Peptide/peptoid hybrid oligomers: The influence of hydrophobicity and relative side-chain length on antibacterial activity and cell selectivity

Article abstract of DOI:10.3390/molecules24244429

Previous optimisation studies of peptide/peptoid hybrids typically comprise comparison of structurally related analogues displaying different oligomer length and diverse side chains. The present work concerns a systematically constructed series of 16 closely related 12-mer oligomers with an alternating cationic/hydrophobic design, representing a wide range of hydrophobicity and differences in relative side-chain lengths. The aim was to explore and rationalise the structure-activity relationships within a subclass of oligomers displaying variation of three structural features: (i) cationic side-chain length, (ii) hydrophobic side-chain length, and (iii) type of residue that is of a flexible peptoid nature. Increased side-chain length of cationic residues led to reduced hydrophobicity till the side chains became more extended than the aromatic/hydrophobic side chains, at which point hydrophobicity increased slightly. Evaluation of antibacterial activity revealed that analogues with lowest hydrophobicity exhibited reduced activity against E. coli, while oligomers with the shortest cationic side chains were most potent against P. aeruginosa. Thus, membrane-disruptive interaction with P. aeruginosa appears to be promoted by a hydrophobic surface of the oligomers (comprised of the aromatic groups shielding the cationic side chains). Peptidomimetics with short cationic side chains exhibit increased hemolytic properties as well as give rise to decreased HepG2 (hepatoblastoma G2 cell line) cell viability. An optimal hydrophobicity window could be defined by a threshold of minimal hydrophobicity conferring activity toward E. coli and a threshold for maximal hydrophobicity, beyond which cell selectivity was lost.

Full text of DOI:10.3390/molecules24244429

molecules
Article
Peptide/Peptoid Hybrid Oligomers: The Influence of
Hydrophobicity and Relative Side-Chain Length on
Antibacterial Activity and Cell Selectivity
Nicki Frederiksen , Paul R. Hansen , Fredrik Björkling and Henrik Franzyk *
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of
Copenhagen, Jagtvej 162, DK-2100 Copenhagen, Denmark; nicki.frederiksen@sund.ku.dk (N.F.);
prh@sund.ku.dk (P.R.H.); fb@sund.ku.dk (F.B.)
*
Correspondence: henrik.franzyk@sund.ku.dk; Tel.: +45-3533-6255
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 29 October 2019; Accepted: 26 November 2019; Published: 4 December 2019
Abstract: Previous optimisation studies of peptide/peptoid hybrids typically comprise comparison
of structurally related analogues displaying different oligomer length and diverse side chains.
The present work concerns a systematically constructed series of 16 closely related 12-mer oligomers
with an alternating cationic/hydrophobic design, representing a wide range of hydrophobicity and
differences in relative side-chain lengths. The aim was to explore and rationalise the structure–activity
relationships within a subclass of oligomers displaying variation of three structural features: (i) cationic
side-chain length, (ii) hydrophobic side-chain length, and (iii) type of residue that is of a flexible
peptoid nature. Increased side-chain length of cationic residues led to reduced hydrophobicity till
the side chains became more extended than the aromatic/hydrophobic side chains, at which point
hydrophobicity increased slightly. Evaluation of antibacterial activity revealed that analogues with
lowest hydrophobicity exhibited reduced activity against E. coli, while oligomers with the shortest
cationic side chains were most potent against P. aeruginosa. Thus, membrane-disruptive interaction
with P. aeruginosa appears to be promoted by a hydrophobic surface of the oligomers (comprised of the
aromatic groups shielding the cationic side chains). Peptidomimetics with short cationic side chains
exhibit increased hemolytic properties as well as give rise to decreased HepG2 (hepatoblastoma G2
cell line) cell viability. An optimal hydrophobicity window could be defined by a threshold of minimal
hydrophobicity conferring activity toward E. coli and a threshold for maximal hydrophobicity, beyond
which cell selectivity was lost.
Keywords: antimicrobial peptides; peptoids; peptidomimetics; solid-phase synthesis; antibacterial
activity; structure-activity study; hemolysis; cell viability; cell selectivity; hydrophobicity
1
. Introduction
Increased emergence of bacterial strains resistant to antibiotics is a serious worldwide concern
for human health. Major factors contributing to the selection for multidrug-resistant (MDR) strains
comprise the widespread non-prescription sales of antibiotics in some countries [ ] and lack of
rapid methods for identification of the specific bacteria causing an infection—with the ensuing
prescription of inefficacious antibiotics [ ]. Together, these issues promote development of resistance,
which ultimately may give rise to pan-resistant strains due to inappropriate use of “last-resort”
antibiotics [ ], and consequently, there is an urgent need for the continuous discovery of new
1
2
3,4
antibiotics. Mechanisms of antimicrobial resistance (AMR) typically confer resistance toward a group
of related drugs belonging to the same class of antibiotics. Hence, it is not a viable long-term approach
merely to develop analogues of existing drugs, since only entirely new classes of antibiotics may retain
a prolonged usage time without the development of severe resistance problems.
Molecules 2019, 24, 4429; doi:10.3390/molecules24244429
www.mdpi.com/journal/molecules

Molecules 2019, 24, 4429
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Antimicrobial peptides (AMPs) constitute a class of potential antibiotics with an alternative mode
of action, and they have evolved to play important roles in the innate immune systems of nearly all
living organisms [
very diverse, but the majority of known AMPs adopt amphipathic structures and possess a characteristic
high overall positive charge, which confers selectivity for bacterial cells over mammalian cells [ ].
A major challenge in the development of drugs from AMP-based leads is their low proteolytic stability.
Approaches that confer increased stability comprise incorporation of D-amino acids [ 10] or design of
-peptoids (N-alkylated
-peptoid hybrids [16],
5], often exhibiting broad-spectrum antibacterial activity [6]. Structurally, AMPs are
7,8
9,
unnatural mimetics, e.g.,
α
-peptoids (N-alkylated glycine oligomers) [11
,
12],
β
β
3
β-alanine oligomers) [13],
β
-peptides [14],
α
/β
-peptides [15],
α
-peptide/
α/γ
N-acylated N-aminoethylpeptides (AApeptides) [17,18], and oligo-acyl-lysyls (OAKs) [19].
Many AMPs fold into -helices or adopt secondary structures that are amphipathic and considered
essential for high antibacterial activity [ 20]. In several studies a correlation between degree of
α
8,
helicity and undesired toxicity of AMPs toward mammalian cells was found [20
–
29]. Furthermore,
3
α
/
β
-peptides and peptide/peptoid hybrids with a low degree of amphipathicity and/or secondary
30]. Interestingly, CD (circular
structure were shown to possess advantageous activity profiles [15
,
dichroism) spectroscopy proved to be of little value in the characterization of peptide/peptoid hybrid
oligomers with an alternating cationic/hydrophobic design, since it was not feasible to assign a
secondary structure (e.g.,
not correspond to any known type of folding [30
hybrid oligomers have failed so far. Analogues displaying
gave rise to the most distinct CD spectra inferring some degree of secondary structure in the presence
of liposomes mimicking bacterial or mammalian membranes [30 31]. This in combination with the
finding that oligomers devoid of -chiral peptoid residues possess the most favourable activity profiles
α
-helical,
β
-sheet or random coil) based on their CD spectra, which did
31]. In addition, crystallisation attempts for such
-chiral side chains in the peptoid residues
,
α
,
α
indicate that for these oligomers secondary structure is not a prerequisite for antibacterial activity—in
fact it appears only to confer undesirable side effects in the presence of mammalian cells. Moreover,
these hybrid oligomers did not exhibit a similar characteristic CD behaviour as reported for peptoid
oligomers displaying α-chiral side chains [32].
Both for AMPs and helix-forming
correlated with higher antibacterial potency and hemolytic properties [15
peptide/ -peptoid hybrids and peptoid oligomers, it was found that, above a certain threshold for
hydrophobicity, the compounds lost selectivity for bacteria over mammalian erythrocytes [32 38 39].
α
/β
3-peptide hybrids, increased hydrophobicity has been
33 37]. For lysine-based
,
–
β
,
,
Various structural modifications that change the overall hydrophobicity of AMPs and peptidomimetics
as well as their effects on the biological activity profiles of the resulting analogues have been reported in
several studies. These modifications include single amino acid substitutions [40], multiple amino acid
substitutions [41], alteration of net charge [34], incorporation of fluorinated residues [42], and appending
end groups influencing polarity (i.e., end-tagging with oligopeptides, hydrophobic, polar, or cationic
moieties) [43–52].
Inspired by the above-mentioned observations regarding the putative interplay between activity
and hydrophobicity, we decided to perform a more comprehensive study also including the so far
poorly explored influence of relative side-chain length as a potential determining factor for antibacterial
activity, hemolysis, and effect on cell viability. Previous structure-activity studies of peptide/peptoid
hybrids with an alternating cationic/hydrophobic design [53] have shown that such oligomers exert
promising antibacterial activity while exhibiting weak hemolytic activity and low effect on mammalian
cell viability as compared to the corresponding peptides [31], and that 12-mer oligomers display the
optimal cell selectivity [30]. Thus, a series of related 12-mer peptide/peptoid hybrids, displaying
elongated/truncated side chains via the incorporation of homologues of lysine/phenylalanine and
the corresponding peptoid residues, was investigated in order to identify trends for how structural
features influence their activity profile. These peptidomimetics were tested against a panel of seven
bacterial strains comprising representatives of the ESKAPE bacteria (E. faecalis, S. aureus, K. pneumoniae,
A. baumannii, P. aeruginosa, and E. coli) [54].

