Antimicrobial and antitumor activity of peptidomimetics synthesized from amino acids

Article abstract of DOI:10.1016/j.bioorg.2020.104506

Thirteen cationic peptidomimetics derived from amino acids bearing an alkyl or ethynylphenyl moiety that mimic the structure of cationic antibacterial peptides were designed and synthesized using a simple coupling reaction of an amino acid with a substitu

Full text of DOI:10.1016/j.bioorg.2020.104506

Journal Pre-proofs
Antimicrobial and Antitumor Activity of Peptidomimetics Synthesized from
Amino Acids
Huan Li, Shuang Fu, Yudan Wang, Xuan Yuan, Lijia Liu, Hongxing Dong,
Qiang Wang, Zhijia Zhang
PII:
S0045-2068(20)31804-6
DOI:
https://doi.org/10.1016/j.bioorg.2020.104506
YBIOO 104506
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
2 June 2020
24 September 2020
19 November 2020
Please cite this article as: H. Li, S. Fu, Y. Wang, X. Yuan, L. Liu, H. Dong, Q. Wang, Z. Zhang, Antimicrobial
and Antitumor Activity of Peptidomimetics Synthesized from Amino Acids, Bioorganic Chemistry (2020), doi:
https://doi.org/10.1016/j.bioorg.2020.104506
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Elsevier Inc. All rights reserved.

Antimicrobial and Antitumor Activity of Peptidomimetics
Synthesized from Amino Acids
Huan Li1, Shuang Fu2, Yudan Wang1*, Xuan Yuan1, Lijia Liu1*, Hongxing Dong1, Qiang
Wang1*, Zhijia Zhang1
1Key Laboratory of Superlight Materials & Surface Technology, Ministry of Education, Institute
of Advanced Marine Materials, College of Materials Science and Chemical Engineering, Harbin
Engineering University, Harbin 150001, China;2 College of pharmacy, Qiqihar Medical
University, Qiqihar, 161000, China;
Corresponding authors: dqllj2000@163.com (Y. Wang); liulijia@hrbeu.edu.cn (L. Liu)
13864285521@126.com (Q. Wang)
Abstract
Thirteen cationic peptidomimetics derived from amino acids bearing an alkyl or
ethynylphenyl moiety that mimic the structure of cationic antibacterial peptides were designed and
synthesized using a simple coupling reaction of an amino acid with a substituted amine.
Antibacterial activities of the resulting peptidomimetics against drug-sensitive bacteria, such as
Gram-positive Staphylococcus aureus (S. aureus) and Bacillus subtilis, Gram-negative
Escherichia coli (E. coli) and Salmonella enterica, and a drug-resistant bacterium, methicillin-
resistant S. aureus (MRSA), were systematically evaluated. Most peptidomimetics show
significant broad-spectrum antibacterial activity. A-L-Iso-C12 (isoleucine derivative bearing a
dodecyl moiety) show MICs of 2.5 μg/mL against S. aureus and 4 μg/mL against MRSA and A-
1

L-Val-C12 (valine derivative bearing a dodecyl moiety) show MICs of 1.67 μg/mL against E. coli
and 8.3 μg/mL against MRSA. A-L-Val-C12 showed low cytotoxicity toward L929 cells in
comparison with SGC 7901 cells, indicating tumor-directed killing by peptidomimetics while
avoiding toxicity to normal cells. The influences of type of amino acid and substituent, length of
substituent, and stereochemistry of amino acids on antibacterial activity and cytotoxicity of
peptidomimetics were systematically investigated. The results indicate that this series of cationic
peptidomimetics derived from amino acids display antitumor activity and may be useful for
treatment of bacterial infections.
Keywords: amino acid; antibacterial; antitumor; low cytotoxicity; peptide
Graphical abstract
1. Introduction
Bacterial resistance to antibiotic agents is increasing, making human infections increasingly
difficult to treat [1]. Drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus
(MRSA) [2], Extended-Spectrum β-lactamase-producing bacteria (ESBLs) [3, 4], and drug-
resistant Mycobacterium tuberculosis [5] are now spreading worldwide [6-8]. In addition to
clinically excessive use and misuse of antibiotics, bacteria adhere and accumulate on biofilm
2

formed under appropriate conditions, and attached bacteria can obtain drug resistance genes
through horizontal gene transfer [9, 10]. The increase in bacterial resistance to antibiotics and
stagnation of research on novel agents has led to an urgent need to develop new antibacterial drugs.
Natural biofilm-active compounds, such as antibacterial peptides (AMPs) and cationic
liposomes, and other lipophilic biofilm-active compounds that kill bacteria, fungi, protozoa,
viruses, and even cancer cells are considered as antibacterial agents of the future [11-13]. However,
limited selectivity, high in vivo toxicity, and high manufacturing costs of antibacterial peptides
limit their use as clinical antibacterial agents [14]. Synthesis of a variety of peptide mimics to
replace natural antibacterial peptides may address these issues [15, 16]. Amino acids have the
advantages of good biocompatibility, low toxicity, wide range of sources, and low costs.
Derivatives of these compounds can be used for the development of new drugs. Using amino acids
to construct amphiphilic molecules that mimic structure and mechanism of antibacterial peptides,
new antibacterial peptidomimetics have been developed, such as α-peptide [17], β-peptide [14,
18], oligoyl lysine [8], α-AA peptide [19], cationic liposomes, and cationic amphiphilic
compounds [20, 21]. Amino acids in these molecules can recognize the outer membrane of bacteria
and can quickly kill bacteria by destroying this membrane. Some new compounds show rapid and
effective antibacterial properties against both Gram-positive and Gram-negative bacteria. Bacteria
show low resistance to these agents [16, 20].
Peptidomimetics can be used as antimicrobial agents and as carriers for gene therapy and
drugs for cancer treatment. As chemotherapeutic substances, cationic liposomes are naturally
positively charged or positively charged by protonation at normal physiological pH. These agents
electrostatically combine with negatively charged phosphates on the surface of DNA to inhibit
DNA formation and growth of tumor cells [22, 23]. Cation and amphiphilic anti-cancer active
3

