C-Terminal Modifications Broaden Activity of the Proline-Rich Antimicrobial Peptide, Chex1-Arg20

Article abstract of DOI:10.1071/CH15169

A series of N- and C-terminal modifications of the monomeric proline-rich antimicrobial peptide, Chex1-Arg20, was obtained via different chemical strategies using Fmoc/tBu solid-phase peptide synthesis in order to study their effects on a panel of Gram-negative bacteria. In particular, C-terminal modifications with hydrazide or alcohol functions extended their antibacterial activity from E. coli and K. pneumoniae to other Gram-negative species, A. baumannii and P. aeruginosa. Furthermore, these analogues did not show cytotoxicity towards mammalian cells. Hence, such modifications may aid in the development of more potent proline-rich antimicrobial peptides with a greater spectrum of activity against Gram-negative bacteria than the parent peptide.

Full text of DOI:10.1071/CH15169

RESEARCH FRONT
CSIRO PUBLISHING
Aust. J. Chem. 2015, 68, 1373–1378
Full Paper
http://dx.doi.org/10.1071/CH15169
C-Terminal Modifications Broaden Activity of the
Proline-Rich Antimicrobial Peptide, Chex1-Arg20
A B
,
B
A B
,
Wenyi Li,
Julien Tailhades, M. Akhter Hossain,
C D
,
C D
,
E
Neil M. O’Brien-Simpson,
Frances Separovic,
Eric C. Reynolds,
and John D. Wade
Laszlo Otvos Jr,
A C F
, ,
A B F
, ,
A
School of Chemistry, University of Melbourne, Melbourne, Vic. 3010, Australia.
B
The Florey Institute of Neuroscience and Mental Health, University of Melbourne,
Melbourne, Vic. 3010, Australia.
C
Bio21 Institute, University of Melbourne, Melbourne, Vic. 3010, Australia.
D
Oral Health CRC, Melbourne Dental School, University of Melbourne,
Melbourne, Vic. 3010, Australia.
E
Department of Biology, Temple University, Philadelphia, PA 19122, USA.
Corresponding authors. Email: fs@unimelb.edu.au; john.wade@florey.edu.au
F
A series of N- and C-terminal modifications of the monomeric proline-rich antimicrobial peptide, Chex1-Arg20, was
obtained via different chemical strategies using Fmoc/tBu solid-phase peptide synthesis in order to study their effects on a
panel of Gram-negative bacteria. In particular, C-terminal modifications with hydrazide or alcohol functions extended
their antibacterial activity from E. coli and K. pneumoniae to other Gram-negative species, A. baumannii and
P. aeruginosa. Furthermore, these analogues did not show cytotoxicity towards mammalian cells. Hence, such
modifications may aid in the development of more potent proline-rich antimicrobial peptides with a greater spectrum
of activity against Gram-negative bacteria than the parent peptide.
Manuscript received: 8 April 2015.
Manuscript accepted: 21 May 2015.
Published online: 12 June 2015.
isolates of Enterobacteriaceae.[9] Both A3-APO and Chex1-
Arg20 were assessed for their ability to induce bacterial
resistance upon repeated incubation of E. coli and K. pneu-
moniae strains in sub-lethal concentrations. While no resistant
E. coli phenotype emerged to either peptide, after 10 passages
the K. pneumoniae became resistant to the monomer but not
the dimer.[10]