Molecules 2019, 24, 4429
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2
. Results and Discussion
2
.1. Selection and Synthesis of Peptidomimetics
Recently, we reported that a peptide/β-peptoid hybrid oligomer with elongated side chains
display equal or slightly higher antibacterial activity against S. pseudintermedius and P. aeruginosa
as compared to the Lys/ NPhe template with identical oligomer length [42 55]. Intrigued by these
findings we designed 16 similarly related peptide/ -peptoid oligomers 16, see Figure 1, in order
β
,
α
1–
to determine whether hydrophobicity and/or relative side-chain length may be clearly correlated to
antibacterial potency and/or effect on mammalian cell viability. Comparison with the corresponding
peptide was omitted since, for a very closely related 16-mer, both the all-d- and all-l-peptide exhibited
exceedingly high toxicity toward mammalian cell lines (i.e., IC50 values of ~20 µM) while exhibiting
poor antibacterial activity (i.e., MICs of 128 µM or above) [31].
Figure 1. Structures of α-peptide/α-peptoid hybrid peptidomimetics.
The peptidomimetics were synthesised by using Fmoc-based solid-phase peptide synthesis
SPPS) methodology for assembly of commercial amino acid and preformed peptoid building blocks
(
(
i.e., 17
–
22). The Fmoc-protected peptoid building blocks were prepared essentially as previously
56]: The primary amine corresponding to the desired side chain was alkylated with
described [30
,
ethyl 2-bromoacetate or tert-butyl 2-bromoacetate to provide an intermediate that subsequently was
hydrolysed and Fmoc-protected to yield the desired peptoid building block (see Figure 2). Importantly,
the cationic peptoid building blocks 17
–20 could all be obtained in pure state on a multigram scale via
an extraction work-up process followed by crystallisation.
Figure 2. Structures of peptoid building blocks.
.2. Effects of Relative Side-Chain Length and Flexibility on Hydrophobicity
The investigated peptidomimetics (see Figure 1) may be considered to comprise two main groups,
consisting either of alternating cationic peptoid residues and hydrophobic amino acid residues (i.e.,
) or of alternating cationic amino acid residues and hydrophobic peptoid residues (i.e., 16).
2
1
–8
9–
Four different lengths of the cationic side chains and two different lengths of the hydrophobic side
chains were selected in order to obtain analogues within a sufficiently wide range of hydrophobicity so

Molecules 2019, 24, 4429
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that the entire series was expected to comprise compounds with a substantial variation in antibacterial
and/or hemolytic properties. Analogues displaying further extended hydrophobic side chains were
not included since analogous peptide/β-peptoid hybrids were found to be poorly soluble in the test
media and were found to be devoid of cell selectivity [55].
Thus, the peptidomimetics included in the present study represent variation of three structural
features: (i) Cationic side-chain length (2-5 carbon atoms), (ii) hydrophobic side-chain length (benzyl
or phenylethyl), and (iii) the type of residue that is of a flexible peptoid nature (i.e., cationic versus
hydrophobic). Accordingly, the peptidomimetics can be further subdivided into four subgroups
(I–IV), each having similar type of hydrophobic residues and nature of peptoid residues but with
varying cationic side-chain length. Thus, subgroups I–IV comprise peptidomimetics 1–4, 5–8, 9–12,
and 13–16, respectively.
The relative hydrophobicity of each peptidomimetic was measured by reverse-phase
high-performance liquid chromatography (RP-HPLC). Typically, the RP-HPLC retention time
(
or percentage acetonitrile at peak elution) constitutes the preferred measure that allows for comparison
of relative hydrophobicity, while log P values of polycationic peptides or peptidomimetics normally
are considered unsuitable due to the extreme hydrophilic nature of such compounds [11 45 57 60].
Comparison of the hydrophobicity of the members of all four subgroups (see Table 1) indicates that
subgroup I (with cationic peptoid residues and Phe, i.e., ) and subgroup II with cationic peptoid
residues and homophenylalanine (hPhe) (i.e., ) display the largest differences in hydrophobicity
between the least and most hydrophobic representative within each subgroup. For both subgroups
III (with cationic amino acid residues and the peptoid analogue of Phe, NPhe, i.e., 12) and IV
with cationic amino acid residues and the peptoid analogue of hPhe, NhPhe, i.e., 13 16) only minor
,
, –
1–4
5–8
9–
(
–
differences in hydrophobicity were seen. These observations infer that, when the cationic residues
are of a peptoid nature, the side-chain length of these has a strong influence on the hydrophobicity.
Expectedly, subgroups I and III, containing either Phe or NPhe, had a relatively low hydrophobicity
(
~ peak elution within the ranges 35.6–37.5% and 36.9–38.0% MeCN, respectively), while subgroups II
and IV, containing hPhe and NhPhe, both displayed a comparably higher hydrophobicity (eluting
within the ranges 40.6–43.5% and 40.5–41.4% MeCN, respectively). Thus, introduction of additional six
methylene groups results, as expected, in increased hydrophobicity.
Within each subgroup, a somewhat surprising trend is seen — increased cationic side-chain length
leads to reduced hydrophobicity. This may be explained by the cationic amino groups becoming
gradually more accessible to the surroundings (due to lower shielding by the aromatic groups of the
hydrophobic residues) when extending the corresponding side-chain length. In particular, for the
peptidomimetics containing 2,4-diaminobutyric acid (Dab) or its peptoid analogue (i.e., NDab) this
effect was pronounced, and these analogues was the most hydrophobic ones within each subgroup.
When comparing these analogues with those containing ornithine (Orn) or its peptoid analogue
(i.e., NOrn), the incorporation of additional methylene groups, expected to confer an increase in
hydrophobicity, was clearly counteracted by a larger decrease in hydrophobicity induced by a reduced
shielding of the amino functionalities by the aromatic groups, resulting in an overall decrease in
hydrophobicity. A similar trend was seen for the hydrophobicity when comparing the peptidomimetics
containing Orn or NOrn with the corresponding analogues displaying Lys or the peptoid analogue of
Lys (i.e., NLys). By contrast, the peptidomimetic in each group that contained homolysine (hLys) or
its peptoid analogue (i.e., NhLys) did not follow this trend, as they consistently possessed a higher
hydrophobicity than the corresponding Lys/NLys analogues. For these peptidomimetics, the shielding
of the cationic amino groups by the aromatic side chains appears to be surpassed already, and hence
the opposite hydrophobicity enhancement conferred by the incorporation of additional methylene
groups in the cationic side chains dominates.