peptides also interact with cancer cells through electrostatic and hydrophobic interactions, leading
to cancer cell necrosis or apoptosis [24-29].
A series of cationic antibacterial peptidomimetics with alkyl or phenylethynyl groups were
synthesized using amino acids as raw materials. These peptidomimetics exhibit excellent broad-
spectrum antibacterial activity and notable killing of SGC 7901 tumor cells. Further, these agents
show low cytotoxicity to L929 cells and good biocompatibility. The effects of the type and length
of substituent groups and the type and stereo configuration of the amino acid residues on
antibacterial properties and cytotoxicity of peptidomimetics were systematically investigated. The
mechanism of antibacterial action was also explored.
2. Results and discussion
2.1. Synthesis of the peptidomimetics
Several amino acids were separately reacted with aliphatic amines with different alkyl chain
lengths, ranging from 4 to 16 carbon atoms, or with an ethynylanline to produce thirteen cationic
peptidomimetics (Scheme 1). Reaction products were characterized with 1H-NMR, 13C-NMR, and
FTIR [30].
4

Scheme 1. Synthesis of the peptidomimetics
2.2. Antibacterial Activity
2.2.1. Minimum inhibitory concentrations (MIC)
Antibacterial efficacy was evaluated by minimum inhibitory concentrations (MIC) of
peptidomimetics (Table 1). Peptidomimetics (entries 5, 6, 8-11 in Table 1) show substantial
activity against both Gram-positive and Gram-negative bacteria, but a higher activity against the
latter. For instance, peptidomimetics with dodecyl chains (entries 5, 6, 8-11 in Table 1) display a
range of MIC values against the Gram-positive bacteria of 1 μg/mL - 20 μg/mL, compared to MICs
of 1.67 μg/mL - 6.5 μg/mL against the Gram-negative bacteria.
The length of alkyl chains is crucial to antibacterial activity. A-L-Phe-C4, with the shortest
aliphatic chain (butyl chain) shows poor antibacterial activity against both Gram-positive S. aureus
5

and Gram-negative E. coli MIC values of 1250 and 312 μg/mL, respectively. The activity of
peptidomimetics is substantially promoted by changing the short butyl chain to a longer octanoyl
chain (A-L-Phe-C8, MIC=39 μg/mL against both S. aureus and E. coli). Further increase of alkyl
chain length led to additional reduction in MIC. For instance, A-L-Phe-C12 with a dodecyl chain
shows MIC values of 5 μg/mL and 2.5 μg/mL against S. aureus and E. coli, respectively. However,
any greater chain length resulted in decreased activity, as observed for the A-L-Phe-C16
hexadecanoyl compound, MIC =1250 μg/mL against E. coli. A parabolic chain length response
curve was thus characteristic for structure−activity relationship (SAR) for peptidomimetics.
Optimum lipophilicity lies somewhere between dodecanoyl and hexadecanoyl chain length.
Amino acid residues in peptidomimetics also have an important effect on the antibacterial
activity. Peptidomimetics with the same dodecyl chain (entries 5, 7-11 in Table 1) show significant
differences in antibacterial activity among in amino acid residues. Peptidomimetics derived from
lipophilic amino acids, such as A-L-Val-C12, A-L-Leu-C12, and A-L-Iso-C12 show significant
activity, especially against MRSA, MIC=4-8.3 μg/mL. MICs of these three mimetics are
comparable to the MIC against MRSA of MSI-78, an AMP currently undergoing phase III clinical
trials as a topical antibiotic. Antibacterial activity of peptidomimetics based on aromatic amino
acids, A-L-Phe-C12, and A-L-PG-C12, is reduced, MIC=15.6-16.6 μg/mL. Further, A-L-Leu-mAA
with ethynyl aniline and L-leucine residues shows activity against all tested bacteria. Similar MICs
were found for A-L-Phe-C4. Peptidomimetics (entries 12, 13 in Table 1) with the same ethynyl
aniline residues derived from different amino acids also display differences in antibacterial activity.
The activity of A-L-Iso-mAA with a L-isoleucine residue is lower than that of A-L-Leu-mAA with
an L-leucine residue.
The structure-activity relationships peptidomimetics indicate that structure and chain lengths
6

of hydrophobic moieties and the structure of the base amino acid are important for antibacterial
performance. Substantial antibacterial activity of the peptidomimetics against MRSA suggests
potential for antibacterial medicine.
7

Table 1. Antibacterial of the peptidomimetics
MIC (μg/mL)
Gram-positive
Gram-negative
Entr
Drug resistant
bacteria
Compound
y
E.coli
Salmonell
S.aureu
s
Bacillus subtilis
MRSA
a
A-L-Phe-C
A-L-Phe-C
A-L-Phe-C
A-L-Phe-C
1
2
312
156
39
416
1250
624
39
416
156
78
624
312
52
4
6
8
156
62.5
15.6
5
3
4
10
13.3
2.5
5
39
20
10
A-L-Phe-C
5
5
10
16.6
20
12
A-D-Phe-C
A-L-Phe-C
6
5
10
8.3
625
4.2
10
12
7
1250
1.67
2.5
2
625
6.5
4.17
5
1250
3.3
3.3
2.5
7.8
1250
8.3
16
A-L-Val-C
8
12
A-L-Leu-C
9
6.25
4
12
A-L-Iso-C
10
11
5
12
12
A-L-PG-C
2.5
3.9
10
15.6
A-L-Leu-
mAA
12
10.6
15.6
15.6
10.6
31.2
15.6
31.2
31.2
62.4
13
14
15
16
A-L-Iso-mAA
LEVa
15.6
20
b
b
b
b
-
-
-
-
GENc
-
-
2
-
-
b
b
b
b
b
b
MSI-78
16-32d
-
8-16 d
-
16-32 d
a LEV (levofloxacin).
b Not determined.
c GEN (gentamycin).
d Literature values [33].
8