Introduction
Increasing resistance to conventional antibiotics has led to a
higher incidence of multi-drug resistant bacterial infections and
has inspired the search for new antimicrobial agents.[1] With
their broad spectrum activity and various modes of actions,
antimicrobial peptides (AMPs) have emerged as possible
alternatives to conventional antibiotics.[2] In addition, proline-
rich antimicrobial peptides (PrAMPs), a major family of insect
AMPs, display the ability to penetrate bacterial cell membranes,
inhibit intracellular components, and stimulate immunomodu-
lation.[3] Additionally, PrAMPs show multi-modal action, such
as ribosome inhibition,[4] protein biosynthesis,[5] and bacterial
membrane disruption (W. Li, N. O’Brien-Simpson, J. Tailhades,
N. Pantarat, R. Dawson, E. Reynolds, F. Separovic, M. A.
Hossain, J. D. Wade, unpubl. data). An important consequence
of this diverse PrAMP activity is that target pathogens are either
unable or less able to develop resistance, a feature not shared
with conventional antibiotics.[6]
The PrAMP, A3-APO, was rationally designed based on
native insect peptides and it displays potent and broad activity
against Gram-negative bacteria.[7] The pharmacokinetics of
A3-APO has been investigated in vivo, and several degrada-
tion fragments have been identified including the major
metabolite, the monomer Chex1-Arg20.[8] The monomer
showed similar activity to the parent peptide when studied
against b-lactam and fluoroquinolone-resistant clinical
To some extent, AMPs generally show lower potency and are
less stable compared with conventional antibiotics. One approach
to improve their stability or activity is by masking both N- and
C-termini for protection against amino- and carboxypeptidases.
Generally, the N-terminus of peptides is a favourable position for
chemical modification as this can be readily achieved using
various N-terminal groups following automated solid-phase pep-
tide synthesis (SPPS).[11] The N-acetyl group is the most com-
monly used moiety to prevent aminopeptidase cleavage as well as
the poly-Pro sequence which could confer additional stability to
the sequence from degradation. A variety of other groups have
been employed for modification such as fatty acetylation[12] and
N,N,N0,N0-tetramethylguanidinylation,[13] the latter of which
resulted in a significant improvement of antibacterial activity
against target pathogens. Alternatively, native peptides normally
bear free carboxylic acids or occasionally an amide at the
C-terminus. Additional C-terminal chemical modifications
include hydrazination which has resulted in the production
of potent antifungal activity.[14] Peptaibols (peptides with a
Journal compilation Ó CSIRO 2015
www.publish.csiro.au/journals/ajc

1374
W. Li et al.
H2N
HN
NH
c, d
aa
NH2
NH
HN
n
N
H
O
2
O
H2N
HN
e, d
H
N
NH
aa
NH2
HN
Pbf
n
N
H
HN
3
O
O
a, b
H2N
NH
Fmoc
Rink amide
HN
aa
n
Fmoc
Rink amide
N
H
O
f, d
aa
NH2
HN
O
n
N
H
O
4
H2N
HN
NH
g, d
aa
NH2
N
N
H
N
O
N
5
Fig. 1. General conditions for the synthesis of N-terminal modified peptides: (a) 20 % piperidine in DMF, 10 min; (b) Fmoc-AA-OH (4 equiv.),
HCTU (3.8 equiv.), DIPEA (4 equiv.), 1 h; (c) acetic anhydride, DIPEA, DMF; (d) TFA/anisole/TIPS (95 : 3 : 2), 2 h; (e) butyric acid, EDCI,
triethylamine; (f) valeric acid, EDCI, triethylamine; and (g) HBTU, NMM.
Table 1. Sequence of PrAMP monomer (Chex1-Arg20) analogues
Chex ¼ 1-aminocyclohexanecarboxylic acetamide, Ac ¼ acetamide, Bu ¼ butyric acetamide, Va ¼ valeric acetamide, Gu ¼ N,N,N0,N0-tetramethylguanidine
(1, previously reported)
Peptide number
Sequence
Chex1-Arg20 analogue
Yield [%]
MWcalc
MWmeas
1
2
3
4
5
6
7
8
Chex-RPDKPRPYLPRPRPPRPVR—NH2
Ac-RPDKPRPYLPRPRPPRPVR—NH2
Bu-RPDKPRPYLPRPRPPRPVR—NH2
Va-RPDKPRPYLPRPRPPRPVR—NH2
Gu-RPDKPRPYLPRPRPPRPVR—NH2
Chex-RPDKPRPYLPRPRPPRPVR—OH
Chex-RPDKPRPYLPRPRPPRPVR—NH–NH2
Chex-RPDKPRPYLPRPRPPRPVR—ol
Monomer
40
38
54
16
46
42
52
31
2475.0
2391.8
2419.9
2433.9
2447.9
2476.0
2490.0
2462.0
2476.8
2392.8
2420.4
2434.4
2448.7
2479.9
2491.0
2465.7
Ac-monomer
Bu-monomer
Va-monomer
Gu-monomer
Monomer-acid
Monomer-hydrazide
Monomer-alcohol
C-terminal alcohol) have also shown antibiotic properties as
well as higher metabolic stability in vitro and in vivo.[15]
Therefore, in view of these positive studies, the effect of similar
N- and C-terminal modifications on the activity of the PrAMP
monomer, Chex1-Arg20, against nosocomial pathogens was
investigated.