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Table 1. Minimal inhibitory concentrations (MICs) for peptidomimetics 1–16.
MIC (µg/mL)
Hydrophobicity
(% MeCN at
Gram-Negative
Gram-Positive
S. aureus E. faecalis
Subgroup
Cmpd
a
E. coli ATCC
K. pneumoniae
ATCC 13883
P. aeruginosa
ATCC 27853
P. aeruginosa
A. baumannii
ATCC 19606
elution )
2
5922
PAO1
ATCC 29213
ATCC 29212
I
I
I
I
1
2
3
4
37.5%
35.7%
35.6%
36.6%
4-8
64
64
32
64
64
>64
>64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
8-16
8-16
4-8
>64
>64
>64
II
II
II
II
5
6
7
8
43.5%
41.2%
40.6%
40.9%
4
4
2-4
2
64
>64
>64
64
4
8
32
64
16
16-32
32-64
64
>64
>64
>64
4
8
16
>64
>64
32
>64
>64
>64
III
III
III
III
9
38.0%
37.2%
36.9%
38.0%
2-4
4-8
4-8
2
32-64
>64
64
1
2-4
4
2
16-32
64->64
>64
>64
>64
>64
>64
>64
>64
>64
>64
10
11
12
4-8
8-16
64
>64
64
16
IV
IV
IV
IV
13
14
15
16
41.4%
40.5%
40.5%
41.1%
4
4-8
4
32
32
>64
>64
2
8
16
16
2-4
8-16
8-16
32
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
4
8
Colistin
0.125-0.25
0.25
0.125-0.25
0.5
0.5
>64
>64
a
RP-HPLC gradient: 0 → 60% B (during 10 min). MeCN: acetonitrile.

Molecules 2019, 24, 4429
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Analysis of similar hydrophobicity data were reported in a previous study concerning 14-mer
AMPs containing hydrophobic, branched amino acids and cationic amino acids [57]. Here the
relative hydrophobicity of H-Gly-(Ile-Ile-Lys-Lys) -Ile-NH and H-Gly-(Ile-Ile-Orn-Orn) -Ile-NH
2
3
2
3
was compared, and the Lys-containing AMP (displaying an additional six methylene groups) was as
expected more hydrophobic than the corresponding Orn analogue. However, when comparing the Orn
analogue with the Dab analogue, and likewise the Dab analogue with the 2,3-diaminopropionic acid
(
Dap) analogue, the peptides with the shorter cationic side chains were more hydrophobic, most likely
due to the cationic charges being partially shielded by the Ile residues. Interestingly, in the present
study, the least hydrophobic compounds within each subgroup were the Lys/NLys analogues ( 11,
3,
7,
and 15), while in the above case of Ile-containing peptides, it was the Orn analogue, which may be
ascribed to the fact that Phe/NPhe and hPhe/NhPhe all are larger in size than Ile.
2
.3. Effects of Relative Side-Chain Length and Flexibility on Antibacterial Activity
The antibacterial activity (see Table 1) of the peptidomimetics was tested against a panel of four
Gram-negative bacteria (E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii) and two Gram-positive
bacteria (E. faecalis and S. aureus). None of the peptidomimetics displayed noteworthy activity
against K. pneumoniae (MIC values
peptide/peptoid and peptide/ -peptoid hybrids as well as other related peptidomimetics generally
lack potency against K. pneumoniae [31 58 61]. Only four peptidomimetics ( 12, and 16) had MIC
values below 32 g/mL against A. baumannii, and three of these compounds contained hLys or NhLys,
while the most potent oligomers (i.e., and 16 having MIC values of 4 and 8 g/mL, respectively)
≥
32 µg/mL), which corroborates previous findings that Lys-based
β
,
–
8, 9,
µ
8
µ
contained hPhe or NhPhe, which implies that extended side chains of both cationic and hydrophobic
residues appear to confer activity toward A. baumannii.
All 16 peptidomimetics were active against E. coli, and most of these had a MIC in the range
4
–8
of 8–16
Comparison of compounds
i.e., and with MIC of 2–4
design in subgroup III (i.e., 10 and 11 with MICs of 4-8
µ
g/mL. Two exceptions were peptidomimetics
g/mL) than the other compounds in subgroup I (i.e.,
and with peptidomimetics with the same cationic residues in subgroup II
g/mL) as well as with the analogues having a reversed amino acid/peptoid
g/mL) showed that compounds and
2
and
3
with slightly lower potency (i.e., MICs
µ
1
and with a MIC of 4-8 g/mL).
4
µ
2
µ
3
(
6
7
µ
2
3
exhibited slightly lower antibacterial activity. Slightly higher MIC values against E. coli were observed
for peptidomimetics in subgroup I as compared to those in subgroup II, possibly due to the additional
methylene groups in the side chains of hPhe (as compared to Phe). By contrast all members of
subgroups III and IV that displayed cationic amino acid residues were almost equipotent (i.e., MICs
within the range 2–8 µg/mL).
Thus, when considering how hydrophobicity influences activity of the analogues against E. coli a
clear pattern could not be discerned (see Figure S1): while the least hydrophobic compounds
(~ peak elution at 35.7% and 35.6% MeCN, respectively) exhibited poorest antibacterial activity,
the most potent peptidomimetics against E. coli (i.e., and 12 with MICs in the range 2–4 g/mL)
were not amongst the analogues with the highest hydrophobicity. Hence, no clear correlation between
structural features and antibacterial potency was evident, since and 12 differed with respect to
2 and
3
7–
9
µ
7–9
both relative side-chain length of cationic and hydrophobic residues as well as the nature of peptoid
residues. This indicated the existence of a hydrophobicity threshold for optimal activity against E. coli
as previously found for end-group modified peptide/β-peptoid hybrids [39].
Comparison of subgroup I peptidomimetics with those in subgroup III showed that their
hydrophobicity was similar, but since all members of subgroup III were more potent, the hydrophobic
nature of the peptoid residues appeared to be a more important contributing factor for high activity
against E. coli than merely the overall hydrophobicity (see Figure S1). When comparing peptidomimetics
from subgroups II and IV their hydrophobicity was similar, but higher than for subgroups I and III,
which appeared to confer improved potency against E. coli.

