Tailoring the Physicochemical Properties of Antimicrobial Peptides onto a Thiazole-Based γ-Peptide Foldamer

Article abstract of DOI:10.1021/acs.jmedchem.0c00077

Antimicrobial peptides (AMPs) are amphipathic molecules displaying broad-spectrum bactericidal activity, providing opportunities to develop a new generation of antibiotics. However, their use is limited either by poor metabolic stability or by high hemolytic activity. We herein addressed the potential of thiazole-based γ-peptide oligomers named ATCs as tunable scaffolds to design polycationic AMP mimetics. Knowing the side chain distribution along the backbone, we rationally designed facially amphiphilic sequences with bactericidal effect in the micromolar range. Since no hemolytic activity was detected up to 100 μM, this class of compounds has shown the potential for therapeutic development.

Full text of DOI:10.1021/acs.jmedchem.0c00077

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Article
Tailoring the Physicochemical Properties of Antimicrobial
Peptides onto Thiazole-based #-Peptide Foldamer
Clément Bonnel, Baptiste Legrand, Matthieu Simon, Margaux Clavié, Agnès Masnou, Estelle Jumas-
Bilak, Young Kee Kang, Paricia Licznar-Fajardo, Ludovic T. Maillard, and MASURIER Nicolas
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00077 • Publication Date (Web): 13 Aug 2020
Downloaded from pubs.acs.org on August 15, 2020
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Journal of Medicinal Chemistry
Tailoring the Physicochemical Properties of Antimicrobial Peptides onto Thiazole-
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based -Peptide Foldamer
Clément Bonnel,[a]‡† Baptiste Legrand,[a] Matthieu Simon,[a] Margaux Clavié,[a] Agnès
Masnou,[b] Estelle Jumas Bilak,[b] Young Kee Kang,[c] Patricia Licznar-Fajardo,* [b]
Ludovic T. Maillard,*[a] Nicolas Masurier
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[a] Institut des Biomolécules Max Mousseron, UMR 5247, CNRS, UM, ENSCM, UFR
des Sciences Pharmaceutiques et Biologiques, 15 Avenue Charles Flahault, 34093
Montpellier Cedex 5, France.
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[b] Laboratoire HydroSciences Montpellier, UMR 5569, CNRS, UM, IRD, Département
d’Hygiène Hospitalière – CHU Montpellier, France.
[c] Department of Chemistry, Chungbuk National University, Cheongju, Chungbuk
28644, Republic of Korea.
KEYWORDS: Antimicrobial peptide, foldamer, thiazole-based -peptide.
ABSTRACT: Antimicrobial peptides (AMPs) are amphipathic molecules displaying
broad-spectrum bactericidal activity providing opportunities to develop new generation
of antibiotics. However their use is limited either by poor metabolic stability or high
hemolytic activity. We herein addressed the potential of thiazole-based -peptide
oligomers named ATCs as tunable scaffolds to design poly-cationic AMPs mimetics.
Knowing the side chain distribution along the backbone, we rationally designed facially
amphiphilic sequences with bactericidal effect in the micromolar range. Since no
hemolytic activity was detected up to 100 µM, this class of compounds has shown the
potential for therapeutic development.
INTRODUCTION
The rapid expansion of drug-resistant infections is a public health issue of
paramount importance. In this race for effective treatments that circumvent
bacterial resistance, antimicrobial peptides (AMPs) provide opportunities to
develop new generation of antibiotics as they display a broad-spectrum
bactericidal activity.1-8 AMPs are typically 12 to 80 residues in length and span
several secondary structures including -sheet, -helix, extended conformation
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and loop. Most are poly-cationic sequences with a spatial segregation of the
cationic and hydrophobic side chains onto distinct regions or faces of the
molecule. These features are crucial to initiate electrostatic interactions with the
negatively charged components of the bacterial envelope9 and to insert into the