For the C-terminal modifications, the peptide with amide
was obtained via standard 9-fluorenylmethoxycarbonyl/tert-
butyl (Fmoc/tBu) SPPS.[9] Recently, Liu and co-workers have
developed a strategy for native chemical ligation of peptide
hydrazides. Such hydrazides can be hydrolysedto free acid under
basic conditions.[16] Therefore, the 2-chlorotrityl chloride resin
was functionalised with hydrazine for the synthesis of peptide
hydrazide 7 by standard Fmoc/tBu SPPS (Fig. 2 upper). Conver-
sion of 7 to the monomer-acid 6 (Table 1) was achieved in
the presence of aqueous basic buffer with NaNO2 pre-activation
(Fig. 2 upper). Since peptides bearing a C-terminal alcohol group
are an interesting class of active compounds, a strategy to
synthesise several peptide alcohols based on O–N acyl-transfer
reaction via Fmoc/tBu SPPS was developed by the Amblard
group.[17] Therefore, N-Fmoc-arginine-b-amino-alcohol 9 was
Results and Discussion
Preparation of Chex1-Arg20 Monomers with Terminal
Modifications
The PrAMP monomers 2–5 (Fig. 1, Table 1) were prepared with
N-terminal modifications by separate reaction with acetic anhy-
dride, butyric acid, valeric acid, and N,N,N0,N0-tetramethyl-O-
(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU).

Termini Modifications Broaden Activity of Chex1-Arg20
1375
O
O
O
N NH2
H
H
N N
H
OH
aa
HN
aa
n
HN
e
H
f
b, c, d
n
aa
HN
n
H2N N
O
NH2
O
NH2
O
NH
Boc
Cl
7
6
Cl
H
H
N
N
NH
H
NH
Pbf
N
NH
Pbf
Pbf
HN
Cl
HN
HN
OH
H
N
e, i
b, c, d
h
O
aa
HN
NH
H
N
n
Pbf
O
O
HN
aan
Fmoc
O
N
H
N
H
HO
HN
O
N
H
N
H
N
H
NH2
O
Boc
O
8
Fmoc
OH
N
H
9
Fig. 2. General conditions for the synthesis of C-terminal modified peptides: (a) hydrazine monohydrate (2 equiv.), triethylamine (3 equiv.); (b) 20 %
piperidine in DMF, 10 min; (c) Fmoc-AA-OH (4 equiv.), HCTU (3.8 equiv.), DIPEA (4 equiv.), 1 h; (d) 1-aminocyclohexanecarboxylic acid (1.5 equiv.),
HCTU (1.4 equiv.), DIPEA (4 equiv.), 1 h; (e) TFA/anisole/TIPS (95 : 3 : 2), 2 h; (f) NaNO2, pH 3, ꢀ158C, 15 min, then adjust to pH 8; (g) 9, 2 % DBU in DMF,
r.t., 12 h; (h) Fmoc-Val-OH (6 equiv.), DIC (3 equiv.), DMAP (0.3 equiv.), DMF/CH2Cl2 (1 : 1), 12 h; and (i) PBS, pH 7.4, 1 h.
H
N
(Table 1), against several Gram-negative and Gram-positive
bacteria strains. The antibacterial activity of the peptides, or
minimal inhibitory concentration (MIC), was determined via
the liquid broth microdilution assay[18] in Muller-Hinton Broth
(MHB).
H
N
NH
NH
Pbf
Pbf
1) IBCF/NMM
DME,Ϫ15ЊC
HN
HN
2) NaBH4/H2O
Ϫ20ЊC
Even at the highest tested concentration, none of these
peptides displayed any activity against the Gram-positive
strains, S. aureus ATCC 29213 and E. faecalis ATCC 29212,
which are considered as high threat pathogens for gut or skin
infections within hospital environments. In contrast, Gram-
negative bacteria displayed different levels of sensitivity to
these monomer analogues. Neither N- nor C-terminal modifica-
tions of 1 had any effect on the activity against E. coli ATCC
29222 (1.6–6.7 mM) and K. pneumoniae ATCC 13883 (0.8–
2.2 mM). N-Terminal modifications neither increased nor
extended their activity (Table 2). However, C-terminal modifi-
cations with either hydrazide or alcohol functionality caused a
significant increase in the antibacterial spectrum and a higher
activity than the monomer having an amide or acid group against
Gram-negative A. baumannii ATCC 19606 (52.5–68.3 mM) and
P. aeruginosa ATCC 47085 (28.9–35.3 mM). The broadened
spectrum of activity may be due to the hydrazide or alcohol
moiety improving stability against bacterial enzyme attack.