Molecules 2019, 24, 4429
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Two different strains of P. aeruginosa were included in the present study. No significant activity
against these was observed for peptidomimetics in subgroup I, which displayed a generally low
hydrophobicity, indicating that this property diminishes activity against P. aeruginosa. For the
remaining subgroups, peptidomimetics with shorter cationic side chains exhibited higher activity
against both strains of P. aeruginosa than peptidomimetics with longer cationic side chains. Generally,
shortening of the cationic side chains by one methylene group conferred a two-fold increased potency.
The compounds that proved most potent against P. aeruginosa contain hydrophobic peptoid residues,
i.e., they belong to subgroups III and IV. Thus, a design with short-chain cationic amino acids and
hydrophobic peptoid residues promotes antipseudomonal activity. These findings indicate that
efficient membrane-disruptive interaction with P. aeruginosa requires that the peptidomimetics possess
a hydrophobic “surface” comprised of the aromatic groups (partially) shielding the cationic side chains
(
see Figure S2). Previously, peptide/
β
-peptoid analogues of peptidomimetics
g/mL) when the cationic
42 55], thus supporting
9–16 were found to
exhibit potent activity against P. aeruginosa (MIC values in the range of 2–8
side chains were short or when the hydrophobic side chains were extended [38
µ
,
,
the present findings.
A putative explanation for the higher activity against E. coli as compared to the very low activity
against K. pneumoniae is the higher overall content of anionic phospholipids in the cell membranes of
E. coli that is likely to make this species more susceptible to the membrane-disruptive actions of these
highly cationic peptidomimetics. Thus, in E. coli, cardiolipin (CL; with an overall charge of
phosphatidylglycerol (PG; with a net charge of 1) comprise 5% and 15%, respectively, of the total
membrane phospholipids, while the content of the zwitterionic phosphatidylethanolamine (PE) is
0% [62]. In contrast, for K. pneumoniae the content of the negatively charged CL and PG is 6% and 5%,
−2) and
−
8
respectively, resulting in an overall less negatively charged membrane, which may be anticipated to
interact less efficiently with these peptidomimetics [63]. Similarly, the phospholipid content in the cell
membrane of P. aeruginosa comprise 11% CL and 21% PG, conferring a considerably higher negative
charge to its membrane as compared to those of both E. coli and K. pneumoniae. Consequently, all
differences in activity cannot readily be explained by their distinct phospholipid compositions alone.
Yet, the outer membranes of P. aeruginosa, E. coli and K. pneumoniae differ somewhat with respect to
the structure of lipid A that constitutes the inner part of lipopolysaccharide, which by far is the most
abundant component of the outer leaflet. While lipid A in E. coli and K. pneumoniae have very similar
structures (only differing in the position and length of a single fatty acid moiety), P. aeruginosa lipid A
contains additional hydroxyl groups that enable increased hydrogen bonding between neighbouring
lipid A molecules, thereby stabilising the outer membrane [64].
The antibacterial activity of the peptidomimetics was also tested against two Gram-positive
bacteria (S. aureus and E. faecalis). Only peptidomimetics
S. aureus, whereas only had weak activity against E. faecalis. Peptidomimetic
hydrophobicity of all analogues, while peptidomimetic was amongst the most hydrophobic
5
and
6
showed moderate activity against
5
5
had the highest
6
members, which is in accordance with previous studies, where a clear relationship was observed
between hydrophobicity of peptoids and their activity against Gram-positive bacteria (in particular
for S. aureus) [47,65]. Earlier studies have found similar trends for peptide/β-peptoid hybrids,
for which increased hydrophobicity could be correlated to higher activity against Gram-positive
bacteria [38,39,42].
2
.4. Effects of Relative Side-Chain Length and Flexibility on Mammalian Cell Viability
The effects of the peptidomimetics on the viability of mammalian cells were tested against
HepG2 cells and human red blood cells (see Table 2). In subgroup I, the combination of cationic
peptoid residues and Phe resulted in a lower hydrophobicity than found for all other subgroups.
Generally, the peptidomimetics in this subgroup exerted minimal effects on HepG2 cell viability, since
all IC50 values were 1280
µg/mL or higher. Similarly, these compounds all exhibited weakly hemolytic
properties (i.e., hemolysis below 10% at 800 µg/mL).

Molecules 2019, 24, 4429
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Table 2. Effects of peptidomimetics on mammalian cell viability.
Hydrophobicity
% MeCN at elution )
IC50
(µg/mL)
Viability at
1280 µg/mL
Hemolytic
Activity
Cell
Selectivity
Subgroup
Cmpd
a
b
c
d
e
(
I
I
I
I
1
2
3
4
37.5%
35.7%
35.6%
36.6%
1280
>1280
1280
49%
62%
52%
49%
7%
5%
8%
9%
160-320
>80
80-160
160-320
1280
II
II
II
II
5
6
7
8
43.5%
41.2%
40.6%
40.9%
307
456
720
266
-
-
-
-
43%
8%
6%
9%
77
114
180-360
133
III
III
III
III
9
38.0%
37.2%
36.9%
38.0%
326
923
>1280
1280
-
-
79%
10%
10%
9%
82-163
115-231
>160
10
11
12
76%
47%
640
IV
IV
IV
IV
a
13
14
15
16
41.4%
40.5%
40.5%
41.1%
261
684
595
410
-
-
-
-
53%
11%
8%
65
86-171
149
9%
103
RP-HPLC gradient: 0
→
60% B (during 10 min). b Toxicity against HepG2 cells is given as IC50 value for inhibiting
growth of HepG2 cells (assay was performed in two biological replicates each with three technical replicates).
The highest concentration tested was 1280
compounds with an IC50 value of 1280
00
µ
g/mL. c The viability of HepG2 cells at 1280
µ
g/mL is only given for
d
µ
g/mL or higher. Percentage hemolysis in human red blood cells (RBCs) at
8
µ
g/mL. e Cell selectivity was calculated as the ratio between IC50 for effect on HepG2 cell viability and MIC
against E. coli.
For subgroup II (i.e.,
5
–8
) there is a clear trend of an increasing effect on cell viability within the
NDab (see Table 2.). For subgroup II further extension of the cationic
side-chain length (from NLys in to NhLys in ) confers a slightly increased hydrophobicity due to
incorporation of additional six methylene groups. This leads to a significantly increased effect of on
cell viability, corresponding to a ca. 2.7-fold lowering of the IC50 value as compared to that found for
subseries NLys
→
NOrn
→
7
8
8
7.
In subgroup II, the most hydrophobic analogue (i.e., 5) was the only representative giving rise to >10%
hemolysis at 800 µg/mL.
Subgroup III (i.e.,
observed a trend of increasing cationic side-chain length conferring gradually decreased hydrophobicity
with an ensuing declining effect on HepG2 cell viability. However, even though and 12 had similar
hydrophobicity, only (with a hydrophobic surface) had a pronounced effect on HepG2 cell viability and
was the most hemolytic compound in the entire array (79% hemolysis at 800 g/mL). Finally, subgroup
IV peptidomimetics (i.e., 13 16) were found to possess relatively high hydrophobicity (see Table 2).
However, no clear trends, attributable to cationic side-chain length or hydrophobicity, were observed
9–12) also had relatively low hydrophobicity (see Table 2), and for 9–11 we
9
9
µ
–
with respect to their effect on HepG2 cell viability or hemolytic activity, except that peptidomimetic 13
,
displaying the shortest cationic side chain, also had pronounced hemolytic properties (53% hemolysis
at 800 µg/mL). The two peptidomimetics with highest hydrophobicity, namely 13 and 16, also exerted
the strongest effect on HepG2 cell viability. In subgroups II and IV, the lowest IC50 values for HepG2
viability were seen for the two peptidomimetics with the shortest and longest cationic side chains,
respectively. Also, in subgroups II–IV, the most distinct hemolytic properties were seen for the members
displaying a hydrophobic “surface” comprised of the aromatic groups shielding the cationic side chains.
These findings regarding the relationships between hydrophobicity and effect on HepG2 cell
viability, dicusssed above for the four subgroups of peptidomimetics, are depicted graphically below
in Figure 3.