phospholipid bilayer leading to depolarization or disruption of the bacterial cell
wall.10 Although other mechanisms of action have been proposed,11 it is widely
believed that such a simple physicochemical destabilization of the membrane
results in a relatively low propensity to elicit bacterial resistance.12, 13 However,
due to poor metabolic stability and frequent hemolytic activity, most AMPs have
faced some difficulties for practical clinical applications.2, 14
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To overcome these issues, one has proposed to tailor the physicochemical
characteristics of AMPs onto new classes of non-natural polymers.15
Peptidomimetic antimicrobials have first been made using folded amphipathic
β-peptides.16-21 Beside a broad-spectrum bactericidal activity, they offer the
additional advantage of being resistant to proteases. However, as for many
AMPs,22, 23 the first generation compounds have low discrimination for bacterial
versus mammalian cells resulting in toxic side effects such as hemolysis.16
Following these pioneer results, other folded architectures such as peptoids,24
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oligo-ureas,25, 26 arylamides27-29 or arylureas30-32 and phenylene ethylenes33 have
been used to template the facially amphiphilic morphology of natural AMPs. In
particular, Brilacidin (PMX-30063) an arylamide foldamer has to date completed
a Phase 2b clinical trial with positive results in serious skin infections,34
demonstrating the therapeutic potential of AMPs-mimicking antibiotic agents.
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Over the last years, our group described a class of constrained heterocyclic γ-
amino acids built around a thiazole ring, named 4-amino(methyl)-1,3-thiazole-5-
carboxylic acids or ATCs (Fig. 1a),35 which were initially used to template an
amphipathic hairpin peptide with antibacterial activities analogous to
gramicidin S.36 Furthermore, ATC containing oligomers showed high propensity
to adopt defined structures in both organic solvents and water.35, 37, 38 The facial
anisotropy of the edifice enables a precise 3D positioning of the appended
functionalities making ATC γ-peptides suitable for material39 and biomedical
applications.40, 41 In line with our research agenda, we herein addressed the
prospects of ATC oligomers as tunable scaffold to design poly-cationic AMPs
mimetics.
RESULT AND DISCUSSION
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Design of ATC-based AMPs mimetics. ATC oligomers are closed to a C3-
symmetry helix with three residues to achieve a whole turn.35 The helical folding
results from a hydrogen-bonding network of the type (i, i+2). The six substituents
per turn define three distinct faces over the γ-peptide (Fig. 1b). As example, side-
chains at γ-position of residues i and i+3 and the one decorating the thiazole ring
of residue i+1 share the same face. On the basis of this topology, we initially
designed four sequences 1-4 with three to twelve ATC units and +2 to +8 cationic
charges, by repeating the same tripeptide pattern. While two faces distributed
methyl substituents, the third one displayed cationic aminobutyl side chains
(Fig. 1c). We also prepared sequence 5, a scrambled isomer of 4 with the height
cationic charges distributed all around the helix axis (See table S1 for the
sequence). Because chain length and hydrophobic/hydrophilic balance were
recognized as crucial parameters for both selectivity and affinity for bacteria,
sequences 6-9 were considered. With one face sharing i-Bu substituents instead
of methyls, the 12-mer 6 was predicted more hydrophobic than its counterparts
4. In oligomers 7 and 8, the weakly basic aminobutyl groups were changed by
guanidinopropyls. Finally, in a last variation 9 the overall charge was decreased
to +4 while a twelve ATCs length was preserved.
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1
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C9
a)
Ri+1
Ri+2
Ri
O
O
O
S
R
O
S
Ac
NH2
N
N


