Article BULLETIN OF THE

DOI: 10.1002/bkcs.10483 T. T. T. Dinh et al. KOREAN CHEMICAL SOCIETY

N-Capping Effects of Stapled Heptapeptides on Antimicrobial

and Hemolytic Activities
Thuy T. T. Dinh,† Do-Hee Kim,‡ Thang Q. Nguyen,† Bong-Jin Lee,‡ and Young-Woo Kim†,*
†
College of Pharmacy, Dongguk University, Seoul 100-715, Republic of Korea.
*E-mail: ywkim730@dongguk.edu
‡
College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
Received June 1, 2015, Accepted June 18, 2015, Published online September 10, 2015

In our previous study, we showcased the potential of an all-hydrocarbon stapled heptapeptide as a privileged
scaffold for the design of artificial antimicrobial peptides. We demonstrated that the amphipathic helicity and
the subtle balance between hydrophobicity and hydrophilicity are important structural features for the antimi-
crobial activities of this class of antimicrobial agents. In this study, we show that elimination of the N-acetyl cap
can further improve the pharmacological properties of the most potent stapled heptapeptides. The structure–
activity relationships newly established in this study would serve as a critical asset for the further development
of a new class of antimicrobial agents to combat the rising problem of antibiotic resistance.
Keywords: Antimicrobial peptides, α-Helix, Stapled peptides, Protease resistance, Peptide drugs

Introduction provided by an additional lipophilic amino acid residue was

Antimicrobial peptides (AMPs) are found in various organ-
isms and play a significant role in the first-line defense
system.1–3 Because of their various distinctive modes of
action, AMPs have recently gained spotlight as a potential
alternative to combat the growing problems of antibiotic-
resistant bacteria.4 To exert their antimicrobial activity, many
natural AMPs need to fold into an amphipathic helical struc-
ture, bearing multiple hydrophobic residues on one face of the
helix and cationic hydrophilic residues on the opposite side.5–7
This unique structural feature is considered to help this spe-
cific class of AMPs effectively interact with bacterial mem-
branes, disrupt their integrity, and thereby kill the bacteria.8–10
In our previous study, we investigated an all-hydrocarbon
stapled heptapeptide as a potential privileged scaffold for
the construction of short, artificial AMPs (Figure 1).11 Ver-
dine’s all-hydrocarbon stapling system12–16 is a highly effec-
tive tool to engineer the heptapeptides with the two structural
requirements of AMPs: amphipathicity and helicity.17 We
have shown that an oct-4-enyl staple can effectively stabilizes
a helical conformation of the short sequence of heptapeptides,
which are otherwise highly difficult to form α-helix. Also, thus
introduced oct-4-enyl cross-link per se can form a hydropho-
bic face of the helix. Therefore, placing several cationic lysine
residues on the opposite side of the staple makes the stapled
helical heptapeptides amphipathic (Figure 1(b)). Amphipathic
helical heptapeptides based on this design rationale showed
moderate antimicrobial activities against both Gram-positive
and Gram-negative bacteria and displayed a clear structure–
activity relationship. For example, the helical conformation
induced by the oct-4-enyl hydrocarbon tether was important
for the antimicrobial activity. The expanded hydrophobic face
also beneficial for the antimicrobial activity.
In the same study, decreasing the number of lysine residues
resulted in a significant loss of the antimicrobial activity, sug-
gesting the importance of the cationic face of the helix for the
antimicrobial activity. This result prompted us to consider
increasing the net charge of these heptapeptides as a potential
strategy to improve the biological activities. However, a sim-
ple replacement of a hydrophobic amino acid with a lysine res-
idue would not be an optimal option as it would disrupt the
proper balance between hydrophobicity and hydrophilicity
of the peptides. Therefore, in this study, we explored the pos-
sibility for improvement of the antimicrobial activity by
increasing the net charge of the stapled heptapeptides via
removal of their N-terminal acetyl cap.
Experimental
General. Fmoc-(S)-α-methyl,α-petenylglycine was purchased
from Okeanos Tech Co. Ltd (Beijing, China). All other Fmoc-
protected α-amino acids, 1-[(1-(cyano-2-ethoxy-2-oxoethyli-
deneaminooxy)-dimethylamino-morpholino)]uranium hexa-
fluorophosphate (COMU), and Rink Amide MBHA resin
were purchased from NovaBiochem (San Diago, CA, USA).
Piperidine, N-methyl-2-pyrrolidinone (NMP), dimethylforma-
mide (DMF), N,N-diisopropylethylamine (DIEA), Grubbs first
generation catalyst (bis(tricyclohexylphosphine)benzylidine
ruthenium (IV) dichloride), 1,2-dichloroethane (DCE), triiso-
propylsilane (TIS), and trifluoroacetic acid (TFA) were pur-
chased from Sigma-Aldrich (Seoul, Korea). Commercially
available solvents and reagents were used as received.
Peptide Synthesis. All the peptides were prepared using the
typical Fmoc/t-Bu strategy on Rink Amide MBHA resin with