Molecules 2019, 24, 4429
9 of 18
Figure 3. %MeCN at RP-HPLC peak elution versus effect on HepG2 cell viability (IC ).
5
0
The correlation between high hydrophobicity and loss of cell selectivity found for the present
series of petidomimetics corroborates findings in a previous study on peptoid oligomers [32]. Here,
a relatively small difference in hydrophobicity (also based on % MeCN at peak elution) of the 12-mer
H-(NLys-Nspe-Nspe) -NH and the corresponding 9-mer was observed (53.6% MeCN versus 51.6%
4
2
MeCN), and the concentration of these peptidomimetics resulting in 10% hemolysis (of human red
blood cells) were 9.1 M and 119.5 M, respectively. Also, two 12-mer analogues with further increased
µ
µ
hydrophobicity (due to incorporation of Nspe-derived residues with either a Me or Cl substituent in
the 4-position; ~ peak elution at 61.1% MeCN and 64.1% MeCN, respectively) proved to be exceedingly
hemolytic with 10% hemolysis reached below 6.25
µM [32]. Also, moieties within a wide range of
polarity have been introduced at the N-terminus of an
α
-peptide/ -peptoid template in order to
β
evaluate the ensuing effects on the activity profile of the resulting analogues [39]. Thus, analogues
with aliphatic or aromatic end-group modifications showed increased antibacterial activity over the
template. However, even though these compounds exhibited similar effects on the viability of NIH
3
T3 (fibroblast cells, IC50 values in the range 35-67
endothelial cells, IC50 values of ~150 M) their hemolytic properties differed considerably (i.e., 50%
hemolysis was reached between 9 M and 602 M) as did their hydrophobicity (~ peak elution
µM) and HUVEC cells (human umbilical vein
µ
µ
µ
between 48.8% and 54.3% MeCN). Two compounds eluting below 50% MeCN displayed the lowest
effect on mammalian cell viability, and in particular, they exhibited significantly reduced hemolytic
properties. Collectively, these findings strongly corroborate the hypothesis that, within a series of
closely related compounds, there exists a threshold for acceptable maximal hydrophobicity, beyond
which cell selectivity is lost completely.
In several studies, two hydrophobicity thresholds have been identified for both AMPs and
antibacterial peptidomimetics [36,41,65–70]. Thus, the hydrophobicity of an AMP or peptidomimetic
must exceed the lower threshold in order to possess potent antibacterial activity. However, when the
higher threshold is surpassed the compound will lose selectivity toward bacteria over mammalian cells.
In the present study, a lower hydrophobicity threshold for activity against susceptible Gram-negative
bacteria (i.e., E. coli and P. aeruginosa) could not be identified for E. coli, whereas against P. aeruginosa, only
peptidomimetics with a hydrophobicity above ~37% MeCN proved active. However, for P. aeruginosa,
this was not the only criteria that should be fulfilled, since peptidomimetics with shorter cationic side
chains proved most active, regardless of hydrophobicity.
For subgroups I and III, which both contained cationic peptoid residues and hydrophobic amino
acid residues, analogues with a low hydrophobicity (~ to peak elution below ~38% MeCN) exerted a
negligible effect on HepG2 cell viability when excluding peptidomimetic
four peptidomimetics containing the shortest cationic side chains (i.e.,
9
9
. Noticeably, three out of
, and 13) had the highest
5,
hydrophobicity in their subgroup and showed pronounced hemolysis in the range 43–79% at 800
Interestingly, although and had very similar hydrophobicity (~ peak elution at 37.5% and 38.0%
MeCN, respectively) they possess quite different hemolytic properties. This deviation from the common
µg/mL.
1
9

Molecules 2019, 24, 4429
10 of 18
trend may be explained by the fact that
1
contains cationic peptoid residues, while the very hemolytic
9
display hydrophobic peptoid residues. Molecular modelling have suggested that peptoids have a
greater diversity of conformational states than peptides [11], which means that the combination of
short cationic side chains and flexible hydrophobic side chains are expected to interact more favorably
with the cell membranes of red blood cells, thus conferring hemolytic properties even below the critical
hydrophobicity threshold for antibacterial activity.
3
. Materials and Methods
3
.1. General Information
Starting materials and solvents were purchased from commercial suppliers (Iris Biotech,
Markredwitz, Germany; Fluorochem, Hadfield, United Kingdom; and Merck, Darmstadt, Germany)
and used without further purification. Water used for analytical and preparative high-performance
liquid chromatography (HPLC) was filtered through a 0.22-
retention time of each peptidomimetic were determined by analytical UHPLC by using a Phenomenex
Luna C18(2) HST column (100 mm 3 mm; particle size: 2.5 m; pore size: 100 Å) on a Shimadzu
Prominence and Shimadzu Nexera system using an aqueous acetonitrile (MeCN) gradient with
.1% trifluoroacetic acid (TFA) added (eluent A: 5:95 MeCN–H O + 0.1% TFA, eluent B: 95:5
µm Millipore membrane filter. Purity and
×
µ
0
2
MeCN–H O + 0.1% TFA); a flow rate of 0.5 mL/min was used. For elution of peptidomimetics,
2
a linear gradient of 0% to 60% B during 10 min was used with UV detection at
λ = 220 nm. All tested
compounds had a purity of at least 97%. For each peptidomimetic, the percentage of MeCN at peak
elution was calculated from the retention time by using the following formula:
ꢀ
ꢁ
ꢀ
ꢁ
Rt
0 min.
Rt
10 min.
%
MeCN = 0.95 0.6
+ 0.05 1 − 0.6
.
(1)
1
Preparative HPLC was performed by using a Phenomenex Luna C18(2) column (250
particle size: 5
Elution was performed with a linear gradient of 0% to 40% B during 20 min at a flow rate of 20 mL/min
with UV detection at = 220 nm. High-resolution mass spectrometry (HRMS) spectra were obtained
by using a Bruker Solarix XR MS detector.
×
21.2 mm;
µ
m) on a Shimadzu Prominence system using the same eluents as for analytical HPLC.
λ
3
.2. Building Block Synthesis
3
.2.1. Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-(5-((tert-
butoxycarbonyl)amino)ethyl)glycine [Fmoc-NDab(Boc)-OH, 17]
Tert-butyl (5-aminoethyl)carbamate (11.7 g) was dissolved in THF (110 mL), and then Et N (16 mL,
3
3
1
.0 equiv) was added to the solution under stirring. The mixture was kept at room temperature (rt) for
0 min. Ethyl bromoacetate (12.24 mL, 1 equiv) was dissolved in THF (100 mL) and added dropwise
to the solution under stirring. The mixture was kept at rt for 16 h. The mixture was concentrated in
vacuo, and then the residue redissolved in Et O (300 mL) and filtered. The filtrate was concentrated
2
in vacuo to afford the intermediate ethyl ester (17.65 g) as a colourless oil, which was dissolved in
dioxane (100 mL) and MeOH (40 mL). Then, 4M NaOH (18 mL) was added dropwise under stirring.
The mixture was kept at rt for 1 h, then concentrated in vacuo, and the residue redissolved in H O
2
◦
(
100 mL). Fmoc-OSu (23.69 g, 1 equiv) was dissolved in warm (45 C) MeCN (170 mL) and added
dropwise to the mixture under stirring. The mixture was kept at rt for 16 h, then concentrated in vacuo
until it turned turbid. Then EtOAc (300 mL) was added, and the resulting mixture was washed with
1
0% citric acid (400 mL). The aqueous phase was extracted with EtOAc (2
organic phases were washed with H O (3 250 mL), and brine (250 mL). The organic phase was
dioxane 3:3:2 (4 400 mL). The combined aqueous phases
were adjusted to pH 2–3 with 4M HCl, extracted with EtOAc (150 mL), then combined, dried over
Na SO and concentrated in vacuo. The resulting solid was recrystallised from EtOAc (200 mL) and
×
150 mL), and the combined
×
2
extracted with 10% NaHCO3
−
10% Na CO3
−
×
2
2
4