2.2.2. Inhibition zone
Inhibition zone tests are used to characterize both the antibacterial activity and permeability
of the peptidomimetics concurrently. Inhibition zone experiments of peptidomimetics with both
Gram-negative and positive bacteria were conducted (Fig. 1). After a 24 h incubation, inhibition
zones were clearly observed for peptidomimetics A-L-Phe-C8, A-L-Phe-C12, A-L-Iso-C12, A-L-
Leu-mAA, and A-L-Iso-mAA against both bacteria types; no significant inhibition zones appeared
for peptidomimetics A-L-Phe-C4, A-L-Phe-C12, A-D-Phe-C12, A-L-Val-C12 (Fig. 1b and 1c).
Among peptidomimetics with dodecyl chains, A-L-Iso-C12 derived from isoleucine showed the
largest inhibition zone against S. aureus and E. coli, consistent with its low MIC. A-L-Iso-C12
displays both high antibacterial activity and notable permeability. Testing of aromatic
peptidomimetics with different chain lengths (A-L-Phe-Cn, n=4, 6, 8, 10, 12, 16) shows that A-L-
Phe-C8 induced the largest zone of inhibition. Also, peptidomimetics, A-L-Leu-mAA, and A-L-
Iso-mAA with ethnyl aniline residues, showed large inhibition zones against both bacteria types
(Fig. 1b and 1c). Thus, a combination of antibacterial activity and permeability may result in
synergistic antibacterial activity.
9

Fig. 1. Antibacterial activity of peptidomimetics: (a) images of punch well agar diffusion test
against E. coli and S. aureus; (b) zone of inhibition diameter against E. coli; (c) zone of inhibition
diameter against S. aureus.
2.3. Inhibition of bacterial growth
Growth rates of bacteria were evaluated using OD600 to evaluate inhibition of bacterial growth
by the peptidomimetics. Higher OD600 indicates higher concentrations of bacteria and poorer
antibacterial activity. Compared with controls, concentrations of A-L-Phe-C12 less than 2 did not
inhibit the growth of E. coli. Growth of bacteria was significantly inhibited at a concentration of 5
μg/mL and almost stopped when the concentration increased to 10 μg/mL (Fig. 2a and 2b). A
concentration of A-L-Val-C12 of 1 μg/mL significantly inhibited E. coli growth. At 10 μg/mL,
10

bacteria were dead. Both peptidomimetics exhibited excellent antibacterial activity. A-L-Val-C12
exhibited better antibacterial activity than A-L-Phe-C12, despite identical dodecyl chains. Amino
acid residues significantly affect the antibacterial activity of peptidomimetics.
Fig. 2. Bacterial growth curves of E. coli treated with peptidomimetics: (a) A-L-Phe-C12; (b) A-
L-Val-C12.
2.4. Bactericidal kinetics
Time-kill kinetics of peptidomimetics against E. coli provide a rate for bactericidal action. E.
coli was cultured to stationary phase, then treated with peptidomimetics, A-L-Phe-C12, and A-L-
Val-C12, at concentrations of 5 μg/mL, 10 μg/mL, 15 μg/mL and 20 μg/mL. These peptidomimetics
show excellent bactericidal activity and rates of bactericidal action increases with increasing
concentration. (Fig. 3).
At concentrations below 10 μg/mL of A-L-Phe-C12, the growth of stationary-phase bacteria
was inhibited, and bacterial reproduction was slow. When the concentration increased to 20 μg/mL,
the bacterial population was significantly reduced, and almost all bacteria were killed after 4 hours
(Fig. 3a). A-L-Val-C12 exhibited more rapid biocidal action reducing the bacterial population by
~7 log10 (CFU/mL) at a concentration of 20 μg/mL in only 10 min. This treatment killed almost all
11

stationary-phase bacteria. (Fig. 3b).
Fig. 3. Bactericidal kinetics of peptidomimetics against stationary phase E. coli: (a) A-L-Phe-
C12; (b) A-L-Val-C12.
2.5. Biofilm disruption
Biofilm is an aggregate of bacteria held together by a mucus-like matrix of carbohydrate that
adheres to a surface. This matrix is the basis for bacterial reproduction and spread. This bacterial
community is an effective defense against antibiotics, making bacteria difficult to kill. Biofilm
activity experiments were performed on biofilms formed by Gram-positive S. aureus and Gram-
negative E. coli using peptidomimetic A-L-Val-C12. The destructive activity of A-L-Val-C12
against biofilm and bacteria in the biofilm was evaluated by measuring survival rate bacteria after
peptidomimetic treatment.
Bacteria were statically incubated until a biofilm was formed, and A-L-Val-C12 at different
concentrations were used to treat the formed biofilm. As the concentration of A-L-Val-C12
increased, the number of bacteria in the mature E. coli biofilm gradually decreased. At
peptidomimetic concentrations greater than 16 μg/mL, the number of bacteria was significantly
reduced to 13.7 log10 CFU. A further increase in peptidomimetic concentration to 256 μg/mL
12

reduced bacterial numbers to 2.5 log10 CFU. As a control, the number of bacteria in untreated
biofilm was 22.7 log10 CFU per well–significantly higher than treated biofilm. A-L-Val-C12 also
showed bactericidal effects on bacteria in the S. aureus biofilm. At a peptidomimetic concentration
of 256 μg/mL, the number of bacteria was 5.5 log10 CFU; the number in untreated biofilm was
20.5 log10 CFU per well. The above results indicate that peptidomimetic A-L-Val-C12 can
effectively destroy bacterial biofilm and kill embedded bacteria. A-L-Val-C12 is more effective
against the E. coli biofilm.
Fig. 4. Antibiofilm activity of A-L-Val-C12 at different concentrations: (a) cell viability in non-
treated and treated biofilms of E. coli grown on cover slips for 72 h; (b) cell viability in non-
treated and treated biofilms of S. aureus grown on cover slips for 24 h.
2.6. Mechanism of action
Excellent and rapid bactericidal activity of peptidomimetics may be due to disruption of
bacterial cell membrane integrity due to electrostatic interactions between negatively charged
bacterial cell membrane with cationized peptidomimetics. Peptidomimetics, A-L-Phe-C8, A-L-
Phe-C12, and A-L-Val-C12, were separately incubated with Gram-negative E. coli and antibacterial
mechanisms investigated using SEM, inverted fluorescence microscopy and flow cytometry (Fig.
5).
13

