation
Major Special Projects of Guangzhou City (201604020007), the Guangdong Provincial
Science and Technology Plan Projects (2015A020209016, 2016A040403040), the
International and Hong Kong – Macau - Taiwan Collaborative Innovation Platform of
Guangdong Province on Intelligent Food Quality Control and Process Technology &
Equipment (2015KGJHZ001), the Guangdong Provincial R & D Centre for the Modern
Agricultural Industry on Non-destructive Detection and Intensive Processing of
Agricultural Products, the Common Technical Innovation Team of Guangdong
Province on Preservation and Logistics of Agricultural Products (2016LM2154) and
the Innovation Centre of Guangdong Province for Modern Agricultural Science and
Technology on Intelligent Sensing and Precision Control of Agricultural Product
Qualities.
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23

Table 1 Antimicrobial activity of tested compounds against bacterial strains and fungal strains
determined as the minimal inhibitory concentration (MIC, in mM) *
Compounds
Gram positive bacteria
Gram negative bacteria
Mixed bacteria
Fungal strains
S. cerevisiae
B. subtilis
7.61
S. aureus
10.14
1.42
E.coli
10.14
2.37
A. niger
>7.61
5.68
a1
>7.61
2.37
>7.61
6.15
a2
1.42
a3
1.18
3.08
3.31
6.63
>7.10
>5.49
>5.22
>4.98
1.33
>7.10
>5.49
>5.22
>4.98
3.98
a4
2.20
2.93
3.29
>5.49
2.44
a5
0.70
2.09
2.44
a6
0.17
2.99
>4.98
1.66
>4.98
1.66
a7
0.17
0.50
b1
>7.24
>6.78
>6.78
>5.30
>5.04
1.45
>7.24
>6.78
>6.78
>5.30
>5.04
>4.82
2.68
>7.24
>6.78
>6.78
>5.30
>5.04
>4.82
2.68
>7.24
>6.78
>6.78
>5.30
>5.04
>4.82
2.68
>7.24
>6.78
>6.78
>5.30
>5.04
>4.82
4.46
>7.24
>6.78
>6.78
>5.30
>5.04
>4.82
4.01
b2
b3
b4
b5
b6
Sorbic acid
Benzoic acid
Ethanol*
2.23
3.28
3.28
3.28
3.28
5.73
5.73
—
—
—
—
—
—
* The mixed bacteria was from rancid milk; —: no activity.
24

1
2
Figure captions
3
Fig. 1. Chemical structures of sorbic (SAAD, a1-a7) and benzoic (BAAD, b1-b6) acid amide derivatives.
Fig. 2. Synthesis of etheyl N-[1-oxo-2, 4-hexadien-1-yl] glycinate (a1). Reagents and conditions: i)
4
o
5
SOCl2, CH3CH2OH, reflux at 0 C; ii) SOCl2, reflux at room temperature; iii) chloroform,
6
diethylamine, reflux stirring at 0 oC for 3 h and then at 60 oC for 4 h.
Fig. 3. Antimicrobial activities of compounds a2, a5 and a7, and sorbic acid incubated with the mixed
bacteria from rancid milk at (a) pH 5.0, (b) pH 7.0 and (c) pH 9.0. The concentration of a2, a5
and a7, and sorbic acid was 2.5 mmol/l. The growth of the mixed bacteria was evaluated
every 6 h.
7
8
9
10
11
12
13
14
Fig. 4. Inhibitory effect of a2, a5, a7 and sorbic acid on MCF-7 cells. MCF-7 cells were treated with
tested compounds for 48 h and the percent viability was determined by the MTT assay.
Values are the mean±SD for four independent experiments.
Fig. 5. 1H NMR spectra of the synthesized compounds a1-a7 and b1-b6.
1

15
16
17
Fig. 1
2

18
19
20
Fig. 2
1

(a)
(b)
2

(c)
Fig. 3
3