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Article
ISSN (Print) 0253-2964 | (Online) 1229-5949
Figure 1. (a) Schematic presentation of all hydrocarbon stapling
chemistry, tethering two pentenyl side-chains at positions 2 and
6 for the preparation of stapled heptapeptides. (b) A helical wheel pro-
jection of a typical stapled heptapeptide. The peptide was designed to
be an amphipathic helix upon stapling. Lines connecting positions
2 and 6 represent the oct-4-enyl hydrocarbon cross-link.
a loading capacity of 0.6 mmol/g.13 The dry resin (50 mg, 30 μ
mol) was swelled in NMP for 10 min before using. The Fmoc
protecting group was removed by treatment with 25% piper-
idine in NMP (2 × 10 min). Fmoc-protected amino acids were
coupled for 30 min using COMU as an activating agent (4.75
equiv), 5 equiv of Fmoc-protected amino acid, and 10 equiv of
DIEA in NMP. The coupling of Fmoc-(S)-α-methyl,α-pete-
nylglycine was conducted for 2 h with the amino acid (3 equiv),
COMU (2.85 equiv), and DIEA (6 equiv). After each coupling
or deprotecting reaction, the resin was thoroughly washed
with dichloromethane (DCM) (1 × 2 min), NMP (1 × 2 min),
DCM (1 × 2 min), and NMP (1 × 2 min).
Metathesis and Purification. Ring-closing metathesis of
resin-bound N-Fmoc, side-chain protected peptides was per-
formed using 20 mol% of Grubbs first generation catalyst in
degassed DCE for 2 h at room temperature. The reactions were
monitored by liquid chromatography–mass spectrometry after
cleavage of the peptides from a resin aliquot. After draining the
reaction solution, the resin was washed with DCE (3 × 2 min)
and then with DCM (3 × 2 min). After the Fmoc protecting
group was removed by treatment with 25% piperidine in
NMP (2 × 10 min), the resin was washed with DCM (3 × 2
min), NMP (2 × 2 min), and DCM (3 × 2 min) and dried in
vacuo overnight. The peptides were deprotected and cleaved
from the resin by treating them with a mixture of TFA/TIS/
water (95/2.5/2.5) for 2 h, and precipitated by adding a 1:1
mixture of n-pentane and diethyl ether. The precipitate was
collected by centrifugation, dissolved in a 1:1 mixture of ace-
tonitrile and water, and filtered to remove resin. The products
were purified through reverse-phase high-performance liquid
chromatography using a Zorbax C18 column (5 μm, 9.4 × 250
mm; Agilent, Palo Alto, CA, USA), and then LC/MS
(API4000; Agilent).
Peptide Ac-S1. ESIMS m/z for C48H79N12O8 [M + H]+
calcd 951.61, found 951.50.
Peptide Ac-S2. ESIMS m/z for C48H79N12O8 [M + H]+
calcd 951.61, found 951.35.
Peptide Ac-S3. ESIMS m/z for C51H85N12O8 [M + H]+
calcd 993.66, found 993.50.
Peptide Ac-S4. ESIMS m/z for C51H85N12O8 [M + H]+
calcd 993.66, found 993.50.
Bull. Korean Chem. Soc. 2015, Vol. 36, 2511–2515 © 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
BULLETIN OF THE
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Peptide H-S1. ESIMS m/z for C46H76N12NaO7 [M + Na]+
calcd 932.60, found +931.60.
Peptide H-S2. ESIMS m/z for C46H76N12NaO7 [M + Na]+
calcd 932.60, found +931.65.
Peptide H-S3. ESIMS m/z for C49H83N12O7 [M + H]+
calcd 951.65, found 951.60.
Peptide H-S4. ESIMS m/z for C49H83N12O7 [M + H]+
calcd 951.65, found 951.65.
Circular Dichroism. The peptides were dissolved in a 25
mM potassium phosphate buffer solution (pH 6.5). The con-
centrations were determined by absorbance spectroscopy at
280 nm (extinction coefficient for tryptophan, λ280 = 5690
cm−1). Circular dichroism (CD) spectra were collected on a
Chirascan HP dual polarization CD spectrometer with a tem-
perature controller using the following standard measurement
parameters: 1 nm step resolution, 3 accumulations, 0.5 s
response, 1 nm bandwidth, and 0.1 cm path length. All spectra
were converted to a uniform scale of molar ellipticity after
background subtraction. The curves were smoothed using
standard parameters.
Antibacterial Assay. Antimicrobial activity was
evaluated by the standard broth microdilution method with