SEM images show that untreated E. coli shows a complete shape and a smooth surface, but
E. coli treated with peptidomimetics exhibits obvious folds in the cell membrane, with some
surface holes (Fig. 5a). Peptidomimetic treatment disrupts the cell membrane of bacteria, forming
pores on its surface and leading to cell death.
The action of the peptidomimetics on bacterial cell membranes was investigated by the "live-
dead staining" (N01 and PI) [34]. Staining of bacteria before and after the peptidomimetic
treatment was observed using fluorescence microscopy. Untreated bacteria showed green and
almost no red fluorescence, indicating normal E. coli cells and few or no dead bacteria. Only red
fluorescence was observed for treated bacteria, indicating bacteria cell membrane rupture and cell
death. Peptidomimetics effectively kill bacteria by destroying their cell membranes.
The cell proliferation was detected with Propidium Iodide (PI) single staining. After staining
of DNA, DNA content of cells was measured by flow cytometry, and DNA replication and cell
death were analyzed based on the distribution of DNA content. Except for the control group, fewer
cells were viable. These results also indicate that peptidomimetics directly damage negatively
charged cell membranes, leading to the leakage of genetic material and cell death.
14

Fig. 5. Mechanism of antibacterial action before and after treatment with A-L-Phe-C8, A-L-Phe-
C12 and A-L-Val-C12 at same concentrations (10 μg/mL) against E. coli: (a) SEM images; (b)
Fluorescence microscopy images after staining N01 and PI; (c) apoptotic rates measured by flow
cytometry.
The antibacterial mechanism of the peptidomimetics is illustrated in Fig. 6. Cationic groups
of peptidomimetics adsorbed to microbial surfaces through electrostatic interactions, thereby
altering the electrochemical properties of the membrane surface. Further, due to the thin
peptidoglycan layer between the inner and outer membranes of E. coli [35], long alkyl chains of
the peptidomimetics more easily penetrate the cell membrane, resulting in perforation, leakage of
intracellular material, and microbial cell death [36].
15

Fig. 6. The antibacterial mechanism of cationic peptidomimetics.
2.7. Antitumor cell activity
The excellent antibacterial and biofilm destruction activity of peptidomimetics, we
investigated potential antitumor activity. Survival of SGC 7901 cells treated with three kinds of
peptidomimetics (A-L-Phe-C12, A-L-Val-C12 and A-L-Phe-C8) at different concentrations was
investigated using the MTT approach (Fig. 7). Compared to controls, survival of SGC 7901 cells
clearly decreased with increasing peptidomimetics concentration. Comparing the three
peptidomimetics at the same concentration, survival of SGC 7901 cells treated with A-L-Phe-C12
and A-L-Val-C12 was less than the survival of the cells treated with A-L-Phe-C8. A-L-Phe-C12 and
A-L-Val-C12 are thus more potent for inhibiting SGC 7901 cells. Meanwhile, IC50 values,
concentrations needed for 50% cell growth inhibition estimated by curve fitting of absorbance
values at different concentrations (Table 2). Peptidomimetics show promising anti-gastric cancer
properties (IC50=23-135 μg/mL) compatible with that of the reference, 5-Fluorouracil (IC50=14
μg/mL).
16

Fig. 7. The viability of SGC 7901 cells incubated with A-L-Phe-C8 (blue), A-L-Phe-C12 (gray) and
A-L-Val-C12 (red) at various concentrations.
Table 2. Antitumor properties of the tested peptidomimetics.
Peptidomimetic
A-L-Phe-C8
135
A-L-Phe-C12
23
A-L-Val-C12
37
5-Fluorouracil
14
IC50 (μg/mL)
Photographs from MTT assays indicate that cell membranes of control cells are structurally
intact, but cells treated with peptidomimetic A-L-Val-C12 are round or oval and show
characteristics of apoptosis (Fig. 8a). Further, PI can cross damaged cell membranes and stain
nuclei red, but cannot penetrate an intact cell membrane. The reagent is used to assess cell viability.
PI staining was used to examine the effect of A-L-Val-C12 on cell apoptosis. Except for the control
group, cells in all the treated groups became more apoptotic in appearance (Fig. 8b). The
percentage of apoptotic SGC 7901 cells treated with A-L-Val-C12 was up to 56% at an A-L-Val-
C12 concentration of 30 μg/mL, much higher than 24.7% at 15 μg/mL and 11.1% at 7.5 μg/mL.
These results indicate that A-L-Val-C12 induces apoptosis in SGC 7901 cells, and the proportion
17

of apoptotic cells increased significantly with increasing concentration.
Fig. 8. Antitumor cell activity of A-L-Val-C12: (a) morphology of the SGC 7901 cells; (b)
apoptosis of SGC 7901 cells analyzed by flow cytometer.
2.8. Biocompatibility
Biocompatibility is an important topic in drug research, and cytotoxicity of the
peptidomimetics was investigated by incubation of normal mammalian L929 cells with
peptidomimetics A-L-Phe-C8, A-L-Phe-C12, and A-L-Val-C12. All peptidomimetics show
relatively low toxicity to L929 cells with a cell survival rate of more than 60%, even for a
concentration of 1 mg/mL. Notably, at a concentration of 62.5 μg/mL, the survival rate of L929
cells incubated with the peptidomimetics was 100%. When a concentration of peptidomimetics is
below 30 μg/mL, it exhibits good activity against gastric cancer SGC 7901 cells and significant
antibacterial properties. Peptidomimetics are non-toxic to mammalian L929 cells at concentrations
that can selectively act on gastric cancer cells SGC 7901 and bacteria. Thus, peptidomimetics
display good biocompatibility.
18