Molecules 2019, 24, 4429
11 of 18
heptane (800 mL) to afford building block 17 (25.0 g, 77.6%) as a white solid; t = 6.53 min. (gradient
R
+
3
∆
0–100% B during 10 min). HRMS: calcd for C H N O [M + Na] 463.18396, found 463.18550;
M = 3.3 ppm. H NMR (600 MHz, methanol-d ) δ 7.79 (m, 2H, ArH-Fmoc), 7.61 (m, 2H, ArH-Fmoc),
4
24
28
2
6
1
7
3
.39 (m, 2H, ArH-Fmoc), 7.31 (m, 2H, ArH-Fmoc), 4.39 (m, 2H, CH -Fmoc), 4.24 (m, 1H, CH-Fmoc),
2
.99 (s, 2H, N-CH -C=O)), 3.43 – 3.25 (m, 2H, CH -CH -N), 3.03 (t, J = 6.2 Hz, 2H, N-CH -CH ), 1.40
2
1
2
2
2
2
3
(s, 9H, CH -Boc). C NMR (151 MHz, methanol-d4)
δ
171.73, 156.77, 156.66, 143.85, 141.20, 127.43,
3
1
26.85, 124.68, 119.55, 78.83, 67.84, 49.09, 48.66, 47.00, 38.27, 27.35.
3
.2.2. Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-(5-((tert-
butoxycarbonyl)amino)propyl)glycine [Fmoc-NOrn(Boc)-OH, 18]
Fmoc-NOrn(Boc)-OH (18) was prepared, analogously to the preparation of 17, from tert-butyl
(
(
5-aminopropyl)carbamate (10.0 g). Crystallisation from EtOAc–heptane afforded building block 18
18.6 g, 61%) as a white solid; t = 6.72 min. (gradient 30–100% B during 10 min). HRMS: calcd for
R
+
1
C H N O [M + Na] 477.19961, found 477.20298; ∆M = 7.1 ppm. H NMR (600 MHz, methanol-d )
4
25
30
2
6
δ
7.79 (m, 2H, ArH-Fmoc), 7.60 (m, 2H, ArH-Fmoc), 7.38 (q, J = 7.1 Hz, 2H, ArH-Fmoc), 7.31 (m, 2H,
ArH-Fmoc), 4.44 (m, 2H, CH -Fmoc), 4.22 (m, 1H, CH-Fmoc), 3.92 (d, J = 4.2 Hz, 2H, N-CH -C=O)),
2
2
3
.34, 3.10 (two m, 2H, N-CH -CH ), 3.03, 2.82 (two m, 2H, CH -CH -CH ), 1.67 (p, J = 6.8 Hz, 2H,
2
2
2
2
2
13
CH -CH -N), 1.41 (s, 9H, CH -Boc). C NMR (151 MHz, methanol-d4)
δ
171.63, 157.02, 156.63, 143.88,
2
2
3
1
41.25, 127.40, 126.83, 124.57, 119.55, 67.39, 48.58, 47.05, 45.94, 37.32, 27.83, 27.36.
3
.2.3. Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-(5-((tert-
butoxycarbonyl)amino)butyl)glycine [Fmoc-NLys(Boc)-OH, 19]
Fmoc-NLys(Boc)-OH (19) was prepared, analogously to to the preparation of 17, from tert-butyl
(
(
5-aminobutyl)carbamate (24.9 g). Crystallisation from EtOAc–heptane afforded building block 19
39.9 g, 43%) as a white solid; t = 6.94 min. (gradient 30–100% B during 10 min). HRMS: calcd for
R
+
1
C H N O [M + Na] 491.21526, found 491.21664; ∆M = 2.8 ppm. H NMR (600 MHz, methanol-d )
4
26
32
2
6
δ
7.80 (m, 2H, ArH-Fmoc), 7.60 (m, 2H, ArH-Fmoc), 7.38 (m, 2H, ArH-Fmoc), 7.31 (m, 2H, ArH-Fmoc),
.46 (m, 2H, CH -Fmoc), 4.22 (m, 1H, CH-Fmoc), 3.90 (m, 2H, N-CH -C=O)), 3.35–3.27 (m, 2H,
4
2
2
CH -CH -N), 3.04, 2.90 (two t, J = 6.8 Hz, 2H, N-CH -CH ), 3.01, 1.19 (two m, 2H, N-CH -CH -CH ),
2
2
2
2
2
2
2
13
1
δ
4
.52, 1.45 (q, J = 7.4 Hz, 1H, CH -CH -CH -N), 1.43 (s, 9H, CH -Boc). C NMR (151 MHz, methanol-d4)
2 2 2 3
171.56, 156.75, 156.53, 143.83, 141.35, 127.44, 126.84, 124.70, 119.60, 78.46, 66.85, 48.08, 47.92, 47.82,
7.08, 39.54, 27.40, 26.66, 24.67.
3
.2.4. Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-(5-((tert-
butoxycarbonyl)amino)pentyl)glycine [Fmoc-NhLys(Boc)-OH, 20]
Fmoc-NhLys(Boc)-OH (20) was prepared, analogously to to the preparation of 17, from tert-butyl
(
(
5-aminopentyl)carbamate (5.0 g). Crystallisation from EtOAc–heptane afforded building block 20
5.4 g, 45%) as a white solid; t = 7.27 min. (gradient 30–100% B during 10 min). HRMS: calcd for
R
+
1
C H N O [M + Na] 505.23091, found 505.23220; ∆M = 2.6 ppm. H NMR (600 MHz, CDCl ) δ 7.74
3
27
34
2
6
(
(
m, 2H, ArH-Fmoc), 7.55 (m, 2H, ArH-Fmoc), 7.37 (m, 2H, ArH-Fmoc), 7.29 (m, 2H, ArH-Fmoc), 4.48
m, 2H, CH -Fmoc), 4.21 (m, 1H, CH-Fmoc), 3.93 (m, 2H, N-CH -C(O)), 3.31, 3.04 (two t, J = 7.3 Hz, 2H,
2
2
CH -CH -N), 3.08 (q, J = 8.8, 8.2 Hz, 2H, N-CH -CH ), 1.52 (p, J = 7.5 Hz, 1H, CH -CH -CH -N), 1.46 (s,
2
2
2
2
2
2
2
9
H, CH -Boc), 1.36 (p, J = 7.4 Hz, 2H, N-CH -CH -CH ), 1.30 (p, J = 7.7 Hz, 2H, CH -CH -CH -N), 1.27,
3 2 2 2 2 2 2
13
1
.09 (two m, 2H, CH -CH -CH -CH -CH ). C NMR (151 MHz, CDCl3)
δ
173.69, 156.79, 156.14, 144.05,
2
2
2
2
2
1
41.48, 127.79, 127.20, 124.98, 120.06, 79.54, 67.81, 48.82, 48.66, 47.40, 40.64, 29.71, 28.56, 27.76, 23.89.
3
.2.5. Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-benzylglycine [Fmoc-NPhe-OH, 21]
Fmoc-NPhe-OH (21) was prepared, analogously to the preparation of 17, from benzylamine
(
(
14.0 g). Crystallisation from EtOAc–heptane afforded building block 21 (24.5 g, 48.3%); t = 7.18 min.
R
+
gradient 30–100% B during 10 min) HRMS: calcd for C H NO [M + Na] 410.13628, found 410.13852;
24
21
4