Based on previous studies of the mechanism on PrAMPs, the
monomer is associated with the inner membrane resulting in a
membrane potential change (W. Li, N. O’Brien-Simpson,
J. Tailhades, N. Pantarat, R. Dawson, E. Reynolds, F. Separovic,
M. A. Hossain, J. D. Wade, unpubl. data). The monomer-
hydrazide 7 and monomer-alcohol 8 may also alter the ionic
permeability of the membranes of these Gram-negative bacteria,
in a similar manner to non-lytic traditional antibiotics and peptai-
bols.[19] The stability of the parent peptide 1 has previously been
determined[8] and that of peptides 7 and 8 are likely to be similar.
Therefore, to expand upon the above results, further work is
underway to prepare multimers of C-terminal modified PrAMPs.
Fmoc
OH
OH
Fmoc
N
H
N
H
O
9
Fig. 3. Preparation of N-Fmoc-arginine-b-amino-alcohol 9.
obtained by Fmoc-arginine pre-activation with isobutyl chloro-
formate (IBCF) followed by NaBH4 reduction in an organic/
aqueous solution (Fig. 3). Then, the arginine-b-amino-alcohol
was anchored to 2-chlorotrityl chloride resin by its amine
function in a one pot reaction following Fmoc removal. After-
wards, the free hydroxyl function was acylated with the follow-
ing N-Fmoc-protected amino acid. Further elongation by
standard Fmoc/tBu SPPS strategy gave the solid-anchored
isopeptide (Fig. 2 lower). To generate peptide 8, O–N acyl-
intramolecular transfer was performed in phosphate buffered
saline (PBS) at pH 7.4 for 1 h. However, no retention time change
was observed on reversed-phase-HPLC (RP-HPLC), which
made monitoring of the reaction difficult. As the iso-dipeptide
(ester bond) is sensitive to basic pH condition, 2 M NaOH was
added to the reaction and no mass deletion on matrix-assisted
laser desorption–ionisation-mass spectrometry (MALDI-MS)
was observed. Therefore, O–N intramolecular transfer might
already have occurred during the TFA cleavage or under the
acidic conditions of RP-HPLC analysis. All peptides were
subjected to comprehensive chemical characterisation, including
analytical RP-HPLC and MALDI-time-of-flight/electrospray
ionisation (MALDI-TOF/ESI) MS to confirm their purity and
moderate yields (Fig. 4).
Cytotoxicity
Antimicrobial Activity
Cytotoxicity of AMPs is an important concern in drug design.
The mammalian cell lines, HEK-293 (ATCC CRL 1573)
and H-4-II-E (ATCC CRL-1548), were used to analyse the
As nosocomial pathogens are an increasing public health hazard,
we tested analogues 2–9, including the monomer amide 1

1376
W. Li et al.
2392.809
(a)
5.00
10.00 15.00 20.00 25.00 30.00 35.00 40.00
1600 1800 2000 2200 2400 2600 2800 3000 3200
m/z
Minutes
2420.363
(b)
0
5.00
10.00 15.00 20.00 25.00 30.00 35.00 40.00
1800
2000
2200
2400
2600
2800
Minutes
m/z
2434.355
(c)
(d)
1800
2000
2200
2400
2600
2800
0
5.00
10.00 15.00 20.00 25.00 30.00 35.00 40.00
Minutes
m/z
2448.743
1800 2000
2200 2400
2600 2800 3000
m/z
2479.888
(e)
1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400
5.00
10.00
15.00
20.00
25.00
30.00
m/z
Minutes
2490.992
(f)
1800 2000 2200 2400 2600 2800 3000
2465.777
5.00
10.00
15.00
20.00
25.00
30.00
m/z
Minutes
(g)
2000 2100 2200 2300 2400 2500 2600 2700 2800 2900
0
5.00
10.00
15.00
20.00
25.00
30.00
m/z
Minutes
Fig. 4. RP-HPLC and MALDI-TOF/ESI MS for peptides: (a)–(g) Ac-monomer 2 – monomer-
alcohol 8, respectively. Analysis condition: Phenomenex C18 column (WIDEPORE 3.6u XB-C18,
150 ꢂ 4.6 nm); buffer A, 0.1 % aq. TFA; buffer B, 0.1 % TFA in acetonitrile; gradient, buffer B
10–30 % in 30 min.