a slight modification measuring the minimal inhibitory
concentration (MIC) values. The antimicrobial assay was con-
ducted against three strains of Gram-positive (Bacillus subtilis
ATCC 6633, Staphylococcus aureus ATCC 6538p, and
Staphylococcus epidermis ATCC 12228) and six strains of
Gram-negative bacteria (Escherichia coli ATCC 25922, Shi-
gella dysentariae ATCC 9752, Salmonella typhimurium
ATCC 14028, Klebsiella pneumonia ATCC 10031, Proteus
mirabilis ATCC 25933, and Pseudomonas aeruginosa
ATCC 27853). These strains of bacteria were incubated in
2 mL Luria-Bertani (LB) broth overnight at 37  C. The pep-
tides dissolved in phosphate-buffered saline (PBS) were pre-
pared with twofold dilutions from 0.2 to 100 μM in 96-well
round-bottom microtiter plates. The peptide solution and
the bacterial inoculums were diluted using LB broth.
Bacterial suspension (106–108 colony-forming units [CFU]/
mL) was then dispensed into the peptide-containing wells.
After 24 h of incubation at 37  C, the wells were examined
to determine MICs, the lowest peptide concentration which
completely inhibits cell growth. The antimicrobial assay
was repeated twice. All bacterial strains were obtained
from the Korean Collection for Type Culture (KCTC) at the
Korean Research Institute of Bioscience and Biotechnology
(KRIBB, Daejeon, Korea).
Hemolysis Assay. Peptides were dissolved in PBS. Ten
microliters of serially diluted peptides (0.8–100 μM final con-
centration) was added to 190 μL of suspensions of human
red blood cells (10% v/v in PBS) and incubated for 30 min
at 37  C. After centrifugation, the supernatants were diluted
with 10-fold PBS and the absorbance at 405 nm was measured
for each solution. The blood suspension treated with 0.2%
Triton X-100 was used as a control for 100% hemolysis.
The percentage of hemolysis was determined using following
equation:
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Article Stapled Heptapeptides
OD405nm sample
%Hemolysis = × 100
OD405nm positive control
The tests were performed with duplicate samples, and the
average values of the two independent measurements were
recorded.
Results and Discussion
Figure 2 shows four N-acetylated, all-hydrocarbon stapled
heptapeptides that showed moderate antimicrobial activities
in our previous study.11 Among these heptapeptides, Ac-S3
and Ac-S4 have shown the most potent antimicrobial activities
against both Gram-positive and Gram-negative bacteria.
These heptapeptides contain a tryptophan and a leucine at
positions 3 and 5, respectively (Ac-S3), and vice versa (Ac-
S4). Ac-S1 and Ac-S2, which carry an alanine residue in place
of the leucine, displayed less potent antimicrobial activities
against many types of bacteria. This result indicates that the
hydrophobic surface formed by the oct-4-enyl hydrocarbon
tether per se is not big enough to afford effective interactions
with bacterial membranes and that the leucine residue is an
important element to provide additional hydrophobic interac-
tions for higher antimicrobial activity of this series of
heptapeptides.
Another key structure–activity relationship feature of this
series of peptides is the importance of their cationic interac-
tions for the activities. Replacement of one of three lysine resi-
dues with an alanine gave rise to a significant decrease in
antimicrobial activity, indicating +3 is a minimum net charge
required for the antimicrobial activity of this series of hepta-
peptides. These results alerted us to an idea that expanding cat-
ionic surface of the amphipathic helices of the stapled
heptapeptides could improve their antimicrobial activity.
However, the margin for a sequence modification in the
stapled heptapeptides is highly limited due to their short
sequence. Among the seven residues of the heptapeptide
Figure 2. (a) Sequences of N-acetylated stapled heptapeptides Ac-S1
to Ac-S4 and their free amino terminal analogs H-S1 to H-S4. Aster-