Molecules 2019, 24, 4429
12 of 18
∆
M = 5.5 ppm. 1H NMR (600 MHz, methanol-d4)
δ
7.76 (m, 2H, ArH-Fmoc), 7.62 – 7.48 (m, 2H,
ArH-Ph), 7.37 (m, 2H, ArH-Fmoc), 7.30 (m, 2H, ArH-Fmoc), 7.28–7.24 (m, 2H, ArH-Ph), 7.24–7.15
m, 2H, ArH-Fmoc), 7.00–6.96 (m, 1H, ArH-Ph), 4.51, 4.33 (two s, 2H, N-CH -C=O), 4.51 (m, 2H,
(
2
1
3
ArH-Fmoc), 4.22 (t, J = 6.1 Hz, 1H, CH-Fmoc), 3.87, 3.75 (two s, 2H, CH -Ph). C NMR (151 MHz,
2
methanol-d4)
δ
171.28, 156.83, 143.80, 141.29, 136.84, 128.29, 127.53, 127.43, 127.37, 127.24, 127.13, 126.80,
1
26.79, 124.58, 119.56, 67.51, 50.91, 46.98, 46.85.
3
.2.6. Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-phenethylglycine [Fmoc-NhPhe-OH, 22
]
Phenethylamine (20.8 mL) was dissolved in THF (75 mL). Then Et N (69 mL, 3.0 equiv) was added
3
to the solution. Tert-butyl bromoacetate (32.2 g, 1 equiv) was dissolved in THF (75 mL) and added
dropwise to the solution under stirring. The mixture was kept at rt for 16 h. The mixture was filtered,
the filtrate was concentrated in vacuo, and the residue was redissolved in CH Cl (50 mL). The solution
2
2
was loaded onto a VLC column (height, 7.5 cm; diameter, 12 cm; column material: 15–40
gel, column pretreated with heptane). Gradient elution was carried out with heptane followed by
CH Cl2 MeOH 100:1 and CH Cl2 MeOH 50:10. The appropriate fractions were concentrated in
vacuo to yield the intermediate tert-butyl ester (31.8 g, 82%) as a yellow oil, which was dissolved in
CH Cl (120 mL), and then TFA (280 mL) was added slowly to the solution under stirring. The mixture
µm silica
−
−
2
2
2
2
was kept at rt for 4 h and then concentrated in vacuo. The residue was dissolved in toluene (100 mL)
and concentrated in vacuo. This was repeated twice to give the crude acid (39.76 g) as a yellow solid,
which was dissolved in dioxane (400 mL) and 10% Na CO (800 mL). Then, Fmoc-Cl (72.6 g, 1.7 equiv)
2
3
◦
dissolved in dioxane (400 mL) was added dropwise under stirring at 0 C. The mixture was then kept
at rt for 16 h. The mixture was adjusted to pH 10 by adding 10% Na CO (200 mL) and split into
2
3
four portions (~450 mL). Each portion was diluted with H O (500 mL), washed with Et O (900 mL),
2
2
and filtered. The filtrate was adjusted to pH 1 by adding concentrated HCl (20 mL), and then extracted
with EtOAc (900 mL). The combined organic phases were concentrated in vacuo to yield the crude
product as a solid (55.8 g, 85%) which was dissolved in CH Cl (100 mL) and loaded onto a VLC
2
2
column (height, 11.5 cm; diameter, 12.0 cm; column material: 15–40
with heptane). Gradient elution was carried out with heptane followed by heptane
heptane EtOAc 1:1. The appropriate fractions were concentrated in vacuo to yield building block
(40.6 g, 62%) as a white solid; t = 7.50 min (gradient 30-100% B during 10 min). HRMS: calcd for
µm silica gel, column pretreated
−
EtOAc 4:1 and
−
2
2
R
+
1
C H NO [M + Na] 424.15193, found 424.15548; ∆M = 8.4 ppm. H NMR (600 MHz, methanol-d )
4
2
5
23
3
δ
7.81 – 7.75 (m, 2H, ArH-Fmoc), 7.59 (m, 2H, ArH-Fmoc), 7.38 (m, 2H, ArH-Fmoc), 7.31 (m, 2H,
ArH-Fmoc), 7.23 (m, 2H, ArH-Ph), 7.19–7.13 (m, 2H, ArH-Ph), 6.92–6.87 (m, 1H, ArH-Ph), 4.47 (m,
2
H, CH -Fmoc), 4.18 (dd, J = 6.5, 5.6 Hz, 1H, CH-Fmoc), 3.78 (m, 2H, N-CH -C=O), 3.35 (m, 2H,
2
2
13
N-CH -CH ), 2.62 (m, 2H, CH -CH -Ph). C NMR (151 MHz, methanol-d4)
δ
171.59, 156.53, 143.93,
2
2
2
2
1
41.39, 138.81, 128.49, 128.07, 127.39, 126.81, 125.90, 124.50, 119.59, 66.54, 49.86, 48.65, 47.05, 33.96.
3
.3. General Protocol for Manual Synthesis of Peptidomimetics
Peptidomimetics 10 12 13, and 16 were prepared manually as previously described [38].
4–
,
,
In brief, H-Rink-Amide ChemMatrix resin (PCAS BioMatrix Inc., Saint-Jean-sur-Richelieu, QC,
Canada; loading 0.52 mmol/g, 0.05 mmol) and Teflon vessels (10 mL; fitted with a polypropylene
filter) were used. Coupling conditions used for Fmoc-protected amino acid and peptoid building
0
blocks: 3.0 equiv building block, 3.0 equiv N,N -diisopropylcarbodiimide (DIC), and 3.0 equiv ethyl
(
®
◦
hydroxyimino)cyanoacetate (OxymaPure ) (>1 h under shaking at 40 C; amino acid building
blocks were coupled twice to ensure complete conversion of resin-bound peptoid secondary amines).
Fmoc deprotection conditions: 20% piperidine in DMF (2
×
10 min, each time with 5 mL under shaking
3 min, each time with 5 mL under
◦
at 40 C). Washing conditions: DMF, MeOH, and CH Cl (3
×
2
2
◦
shaking at 40 C). Capping was applied after loading, and after all couplings with amino acid building
◦
blocks (except the last one): Ac O–DIPEA–NMP 1:2:3 (5 mL for 10 min under shaking at 40 C).
2