cytotoxicity of the monomer (Chex1-Arg20) analogues via the
Promega CellTiter 96Ò AQueous Non-Radioactive Cell Prolif-
eration Assay (www.promega.com). The data showed that none
of the analogues displayed any toxicity on either mammal cell
lines at the highest tested concentration (100 mM), with the
exception of peptide 6 (the C-terminal acid analogue,
48.1 ꢁ 3.4 mM), but this was still much higher than the anti-
bacterial activity MIC (Table 2).

Termini Modifications Broaden Activity of Chex1-Arg20
1377
Table 2. Antibacterial activity, MIC (lM), against Gram-negative nosocomial pathogens and cytotoxicity (lM) against HEK-293 ATCC CRL-1573
and H-4-II-E ATCC CRL-1548 of PrAMP monomer (Chex1-Arg20) analogues
No activity was observed against Gram-positive, S. aureus ATCC 29213, and E. faecalis ATCC 29212
Peptide number E. coli ATCC 29222 K. pneumoniae ATCC 13883 A. baumannii ATCC 19606 P. aeruginosa ATCC 47085
Cytotoxicity
HEK-293 H-4-II-E
1
2
3
4
5
6
7
8
2.6 ꢁ 0.7
3.6 ꢁ 0.1
6.5 ꢁ 2.6
5.6 ꢁ 1.8
5.8 ꢁ 2.2
6.7 ꢁ 1.3
4.0 ꢁ 1.5
1.6 ꢁ 0.1
0.8 ꢁ 0.0
2.0 ꢁ 0.2
2.2 ꢁ 0.4
2.1 ꢁ 0.0
1.5 ꢁ 0.4
2.0 ꢁ 0.1
1.7 ꢁ 0.1
1.2 ꢁ 0.3
.100
.100
.100
56.3 ꢁ 4.5
.100
.100
.100
.100
.100
.100
.100
.100
.100
.100
.100
.100
96.3 ꢁ 3.2
82.0 ꢁ 5.0
64.7 ꢁ 2.1
.100
.100
.100
.100
.100
.100
52.5 ꢁ 9.5
68.3 ꢁ 1.9
48.1 ꢁ 3.4
.100
.100
28.9 ꢁ 2.7
35.3 ꢁ 5.2
Conclusion
Agilent 400-MR (400 MHz) spectrometer with the deuterated
solvent as reference and sample concentration of
,10 mg mLꢀ1
a
In summary, various chemical synthesis strategies were
employed to produce a variety of N- or C-terminal analogues of
the potent PrAMP monomer, Chex1-Arg20. The N-terminal
modifications did not alter the antibacterial activity of Chex1-
Arg20. However, compared with natural C-terminal carboxyl
and carboxamide moieties, the hydrazide or alcohol C-terminal
modifications broadened the spectrum of activity of Chex1-
Arg20 while retaining the level of efficacy against Gram-
negative bacterial strains. Furthermore, none of these broader
spectrum and potent Chex1-Arg20 analogues displayed cyto-
toxicity in the presence of mammalian cell lines. These results
bode well for the further development of next-generation ana-
logues of Chex1-Arg20 as potential novel antibiotics.
.