isks at positions 2 and 6 represent cross-linking residues and lines rep-
resent the oct-4-enyl staple. (b) Helical wheel projections of the
stapled heptapeptides. Each peptide was designed to be an amphi-
pathic helix upon stapling. Lines connecting positions 2 and 6 repre-
sent the oct-4-enyl hydrocarbon staple.
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BULLETIN OF THE
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sequence, two are taken to incorporate the oct-4-enyl staple;
one for a tryptophan residue, whose presence is important
for favorable interactions with bacterial membranes; one for
a leucine to provide additional hydrophobic interactions.
Therefore, the maximum number of cationic lysine residues
in this series is three.
To expand the cationic surface of the stapled heptapeptides
without adding additional amino acids or replacing other
essential residues, we therefore decided to remove their N-
acetyl cap and investigated its structural and biological conse-
quences. The N-acetyl group was introduced in this series of
stapled heptapeptides to maximize the possibility of its helix
formation: an N-acetyl cap is known to stabilize the N-terminal
helix by providing an additional hydrogen bond acceptor in
the helix backbone at the N-terminus.18 Although the removal
of the N-acetyl cap may therefore destabilize the helical struc-
tures, the impact may be minimal by virtue of the powerful
helix-stabilizing effect of the hydrocarbon staple. We there-
fore envisioned that the overall antimicrobial activity could
be improved as a result of the expanded cationic surface pro-
vided by the free amino terminus. To test this hypothesis, we
prepared heptapeptide analogs H-S1, H-S2, H-S3, and H-S4,
which carry a free amine group at the N-terminus along with
their N-acetylated counterparts Ac-S1, Ac-S2, Ac-S3, and
Ac-S4, respectively (Figure 2(a)).
Conformational analysis using far ultraviolet CD spectrom-
etry indicates that the removal of the N-acetyl cap from Ac-S2
and Ac-S4 causes a significant loss of their helical contents
(Figure 3(b) and (d)). On the contrary, conformations of
sequences S1 and S3 appear to be not greatly affected by
the presence of the N-acetyl cap (Figure 3(a) and (c)). As
the only difference between these two sets of sequences is
the position of the tryptophan residue, this result suggests a
possible role of the tryptophan residue at position 3 for the
N-terminal helix stabilization.19–21 It should be also noted that
H-S3 displays a CD spectrum that is typically observed from
α-helical peptides even without the N-acetyl cap (Figure 3(c)).
In antimicrobial assay, compared to their N-acetylated
counterparts, H-S2 and H-S4 displayed significantly
decreased antimicrobial activities against most of bacteria
except Pseudomonas aeruginosa (Table 1). The reduced anti-
microbial activities of these analogs are probably attributed to
their decreased helicity after the N-acetyl cap was removed.
This result indicates that increased net charge of the stapled
heptapeptides is not beneficial when their helical structure is
not maintained. However, sequence S1 showed mixed effects:
compared to Ac-S1, H-S1 showed similar or slightly
decreased activities against Gram-positive bacteria whereas
its activities against Gram-negative species were slightly
improved (Table 1). In case of Ac-S3, which was the most
potent antimicrobial analog in our previous study, elimination
of its N-acetyl cap resulted in beneficial effects: H-S3 dis-
played enhanced antimicrobial activities against most of the
bacteria used in this study. In fact, among the eight heptapep-
tide analogs tested in this study, H-S3 showed the most potent
antimicrobial activity against each species. In particular, it is
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Figure 3. CD spectra of stapled heptapeptides (a) H-S1, (b) H-S2, (c) H-S3, and (d) H-S4 in comparison with their respective N-acetylated
analogs.