Molecules 2019, 24, 4429
13 of 18
Cleavage and side-chain deprotection were performed with TFA–H O–CH Cl (95:2.5:2.5; 2
×
1 h,
2
2
2
each with 5 mL under shaking at rt). The filtrates were collected, and the resin was further eluted with
TFA (2 mL) and CH Cl (2 mL). The combined filtrates were concentrated in vacuo and co-evaporated
2
2
with toluene (3
fractions were concentrated in vacuo and lyophilised. Identity was verified by HRMS, and purity
>97%) was determined by analytical UHPLC.
×
5 mL). The crude product was purified by preparative HPLC, and the appropriate
(
3
.4. General Protocol for Microwave-Assisted Automated Synthesis of Peptidomimetics
Peptidomimetics
Fmoc-based SPPS on a CEM
1
–
3
,
11
™
,
14, and 15 were prepared by automated microwave (MW)-assisted
®
Liberty microwave peptide synthesiser. H-Rink-Amide ChemMatrix
−1
resin (PCAS Bio-Matrix Inc., Saint-Jean-sur-Richelieu, QC, Canada; loading 0.52 mmolg , 0.1 mmol)
was used. Fmoc (9-fluorenylmethyloxycarbonyl) deprotection conditions: excess 20% piperidine
◦
◦
in DMF, initially 75 C (MW) for 30 s, subsequently 75 C (MW) for 180 s. Coupling conditions:
®
◦
5
.0 equiv of building block, 5.0 equiv DIC, and 5.0 equiv OxymaPure , DMF, 75 C (MW) for 15 min.
The resin was transferred to a Teflon vessel fitted with a polypropylene filter by using DMF and
CH Cl . Upon draining, the resin was washed with DMF, MeOH, and CH Cl (3 3 min, each time
×
2
2
2
2
with 5 mL under shaking at rt). Cleavage and side-chain deprotection followed by purification were
performed as described above for manual synthesis of peptidomimetics.
3
.5. Determination of Minimum Inhibitory Concentration
MIC values were evaluated by using the modified Hancock lab protocol [71] in non-binding
5
polystyrene microtitre plates, with bacterial suspensions of
(
∼
2
×
10 CFU/mL in Mueller-Hinton broth
Difco, Sparks MD, USA), with Mg2 and Ca concentrations of 4 mg/L each. The compounds were
+
2+
dissolved in H O and diluted in 0.01% acetic acid, 0.2% BSA (final concentration). Aliquots of 11
µL
2
of 10
×
test compounds were then transferred to the appropriate wells with bacterial suspensions.
For preparation and transfer of the solutions of the compounds, low-binding sterile tubes and tips
◦
◦
(
Axygen, Union City CA, USA) were used. After 20 h incubation of covered plates at 35 C ( 2 C) with
±
circular shaking at 220 rpm, the MICs were read. The antibacterial activity of peptidomimetics was
tested against E. coli ATCC 25922, K. pneumoniae ATCC 13883, P. aeruginosa ATCC 27853, P. aeruginosa
PAO1, A. baumannii ATCC 19606, S. aureus ATCC 29213, and E. faecalis ATCC 29212.
3
.6. Determination of Hemolytic Activity
The lysis of human red blood cells was measured as previously described [15], with modifications.
In brief, freshly drawn human red blood cells (hRBCs) were washed three times with PBS buffer and
centrifuged two times for 5 min at 2500 rpm. A two-fold serial dilution of peptidomimetics in PBS
buffer was added to each well in a sterile polypropylene V-bottom 96-well plate to the total volume of
7
5
µ
L. A 0.5% v/v hRBC suspension (75
µ
L in PBS buffer) was added to each well to reach a final volume
◦
of 150
µL in each well. The plate was incubated (37 C) for 60 min, and then the cells were subsequently
pelleted by centrifugation at 1000 rpm for 5 min. The supernatants (50 µL) were transferred to clear,
flat-bottomed plastic 96-well plates. The concentration of hemoglobin was detected by measuring
the OD at 405 nm. The OD of cells incubated with 0.1% SDS defined 100% hemolysis, while the OD
of cells incubated with PBS buffer defined 0% hemolysis. The concentrations tested were 200, 400,
and 800 µg/mL (only average values for the highest concentration are stated in Table 2).
3
.7. Determination of Antiproliferative Activity on HepG2 Cell Line
Side effects affecting cell viability were estimated on the HepG2 cell line ATCC HB-8065. In brief,
HepG2 cells were seeded in flat-bottomed 96-well plates at a concentration of 5000 cells per well.
◦
At 80
−
90% confluency, the cells were incubated for 24 h in a humidified incubator (5% CO , 37 C).
2
The medium was removed, and the cells were incubated for 48 h in a humidified incubator (5% CO ,
7 C) with compounds in two-fold serial dilutions. An MTT assay was then performed as previously
2
◦
3

Molecules 2019, 24, 4429
14 of 18
described [72]. In brief, after incubation with the test compounds, the culture medium was removed
◦
and fresh medium with 0.2 mg/mL MTT was added in each well of the plate. After incubation (3 h, 37 C,
5
% CO ), the medium with MTT was removed, and 200
µL dimethyl sulfoxide were added at once to
2
each sample. Absorbance of MTT was measured using spectrophotometer TECAN Infinite M1000 at
5
40 nm. The relative viability was calculated by using the formula: OD
×
100/ODcontrol cells.
treated cells
The IC50 values are calculated by using the program Graph Pad Prism® 5.0. For all compounds, the test
range was 10–1280 µg/mL.
4
. Conclusions
In the present work, we explored how the hydrophobicity and activity profiles (i.e., antibacterial
activity versus effect on viability of mammalian cells) of a series of peptide/peptoid hybrid
peptidomimetics depend on the structural features termed “relative side-chain length” and “nature of
the more flexible residue” (i.e., whether the peptoid units are cationic or hydrophobic).
A general trend within each subgroup was that increased cationic side-chain length confers
gradually reduced hydrophobicity, since the amino functionalities become increasingly more accessible
to the surroundings due to a lowered shielding by the aromatic groups of the hydrophobic residues.
This effect was most distinct for analogues displaying the short cationic Dab or NDab residues, as
this feature conferred the highest hydrophobicity within each subgroup. However, extension of
the cationic side-chain length beyond that of lysine resulted in a slightly increased hydrophobicity
in accordance with an expected hydrophobicity enhancement resulting from the incorporation of
additional methylene groups in the cationic side chains, thereby increasing the carbon content relative
to the nitrogen/oxygen content.
The peptidomimetics were tested against a panel of seven bacterial strains. None of the
peptidomimetics displayed significant activity against K. pneumoniae, and only four displayed activity
against A. baumannii, while only a few analogues exhibited moderate to low activity against S. aureus
and E. faecalis, respectively. By contrast, all analogues were active against E. coli, with the two least
hydrophobic peptidomimetics being the least active, indicating the existence of a hydrophobicity
threshold for optimal activity against E. coli. Otherwise, no clear design rules were identified except for
the observation that in two of four subgroups the most hydrophobic member was slightly more potent.
Most analogues were active against P. aeruginosa, and generally a shortening of the cationic side chains
by one methylene group gave rise to a two-fold increased potency. This infers that peptidomimetics
displaying a hydrophobic surface, comprised of the aromatic groups (partially) shielding the shorter
cationic side chains, display more efficient membrane-disruptive interaction with P. aeruginosa.
Testing for hemolytic properties as well as effect on HepG2 cell viability revealed that
peptidomimetics displaying the above-mentioned hydrophobic surface proved to be the most hemolytic
and affect HepG2 cell viability to the highest degree. Expectedly, high hydrophobicity was clearly
correlated to increased effect on HepG2 cell viability, thereby corroborating that for a series of closely
related compounds a threshold for acceptable maximal hydrophobicity may typically be identified.
Consequently, it is important to: (i) ensure that the surface does not become too hydrophobic (i.e.,
considerably shorter cationic side chains as compared to the length of the hydrophobic side chains),
and (ii) keep the overall hydrophobicity below the threshold that confers extensive toxicity toward
mammalian cells regardless of the effects caused by relative side-chain length.
Supplementary Materials: The following are available online, correlations between cationic side-chain length,
hydrophobicity, and MIC or IC , HRMS spectra, HPLC chromatograms for all peptidomimetics and building
50
blocks, and HepG2 cell viability curves for peptidomimetics.
Author Contributions: Conceptualisation, N.F., P.R.H., F.B. and H.F; investigation, N.F.; writing—original
draft, N.F.; writing—review and editing, N.F., P.R.H., F.B. and H.F.; visualisation, N.F., P.R.H., F.B. and H.F;
supervision, H.F.
Funding: Nicki Frederiksen was supported by a PhD grant from the Center for Peptide-Based Antibiotics (CEPAN)
financed by the Novo Nordisk Foundation. NMR equipment used in this work was purchased via grant #10-085264
from the Danish Research Council for Independent Research—Nature and Universe.

Molecules 2019, 24, 4429
15 of 18
Acknowledgments: We would like to thank Birgitte Simonsen and Uraiwan N. Adamsen for practical help with
compound purification and characterisation.
Conflicts of Interest: The authors declare no conflict of interest.
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