Preparation of N-Fmoc-arginine-b-amino-alcohol 9
Fmoc-Arg(Pbf)-OH (1.5 mmol, 1 g) was dissolved in dime-
thoxyethane (DME, 10 mL) in a 50 mL round bottom flask,
and then NMM (1.1 equiv., 1.65 mmol, 0.187 mL) and IBCF
(1.1 equiv., 1.65 mmol, 0.219 mL) were added to the mixture at
ꢀ158C. The reaction was left for 30 min and filtered into another
round bottom flask. NaBH4 (3 equiv.) was added to the filtrate at
ꢀ158C and stirred for 15 min. Saturated NaHCO3 was then used
to quench the reduction and the mixture was extracted three
times with ethyl acetate. The combined organic phase was
washed with 0.5 M HCl and saturated NaCl solution. The
organic phase was dried with magnesium sulfate and concen-
trated to afford the product 9 (0.8 g, 81 % yield) without further
purification (Fig. 3), mp 101.2–103.58C. nmax (KBr)/cmꢀ1
3439.9, 2926.7, 1552.0, 1450.2, 1251.5, 1104.7, 734.8, 568.5.
dH (CDCl3) 7.76–7.68 (2H, m), 7.54 (2H, d, J 7.3), 7.34 (2H, t, J
7.4), 7.22 (2H, d, J 7.0), 6.25 (3H, t, J 32.1), 6.08 (1H, s), 5.60
(1H, s), 4.34 (2H, d, J 6.4), 4.19–4.06 (2H, m), 3.75–3.55 (3H,
m), 3.39 (1H, s), 3.21 (2H, s), 2.89 (2H, s), 2.56 (3H, s), 2.49 (3H,
s), 2.06 (3H, s), 1.55 (4H, d, J 15.1), 1.41 (6H, s). dC (75 MHz,
CDCl3): 158.8, 156.9, 156.3, 143.8, 143.7, 141.2, 138.3, 132.6,
132.2, 128.7, 127.6, 127.0, 125.1, 124.7, 121.0, 119.9, 117.6,
86.4, 66.7, 47.2, 43.1, 41.0, 28.5, 25.2, 19.3, 17.9, 12.4, 0.0.
m/z 635.2942. HRMS (ESIþ) Anal. Calc. for C34H42N4O6S
[M þ H]þ 635.2825.
Methods
Peptide Synthesis
The peptides were synthesised by Fmoc/tBu solid-phase meth-
ods.[20] Peptide chains 1–5 assemblies were carried out on a
CEM Liberty microwave-assisted synthesizer using TentaGel
Rink amide resins (RAM). Standard Fmoc-chemistry was used
throughout with a 4-fold molar excess of the Fmoc-protected
amino acids in the presence of 4-fold (2-(6-chloro-1H-
benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluoropho-
sphate (HCTU) and 8-fold N,N-diisopropylethylamine
(DIPEA). Final N-terminal modifications were obtained by
incubating the N-terminally deprotected peptides with 10 equiv.
acetic anhydride/DIPEA for 2, 4 equiv. butyric acid/1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (EDCI)/triethylamine
for 3, 4 equiv. valeric acid/EDCI/triethylamine for 4, and
10 equiv. HBTU/N-methylmorpholine (NMM) for 5.
The assembly of peptide chains 6–8 was carried out by
using hydrazide or N-Fmoc-amino acid alcohol functionalised
2-chlorotrityl chloride resin followed by manual standard Fmoc/
tBu SPPS. The peptides were cleaved from the solid support
resin with TFA in the presence of anisole and triisopropylsilane
(TIPS) as scavenger (ratio 95 : 3 : 2) for 2 h at room temperature
(r.t.). After cleavage, the resin was removed by filtration, the
filtrate was concentrated under a stream of nitrogen, and the
peptide products were precipitated in ice-cold diethyl ether,
washed and centrifuged three times. The peptides were then
purified with moderate yield by RP-HPLC in water and aceto-
nitrile with 0.1 % TFA. The final products were monitored
and characterised by RP-HPLC and MALDI-TOF MS. 1H and
13C NMR spectra were obtained at room temperature using an
Antibacterial Assay
Antibacterial assays were undertaken to determine the MIC. E. coli
ATCC 29222, K. pneumoniae ATCC13883, A. baumannii ATCC
19606, P. aeruginosa ATCC 47085, S. aureus ATCC 29213, and
E. faecalis ATCC 29212 were grown and maintained at 378C on
Luria-Bertani agar plates. Single colonies from the agar plates
were used to inoculate MHB, and the growth was monitored at
650 nm using a spectrophotometer (model 275E; Perkin-Elmer,
Sydney, NSW) with culture purity checked by microscopic
examination and culture. Batch-grown cells were harvested
during late exponential growth phase and counted. Their via-
bility was determined using a BacLight viability kit (Invitrogen,
Sydney, NSW) and a QuantaSC-MPLflow cytometer(Beckman
Coulter Pty Ltd, Sydney, NSW).