Fig. 9. Cell viability of L929 cells incubated with A-L-Phe-C8 (blue), A-L-Phe-C12 (gray) and A-
L-Val-C12 (red) at various concentrations.
3. Conclusions
A series of peptidomimetic antibacterial agents were successfully synthesized using amino
acids as raw materials. The obtained peptidomimetics show excellent broad-spectrum antibacterial
and biofilm destruction activity against both Gram-positive (including MRSA) and Gram-negative
bacteria. These peptidomimetics are rapidly bactericidal via disrupting bacterial cell membranes
and causing leakage of intracellular material. Further, peptidomimetics showed substantial
inhibitory capacity on the growth of gastric cancer SGC 7901cells, and low toxicity to mammalian
normal L929cells. These peptidomimetics have great potential to be developed as novel
antibacterial and antineoplastic agents.
19

4. Experimental
4.1. Materials
Isobutyl chlorocarbonate, 3-aminophenylacetylene, L-phenylalanine, D-phenylalanine L-
alanine, L-leucine, L-isoleucine, and L-phenylglycine were supplied by Saen Chemical
Technology (Shanghai) Co., Ltd. 1-aminobutane, hexylamine, octylamine, decanamine,
dodecylamine and hexadecylamine were purchased from Aladdin (China). Dichloromethane,
Diethyl ether, Ethyl acetate, Triethylamine, Chloroform, Methanol, Hexane, NaOH, NaHCO3,
KHSO4, anhydrous MgSO4, beef, agar, PBS buffer and other solvents were supplied by Shanghai
Macklin Biochemical Co., Ltd (China). The bacterial strains of Staphylococcus aureus (S. aureus)
and Bacillus subtilis, Gram-negative Escherichia coli (E. coli) and Salmonella enterica and drug-
resistant bacteria, methicillin-resistant S. aureus (MRSA) were provided by Shanghai Luwei
Technology Co., Ltd. In this study, all reagents were analytical grade.
4.2. Measurements
1H NMR (400MHz) spectra were recorded on a JEOL LEOLEX-400 spectrometer. The
surface functional groups and chemical compositions of products were investigated using Fourier
transform infrared spectroscopy (FT-IR, Nicolet Avatar-330) by the KBr pellet pressing method.
The UV–vis absorption spectra were determined by a UV-1601 spectrophotometer. Bacterial
growth was investigated using microplate reader (Mindray MR-96A).The morphological
structures of the microorganisms were measured by scanning electron microscopy (SEM,
Thermoscientific ApreoS LoVac, USA). Cells were observed under a DY5000X inverted
microscope (Chongqing Photoelectric Instrument Co. Ltd., Chongqing, China). Cells and PI
20

single-stained cells for apoptosis were quantitatively assayed on a flow cytometer (Becton
Dickinson, San Jose, CA, USA) using CellQuest software.
4.3. General procedure for the synthesis of amide coupling peptidomimetics
Various N-Boc protected amino acids were separately reacted with an aliphatic amine and
with 3-aminophenylacetylene to produce the corresponding N-Boc protected peptidomimetics, and
then removal of the Boc group provide the free peptidomimetics (Scheme 1). A typical procedure
as follows: To a solution of N-Boc protected amino acids (10 mmol) in CH2Cl2 (20 mL) Et3N (11
mmol) and isobutyl chloroformate (11 mmol) were added at 0 °C under stirring. After 55min, the
amine (12.5 mmol) was added and the reaction was allowed to warm to room temperature and
detected by TLC (hexane/ethyl acetate = 3:1, Rf ≈ 0.5). After 24 h, the mixture was washed with
1M KHSO4, saturated NaHCO3, brine in sequence. The collected organic layer was dried over
anhydrous MgSO4 and then concentrated. The crude product was purified by flash column
chromatograph to give the N-Boc protected peptidomimetics (35-78% yield).
Then, to a solution of N-boc protected peptidomimetics in ethyl acetate (8 mL) a solution of
HCl in ethyl acetate (2 mL) was added and the mixture was stirred until the completion of the
reaction (10 h). The ethyl acetate was evaporated and the mixture was washed with ether (3×20
mL). After concentration and dry in vacuum, the resulting compound was subsequently
characterized by using 1H NMR and 13C NMR. (yield 55-90%)
4.3.1. (S)-2-Amino-N-butyl-3-phenylpropanamide (A-L-Phe-C4)
White foamed solid, yield: 62.5%. 1H NMR (400 MHz, DMSO-d6) δ 8.50-8.37 (s, -CH-NH2,
2H), 7.32-7.17 (m, Ar-H, 5H), 3.93 (dd, -NH-CO-CH-, 1H), 3.10–2.82 (m, -NH-CO-CH2-, Ar-
CH2-, 4H), 1.36–0.95 (dt, -(CH2)2-CH3, 4H), 0.80 (m, -CH2-CH3, 3H). 13C NMR (101 MHz,
21