Table 1. Antimicrobial activities of stapled heptapeptides against selected bacteria.

MIC (μM)a
Bacteria line Ac-S1 H-S1 Ac-S2 H-S2 Ac-S3 H-S3 Ac-S4 H-S4 Cb
Gram (+)
Bacillus subtilis 25 25 25 37.5 18.8 12.5 12.5 25 3.1
Staphylococcus aureus 25 37.5 37.5 37.5 25 12.5 25 50 6.3
Staphylococcus epidermis >100 >100 >100 >100 100 25 100 100 6.3
Gram (−)
Escherichia coli 37.5 25 25 50 25 12.5 25 25 12.5
Shigella dysentariae 100 100 100 >100 50 50 50 75 25
Salmonella typhimurium >100 100 >100 >100 75 50 100 100 25
Klebsiella pneumonia 50 25 37.5 50 37.5 25 37.5 37.5 12.5
Pseudomonas aeruginose 50 25 50 37.5 50 25 50 25 12.5
a
Minimum inhibitory concentration was defined as the lowest peptide concentration (μM) that completely inhibits the cell growth after 24 h of incubation
at 37  C. The experiment was performed in duplicate.
b
Control peptide C is a 11-mer peptide reported in a previous study.22

worth noting that H-S3 even exerted a reasonable antimicro- Conclusion

bial activity against Staphylococcus epidermis, against which
no other analogs showed any measurable activities. Compared
to its N-acetylated analog, H-S3 also showed a notably
reduced hemolytic activity in the concentration range
employed in this study (Table 2). More importantly, H-S3 eli-
cits no observable hemolytic activity at the MIC ranges for
bacteria species used in this study.
In this study, we examined the effects of the N-acetyl cap of the
all-hydrocarbon stapled heptapeptides on the conformational
and biological properties. In this specific series of peptides,
helix-destabilizing effects of the N-acetyl elimination differ
depending on the position of the tryptophan residue. We found
that removal of the N-acetyl cap from the most potent stapled

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 BULLETIN OF THE
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Table 2. Hemolytic activities (%) of stapled heptapeptides against human red blood cells.a
Concentration (μM) Ac-S1 H-S1 Ac-S2 H-S2 Ac-S3 H-S3 Ac-S4 H-S4 Cb
100.0 <1.0 <1.0 <1.0 <1.0 5.53 2.05 5.74 <1.0 50.8
50.0 <1.0 <1.0 <1.0 <1.0 1.59 <1.0 2.84 <1.0 36.6
25.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 1.29 <1.0 11.3
12.5 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 6.9
6.3 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 3.0
3.1 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0
1.6 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0
0.8 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0
a
Percent hemolysis is relative to that by 0.1% Triton X-100. The experiment was performed in duplicate.
b
Control peptide C is a 11-mer peptide reported in a previous study.22

heptapeptide in our previous study, causes a minimal change
in its helical conformation, and thereby affords more potent
analog by virtue of the expanded cationic surface provided
by the free amino terminus. The structure–activity relation-
ships newly established in this study would serve as a critical
asset for the further development of a new class of antimicro-
bial agents to combat the rising problem of antibiotic
resistance.
Acknowledgments. This work was supported by the GRRC
program of Gyeonggi province ([GRRC-DONGGUK2014-
B02], development and discovery of new therapeutic target
modulators). This research was also supported by the Basic
Research Program through the National Research Foundation
of Korea (NRF) funded by the Ministry of Education, Science
and Technology (2011–0022742).
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