Viable cells were diluted to 2.5 ꢂ 105 cells mLꢀ1 in MHB at
378C immediately before the determination of MIC. The MICs

1378
W. Li et al.
were obtained according to the Lambert and Pearson growth
curve analysis method,[21] where instead of reporting the con-
centration at which no growth is observed, an extrapolation of
the maximal growth versus the peptide concentration is used.
Briefly, 2.5 ꢂ 105 mLꢀ1 viable bacterial cells (100 mL of stock
suspension) were added to serial dilutions of peptides in MHB
(100 mL, from initial stock suspensions of 200 mM) in 96-well
flat-bottom microtiter plates (Interpath Service, Melbourne,
Vic.) and incubated at 378C. The bacterial growth was moni-
tored for the determination of MIC at 20 min-intervals over a
16 h period at an optical density at 620 nm (OD620) using an
iEMS microplate reader (Pathtech Pty Ltd, Melbourne, Vic.).
The relative growth at each peptide concentration was compared
with maximal growth (determined as the point when bacteria
incubated in media alone entered the stationary phase of growth,
100 % growth), and the MIC was determined as the lowest
peptide concentration needed to completely inhibit the growth
of the bacteria, i.e. the intersection of the linear curve with the
x-axis.
[4] A. Krizsan, D. Volke, S. Weinert, N. Strater, D. Knappe, R. Hoffmann,
Angew. Chem. Int. Ed. 2014, 53, 12236. doi:10.1002/ANIE.
201407145
[5] M. Mardirossian, R. Grzela, C. Giglione, T. Meinnel, R. Gennaro,
P. Mergaert, M. Scocchi, Chem. Biol. 2014, 21, 1639. doi:10.1016/
J.CHEMBIOL.2014.10.009
[6] M. Cassone, N. Frith, P. Vogiatzi, J. D. Wade, L. Otvos, Jr, Int. J. Pept.
Res. Ther. 2009, 15, 121. doi:10.1007/S10989-009-9176-1
[7] L. Otvos, Jr, J. D. Wade, F. Lin, B. A. Condie, J. Hanrieder,
R. Hoffmann, J. Med. Chem. 2005, 48, 5349. doi:10.1021/JM050347I
[8] P. B. Noto, G. Abbadessa, M. Cassone, G. D. Mateo, A. Agelan,
J. D. Wade, D. Szabo, B. Kocsis, K. Nagy, F. Rozgonyi, L. Otvos, Jr,
Protein Sci. 2008, 17, 1249. doi:10.1110/PS.034330.108
[9] M. Cassone, P. Vogiatzi, R. La Montagna, V. De Olivier Inacio,
P. Cudic, J. D. Wade, L. Otvos Jr, Peptides 2008, 29, 1878.
doi:10.1016/J.PEPTIDES.2008.07.016
[10] M. Cassone, N. Frith, P. Vogiatzi, J. D. Wade, L. Otvos, Jr, Int. J. Pept.
Res. Ther. 2009, 15, 121. doi:10.1007/S10989-009-9176-1
[11] F. Guida, M. Benincasa, S. Zahariev, M. Scocchi, F. Berti, R. Gennaro,
A. Tossi, J. Med. Chem. 2015, 58, 1195. doi:10.1021/JM501367P
[12] D. Oh, A. Nasrolahi Shirazi, K. Northup, B. Sullivan, R. K. Tiwari,
M. Bisoffi, K. Parang, Mol. Pharm. 2014, 11, 2845. doi:10.1021/
MP500203E
Cell Proliferation Test
To determine cell proliferation, 100 mL HEK-293 (ATCCÒ
CRL-1573TM) and H-4-II-E (ATCCÒ CRL-1548TM) cells
(5 ꢂ 103) were seeded into 96-well plates and cultured at 378C,
5 % CO2 incubator. After 48 h, 20 mL of the CellTiter 96Ò
AQueous Non-Radioactive Cell Proliferation Assay (Promega)
were added to each well and the plates were incubated for 1–2 h
at 378C, 5 % CO2 incubator. Absorbance was measured at
490 nm.