DMSO-d6) δ 167.9, 135.6, 129.9, 128.8, 127.4, 53.9, 40.2, 38.7, 37.4, 31.2, 19.8, 14.1. IR (cm-
1,KBr) 3325(N-H), 1653(C=O), 1568(N-H).
4.3.2. (S)-2-Amino-N-hexyl-3-phenylpropanamide(A-L-Phe-C6)
White foamed solid, yield: 57.2%. 1H NMR (400 MHz, DMSO-d6) δ 8.54-8.26 (s, -CH-NH2,
2H), 7.31-7.10 (m, Ar-H, 5H), 3.93 (dd, -NH-CO-CH-, 1H), 3.10–2.82 (m, -NH-CO-CH2-, Ar-
CH2-, 4H), 1.36–0.95 (dt, -(CH2)4-CH3, 8H), 0.80 (m, -CH2-CH3, 3H). 13C NMR (101 MHz,
DMSO-d6) δ 167.9, 135.6, 129.9, 128.8, 127.4, 53.9, 39.9, 37.3, 31.4, 29.1, 26.4, 22.5, 14.4. IR
(cm-1,KBr) 3322(N-H), 1654(C=O), 1563(N-H).
4.3.3. (S)-2-Amino-N-octyl-3-phenylpropanamide(A-L-Phe-C8)
White foamed solid, yield: 59.7%. 1H NMR (400 MHz, DMSO-d6): δ 8.55-8.15 (s, -CH-NH2,
2H), 7.31-7.12 (m, Ar-H, 5H), 3.86 (dd, -NH-CO-CH-, 1H), 3.08–2.80 (dt, -NH-CO-CH2-, Ar-
13
CH2-, 4H), 1.29–1.03 (dt, -(CH2)8-CH3, 16H), 0.82 (m, -CH2-CH3, 3H). C NMR (101 MHz,
DMSO-d6) δ 167.9, 135.6, 130.1, 128.8, 127.4, 53.9, 40.1, 37.4, 31.7, 29.1, 26.7, 22.6, 14.4. IR
(cm-1,KBr) 3323(N-H), 1654(C=O), 1562(N-H).
4.3.4. (S)-2-Amino-N-decyl-3-phenylpropanamide(A-L-Phe-C10)
White foamed solid, yield: 62.8%. 1H NMR (400 MHz, DMSO-d6) δ 8.48-8.25 (s, -CH-NH2,
2H), 7.34-7.17 (m, Ar-H, 5H), 3.92 (dd, -NH-CO-CH-, 1H), 3.14–2.77 (dt, -NH-CO-CH2-, Ar-
13
CH2-, 4H), 1.30–0.95 (dt, -(CH2)6-CH3, 12H), 0.84 (m, -CH2-CH3, 3H). C NMR (101 MHz,
DMSO-d6) δ 167.9, 135.6, 129.9, 128.8, 127.4, 53.9, 39.9, 37.4, 31.8, 29.3, 26.7, 22.6, 14.4. IR
(cm-1,KBr) 3323(N-H), 1654(C=O), 1563(N-H).
22

4.3.5. (S)-2-Amino-N-dodecyl-3-phenylpropanamide(A-L-Phe-C12)
White foamed solid, yield: 64.5%. 1H NMR (400 MHz, DMSO-d6) δ 8.48-8.28 (s, -CH-NH2,
2H), 7.28-7.19 (m, Ar-H, 5H), 3.93 (dd, -NH-CO-CH-, 1H), 3.08–2.82 (dt, -NH-CO-CH2-, Ar-
13
CH2-, 4H), 1.33–1.00 (dt, -(CH2)8-CH3, 16H), 0.83 (m, -CH2-CH3, 3H). C NMR (101 MHz,
DMSO-d6) δ 168.0, 135.6, 129.9, 128.8, 127.4, 53.9, 40.1, 39.0, 37.4, 31.8, 29.5, 26.7, 22.6, 14.4.
IR (cm-1,KBr): 3323(N-H), 1654(C=O), 1563(N-H).
4.3.6. (R)-2-Amino-N-dodecyl-3-phenylpropanamide(A-D-Phe-C12)
White foamed solid, yield: 59.7%. 1H NMR (400 MHz, DMSO-d6): δ 8.55-8.32 (s, -CH-NH2,
2H), 7.31-7.18 (m, Ar-H, 5H), 3.94 (dd, -NH-CO-CH-, 1H), 3.08–2.80 (dt, -NH-CO-CH2-, Ar-
13
CH2-, 4H), 1.29–1.03 (dt, -(CH2)8-CH3, 16H), 0.82 (m, -CH2-CH3, 3H). C NMR (101 MHz,
DMSO-d6): δ 168.0, 135.8, 130.1, 128.9, 127.5, 54.1, 40.1, 37.5, 31.9, 29.5, 26.7, 22.6, 14.4.
IR(cm-1,KBr): 3323(N-H), 1654(C=O), 1563(N-H).
4.3.7. (S)-2-Amino-N-hexadecyl-3-phenylpropanamide(A-L-Phe-C16)
White foamed solid, yield: 59.0%. 1H NMR (400 MHz, DMSO-d6): δ 8.39-8.28 (s, -CH-NH2,
2H), 7.30-7.19 (m, Ar-H, 5H), 3.91 (dd, -NH-CO-CH-, 1H), 3.09–2.84 (dt, -NH-CO-CH2-, Ar-
13
CH2-, 4H), 1.26–1.05 (dt, -(CH2)8-CH3, 16H), 0.82 (m, -CH2-CH3, 3H). C NMR (101 MHz,
DMSO-d6): δ 167.9, 135.7, 130.1, 128.8, 127.4, 53.9, 40.1, 37.4, 31.8, 29.5, 26.8, 22.6, 14.4.
IR(cm-1,KBr): 3322(N-H), 1654(C=O), 1563(N-H).
4.3.8. (S)-2-Amino-N-dodecyl-3-methylbutanamide(A-L-Val-C12)
Yellow viscous liquid, yield: 74.8%. 1H NMR (400 MHz, DMSO-d6): δ 7.73 (s, -CH-NH2,
2H), 6.53 (s, -NH-CO-, 1H), 3.66 (s, -CH-NH2, 1H), 3.06-2.93 (d, -CO-NH--CH2-, 1H), 1.83 (s, -
23

(CH3)2-CH-CH2-, 1H), 1.33,1.19 (s, -(CH2)10-CH3, 20H), 0.78 (m, -CH2-CH3,-(CH3)2-CH-, 9H).
13C NMR (101 MHz, DMSO-d6): δ 167.8, 57.8, 40.1, 31.8, 30.1, 29.5, 26.9, 22.6, 18.6, 14.3.
IR(cm-1,KBr): 3326(N-H), 1658(C=O), 1563(N-H).
4.3.9. (S)-2-Amino-N-dodecyl-4-methylpentanamide(A-L-Leu-C12)
Dark orange viscous liquid, yield: 69.6%. 1H NMR (400 MHz, DMSO-d6): δ 8.63 (s, -NH-
CO-, 1H), 8.29 (s, -CH-NH2, 2H), 3.67 (s, -CH-NH2, 1H), 3.15-2.94 (d, -CO-NH-CH2-, 1H), 1.51
(m, -(CH3)2-CH-CH2-, 1H), 1.20 (s, -CH-CH2-CH-, -(CH2)10-CH3, 22H), 0.81 (m, -CH2-CH3,-
13
(CH3)2-CH-, 9H). C NMR (101 MHz, DMSO-d6): δ 169.0, 71.7, 51.5, 39.8, 31.8, 29.3, 27.6,
26.7, 24.3, 22.7, 19.1, 14.4. IR(cm-1,KBr): 3281(N-H), 1672(C=O), 1564(N-H).
4.3.10. (S)-2-Amino-N-dodecyl-3-methylpentanamide(A-L-Iso-C12)
1
Dark green viscous liquid, yield: 70.6%. H NMR (400 MHz, DMSO-d6) δ 8.53 (s, -NH-
CO-, 1H), 8.22 (s, -CH-NH2, 2H), 3.69 (s, -CH-NH2, 1H), 3.16,2.93 (d, -CO-NH-CH2-, 1H), 1.77
(m, CH3-CH-CH-, 1H), 1.49-1.35 (d, CH3-CH2-CH-), 1.20 (s, -(CH2)10-CH3, 22H), 0.82 (m, -CH2-
CH3, -CH-CH3, 9H). 13C NMR (101 MHz, DMSO-d6): δ 167.8, 65.4, 57.1, 39.8, 36.5, 31.8, 29.5,
26.8, 24.6, 22.6, 14.8, 14.3, 11.5. IR(cm-1,KBr): 3281(N-H), 1672(C=O), 1564(N-H).
4.3.11. (S)-2-Amino-N-dodecyl-2-phenylacetamide(A-L-PG-C12)
1
Dark yellow foamed solid, yield: 58.9%. H NMR (400 MHz, DMSO-d6): δ 7.44-7.02 (d,
Ar-H, 5H), 3.18–2.78 (dt, -NH-CO-CH2-, 2H), 1.33–1.00 (dt, -(CH2)10-CH3, 20H), 0.83 (m, -CH2-
CH3, 3H). 13C NMR (101 MHz, DMSO-d6) δ 167.5, 135.0, 129.2, 128.1, 55.7, 40.1, 31.8, 29.5,
26.6, 22.6, 14.4. IR(cm-1,KBr): 3324(N-H), 1657(C=O), 1561(N-H).
24

4.3.12. (S)-3-Amino-N-(3-ethynylphenyl)-5-methylhexanamide(A-L-Leu-mAA)
Dark yellow foamed solid, yield: 58.9%. 1H NMR (400 MHz, DMSO-d6): δ 8.00, 7.63 (dd,
Ar-H, 4H), 4.21 (m, -NH-CO-CH-, 1H), 2.64 (s, Ph-C≡CH, 1H), 1.93-1.82 (m, (CH3)2-CH-), 1H),
1.81-1.08 (m, (CH3)2-CH-CH2-), 2H), 0.89-0.79 (m, -(CH3)2-CH-, 6H). 13C NMR (101 MHz,
DMSO-d6) δ 197.1, 169.0, 143.0, 132.8, 129.9, 119.3, 52.2, 39.9, 27.0, 24.2, 23.1, 22.7. IR(cm-
1,KBr) 3287(N-H), 1694(C=O), 1553(N-H).
4.3.13. (S)-3-Amino-N-(3-ethynylphenyl)-4-methylhexanamide (A-L-Iso-mAA)
Dark green foamed solid, yield: 73.1%. 1H NMR (400 MHz, DMSO-d6): δ 7.87, 7.41 (m, Ar-
H, 4H), 3.96 (m, -NH-CO-CH-, 1H), 2.52 (s, Ph-C≡CH, 1H), 2.07-1.70 (m, CH3-CH-CH2-), 1H),
1.89-1.25 (m, CH3-CH-CH2-), 2H), 1.02-0.76 (m, -CH3-CH-, -CH2-CH3, 6H). 13C NMR (101 MHz,
DMSO-d6) δ 197.0, 168.0, 142.9, 132.8, 130.0, 119.3, 58.5, 39.9, 30.4, 27.0, 18.9, 18.3. IR(cm-
1,KBr) 3287(N-H), 1693(C=O), 1552(N-H).
4.4. Antibacterial tests
Determination of minimum inhibitory concentration (MIC)
MIC, the minimum concentration required for peptidomimetics to completely inhibit the
bacterial growth. The antibacterial properties of the peptidomimetics against Gram-positive S.
aureus and Bacillus subtilis, Gram-negative E. coli and Salmonella enterica, and a drug-resistant
bacterium, methicillin-resistant S. aureus (MRSA) were assessed by the TTC coloration method
[37]. The bacterial suspensions were incubated in the medium and diluted to the test concentration
of 107 CFU/mL. All the peptidomimetics were dissolved in sterile water and 0.02% TTC coloration
agent prepared in liquid medium. TTC-containing liquid medium (100 μL) was added to each well
25

of the 96-well plate. Next, the peptidomimetics solutions (100 μL) were added to the 96-well plates
and diluted in turn with three parallel experiments were conducted. Finally, the diluted bacterial
suspensions (100 μL) were added to the 96-well plate. The mixtures were incubated at 37 °C
(Bacillus subtilis) for 72 h or 37 °C (other bacteria) for 24 h. Negative contrast was a liquid culture
containing 200 μL of media and a solution containing 200 μL of bacteria at 107 CFU/mL as a
positive contrast. The MIC value is the lowest concentration without obvious bacterial growth.
Therefore, due to the presence of TTC, the concentration of the peptidomimetics without red is
MIC [37].
Inhibition Zone Test
The bacterial suspensions were incubated overnight at 37 °C in a freshly liquid medium. The
diluted bacterial suspensions (107 CFU/mL, 100 μL) and agar medium were poured into a petri
dish in sequence. The dish was shaken slightly before the solid medium solidifies to get
homogeneous mixture of bacteria and solid medium. Finally, various peptidomimetics were placed
in the Oxford cup on a solidified plate and incubated overnight at 37 °C. After incubation, the
inhibition zone around the Oxford cup was measured.
Biofilm disruption assay
S. aureus and E. coli were cultured for 4-6 h to attain the middle logarithmic stage. The
bacterial suspensions (~105 CFU/mL, 100 μL) were added to the 96-well plates, and the biofilm
could be observed at the bottom of the plates for a certain period of time at 37 °C (S. aureus for
24 h, E. coli for 72 h). Then the 96-well plates were centrifuged at 3000 rpm for 5 min. The
suspended liquid was removed and the wells were washed once with 1×PBS. Sterile water was
used to prepare A-L-Val-C12 solutions with different concentrations. For the test group, the A-L-
26