[13] P. Czihal, D. Knappe, S. Fritsche, M. Zahn, N. Berthold,
S. Piantavigna, U. Mu¨ller, S. Van Dorpe, N. Herth, A. Binas,
G. Ko¨hler, B. De Spiegeleer, L. L. Martin, O. Nolte, N. Stra¨ter,
G. Alber, R. Hoffmann, ACS Chem. Biol. 2012, 7, 1281. doi:10.1021/
CB300063V
[14] A. Aboelmagd, I. A. Ali, E. M. Salem, ARKIVOC 2011, 2011, 337.
doi:10.3998/ARK.5550190.0012.925
[15] (a) E. Benedetti, A. Bavoso, B. Di Blasio, V. Pavone, C. Pedone,
C. Toniolo, G. M. Bonora, Proc. Natl. Acad. Sci. USA 1982, 79, 7951.
doi:10.1073/PNAS.79.24.7951
(b) W. Bauer, U. Briner, W. Doepfner, R. Haller, R. Huguenin,
P. Marbach, T. J. Petcher, J. Pless, Life Sci. 1982, 31, 1133.
doi:10.1016/0024-3205(82)90087-X
Supplementary Material
1H and 13C NMR spectra of N-Fmoc-arginine-b-amino-alcohol
9 are available on the Journal’s website.
[16] (a) J.-S. Zheng, S. Tang, Y.-K. Qi, Z.-P. Wang, L. Liu, Nat. Protoc.
2013, 8, 2483. doi:10.1038/NPROT.2013.152
(b) G.-M. Fang, Y.-M. Li, F. Shen, Y.-C. Huang, J.-B. Li, Y. Lin,
H. K. Cui, L. Liu, Angew. Chem. Int. Ed. 2011, 50, 7645. doi:10.1002/
ANIE.201100996
(c) G.-M. Fang, J.-X. Wang, L. Liu, Angew. Chem. Int. Ed. 2012, 51,
10347. doi:10.1002/ANIE.201203843
Acknowledgements
We gratefully acknowledge support of the studies undertaken in the authors’
laboratory by an ARC Discovery Project grant (DP150103522) to JDW and
MAH, and an NHMRC Project grants (APP1029878) to NMOBS and
(APP1008106) to ECR and NMOBS. JDW is an NHMRC (Australia)
Principal Research Fellow. Research at the FNI was also supported by the
Victorian Government’s Operational Infrastructure Support Program. WL
acknowledges the University of Melbourne for a MIRS PhD award. Thank
you to Shu Jie Lam for assistance with NMR spectroscopy.
[17] J. Tailhades, M.-A. Gidel, B. Grossi, J. Le´caillon, L. Brunel, G. Subra,
J. Martinez, M. Amblard, Angew. Chem. Int. Ed. 2010, 49, 117.
doi:10.1002/ANIE.200904276
[18] D. Amsterdam, in Antibiotics in Laboratory Medicine (Ed. V. Lorian)
1996. pp. 52–111 (Williams & Wilkins: Baltimore, MD).
[19] (a) F. Separovic, J. Gehrmann, T. Milne, B. A. Cornell, S. Y. Lin,
R. Smith, Biophys. J. 1994, 67, 1495. doi:10.1016/S0006-3495(94)
80623-X
References
[1] K. Gammon, Nature 2014, 509, S10. doi:10.1038/509S10A
[2] H. Jenssen, P. Hamill, R. E. W. Hancock, Clin. Microbiol. Rev. 2006,
19, 491. doi:10.1128/CMR.00056-05
(b) R. Nagaraj, P. Balaram, Acc. Chem. Res. 1981, 14, 356.
doi:10.1021/AR00071A005
[3] (a) L. Otvos, Jr, J. Pept. Sci. 2005, 11, 697. doi:10.1002/PSC.698
(b) W. Li, J. Tailhades, N. O’Brien-Simpson, F. Separovic, L. Otvos, Jr,
M. A. Hossain, J. D. Wade, Amino Acids 2014, 46, 2287. doi:10.1007/
S00726-014-1820-1
[20] G. B. Fields, R. L. Noble, Int. J. Pept. Protein Res. 1990, 35, 161.
doi:10.1111/J.1399-3011.1990.TB00939.X
[21] R. J. W. Lambert, J. Pearson, J. Appl. Microbiol. 2000, 88, 784.
doi:10.1046/J.1365-2672.2000.01017.X