Val-C12 solutions (100 μL) were added to the 96-well plates, while only 100 μL of 1×PBS solution
was added for the control group. After cultured at 37 °C for 24h, the supernatant was removed and
the suspensions were centrifuged with 3000 rpm for 5min. The bacterial suspensions were diluted
with a gradient of 10 times and dropped into the solid medium agar plate. After the culture of 24
h at 37 °C, colony counting was performed and the results were expressed by log10 (CFU/well).
Bacterial growth inhibiting
E. coli from a single colony was inoculated into the liquid medium at 200 rpm and shaken
overnight at 37 °C. Peptidomimetics with different concentrations and E. coli solution (OD600 =
1, 200 μL) were added to liquid medium (10 mL) in turn, and the final concentration range of the
peptidomimetics was 1 μg/mL to 20 μg/mL. The liquid medium was incubated at 37 °C with 200
rpm. The bacterial suspensions without peptidomimetics were used as the blank control group.
During incubation, the mixture (100 μL) was poured into 96-well plates to measure the OD values
of the bacterial suspensions at the wavelength of 600 nm. The growth inhibition curves of E. coli
were obtained according to the OD values.
Bactericidal kinetics
In order to obtain the action rate of the peptidomimetics against bacteria, the bactericidal
activity was evaluated by time killing kinetics test. The bacterial suspensions were diluted to
concentration of 107 CFU/mL. Then, the peptidomimetics with the concentrations of 5 μg/mL, 10
μg/mL, 15 μg/mL and 20 μg/mL were added respectively. For comparison, a blank group without
antibacterial agent was implemented. After dosing, each group was taken to 96-well plates and
centrifuged at 3000rpm for 5min. The supernatant was removed, and 1×PBS solution (100 μL)
was suspended. The diluted bacteria (100 μL) were dropped into the solid medium agar plate, and
27

the average value of each concentration was calculated. Then the agar plate was cultured overnight
in a constant temperature incubator at 37 °C. The number of colonies was calculated in log
(CFU/mL).
Fluorescence Microscopy
Peptidomimetics dissolved in sterile water were added to the E.coli suspensions (108
CFU/mL). The mixture was incubated for 1.5 h and washed with 0.85% NaCl at 9000 rpm for
5min. The mixture was dissolved in 0.85% NaCl (1 mL), then green fluorescent nucleic acid
staining solution N01 (2 μL) and red fluorescent nucleic acid staining solution PI (0.5 μL) were
added. The mixture was incubated at room temperature and out of light for 15 min and re-
suspended with 0.85% NaCl to remove excess staining solution. The mixture (5 μL) was dropped
on the slide and examined with a fluorescence microscope.
Scanning Electron Microscopy (SEM)
The E. coli cells were cultured in liquid medium at 37 °C for 6 h. The cells were collected
after centrifugation and re-suspended in liquid medium. Peptidomimetics with the concentration
of 10 μg/mL were added to the suspensions. The suspensions were incubated at 37 °C in a shaker
for 2 h at 225 rpm, and the cells were collected by centrifugation at 9000 rpm. After treatment, the
cells were fixed overnight with glutaraldehyde and then gradient dehydrated with 10 %, 30 %,
50 %, 70 %, 90 % and 100 % ethanol for 10 minutes. Dehydrated cells (5 μL) were dropped to the
silicon chip and dried at room temperature. The morphology of E. coli cells were observed by
SEM.
28

4.5. Cell cytotoxicity and biocompatibility assay
MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay was used to
appraise the toxicity of the peptidomimetics to L929 fbroblast cells and SGC 7901 cells. MTT
method is a colorimetric and non-radioactive method to measure cell viability by increasing the
metabolism of tetrazolium salt. The measurements were conducted in a 96-well opaque white plate
with the L929 fbroblast cells and SGC 7901 cells density of about 7500 cells per well. After 24 h
of culture with CO2 (5 %, 37 °C), the cultured cells grew close to the culture bottle wall to form a
monolayer. The cells were washed twice with serum-free medium (100 μL) and then starved for
an hour at 37 °C. After starvation, cells were treated with different concentrations of the
peptidomimetics for 24 h. At the end of the treatment, the as-prepared MTT stock solution (5
mg/mL, 0.02 mL) was added to each well and sequentially cultivated at 37 °C. The cells were
washed with PBS (200 μL) to remove the redundant MTT containing medium. Then the crystals
were dissolved in DMSO (100 μL) and the plate was shaken for 5 min at 125 rpm for the sake of
solvent completely mixed with formazan which was obtained by reducing living cells metabolism
determined by MTT. The optical density of formazan, which was directly proportional to the
amount of living cells, was measured using a microplate reader regulated at 490 nm.
Then flow cytometry was used to detect apoptosis, and Annexin-V/FITC and PI were utilized
to evaluate the apoptosis of SGC 7901 cells after different treatments of the peptidomimetics. The
SGC 7901 cells were treated with different materials. After that, binding buffer (500ꢀμL) and the
staining of Annexin-V/FITC and PI (5ꢀμL) were added to the mixture and the apoptosis was
detected by the flow cytometry analyzer.
29