N
N

H
H
H
H
7
N
S
N
S
N
N
n
8
9
Ti+1
i+1
Ti+2
i+2
Ti
i
T
ATC
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b)
Ti+2
Ti+2
Ri+1
ATCi+2
ATCi+1
Ri+2
Ri+1
Ti+1
Ti
Ri+2
Ri
Ti+1
Ti
ATCi
Ri
N-ter
c)
CH3
CH3
charge
1 n = 1
2 n = 2
3 n = 3
4 n = 4
+2
+4
+6
+8
R1 = T2 = (CH2)4-NH3
T1 = R2 = R3 = T3 = CH3
+
+
NH3
CH3
CH3
+
NH3
5 = scrambled of 4 (see SI)
i-Bu
i-Bu
R1 = T2 = (CH2)4-NH3
+
R2 = T3 = i-Bu
+8
6 n = 4
T1 = R3 = CH3
+
NH3
CH3
CH3
+
NH3
CH
NH2
NH2
C
3 CH3
H
+
R1 = T2 =
(CH2)3
N
+6
+8
7 n = 3
8 n = 4
T1 = R2 = R3 = T3 = CH3
NH
+
NH2
H
HN
NH
CH3
CH3
+
R1
=
(CH )
NH2
N
C
2 3
9 n = 4
+4
HN
T1 = R2 = T2 = R3 = T3 = CH3
NH
HN +
CH3
or
Figure 1. a) Nomenclature, typical H-bonding pattern and schematic view of the position
of the side chains looking down the ATC oligomers (from N-terminus to C-terminus). b)
Helical wheel diagram of canonical ATC -peptides. c) Designed antimicrobial γ-
peptides 1-9.
Syntheses of ATC oligomers. The general route to the N-Fmoc-ATC-OH
monomer units 13 is reported Scheme 1. They were synthesized from the
commercially available N-Fmoc-Ala-OH and N-Fmoc-Lys(Boc)-OH following
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our previously reported methodology.42 The N-Fmoc-amino acids were first
engaged in a cross-Claisen condensation to lead the β-ketoesters whose
bromination of the malonic position was done with NBS and a catalytic amount
of Mg(ClO4)2. The subsequent α-bromo-β-ketoesters 11 were then reacted with
the appropriate thioamides depending on the required side chain at position 2 of
the thiazole ring. Finally, chemoselective deprotection of the dimethylallyl esters
was carried out under Tsuji-Trust conditions to yield the N-Fmoc-ATC-OH 13.
N-Fmoc-ATC-OH 13a-d were previously described.42 The N-Fmoc-ATC-OH
building-blocks 13e-h were prepared as reported in Scheme 2. Isothiovaleramide
was synthesized in two steps starting from isovaleryl chloride in 43% overall
yield. Hantzsch cyclization with 11a led to the fully protected ATC 12e (41%). N-
Boc-5-aminovaleric 14 was reacted with i-butylchloroformate and gaseous
ammonia to quantitatively yield N-Boc-5-aminovaleramide 15, which was
converted to the corresponding thioamide with Lawesson reagent (50%). A two
steps cyclization with either the α-bromo-β-ketoester 11a or 11b and
deprotection of the dimethylallyl ester led to N-Fmoc-ATC-OH 13f-g. Finally
Boc-GABA-OH 19 was considered for the synthesis of the monomer unit 13h
that displayed a di-Boc-Guanidinopropyl side chain. After conversion of Boc-
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GABA-OH into the corresponding thioamide 21, the Boc group was removed
under acidic conditions. The resulting free amine was reacted with the 1,3-Di-
Boc-2-(trifluoromethylsulfonyl) guanidine 25 to yield the thioamide 26, which
was then condensed in basic conditions with the α-bromo-β-ketoesters 11a.
Aromatization of the hydroxythiazoline was obtained by treatment with
trifluoroacetic anhydride (TFAA) DIEA. The N-Fmoc-ATC-OH 13h was finally
obtained after deprotection of the dimethylallylester under Tsuji Trost
conditions.
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Scheme 1. Synthesis of N-Fmoc-ATC-OH and γ-peptides 1-9.
Stepwise elongations of peptides 1-9 were done on Rink amide resin following a
Fmoc/t-Bu SPPS strategy. Couplings were performed at r.t. over night using
DIC/Oxyma Pure as reagent and NMM as base. It was found that decreasing the
time reaction or changing the coupling reagent led to incomplete reactions. The
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N-termini were capped with Ac2O then cleavages from the solid support and
deprotections of the aminobutyl side chains were performed by TFA/TIS/H2O
(95/2.5/2.5). All the sequences were finally purified by reverse-phase HPLC.
Scheme 2. Chemical route to N-Fmoc-ATC-OH 13e-h.
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Circular dichroism. The folding behaviors of oligomers were first assessed by
CD in water (pH 6.0) at 25 µM between 200 and 300 nm. Irrespective of the
peptide, the CD signatures displayed two negative minima at 208 and 220 nm
and a strong positive maximum at 265 nm, which is consistent with a C9-helical
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folding.35 In addition, the molar ellipticity values per residue (73600 deg.cm-2
dmol-1 at 265 nm) were similar to the previously reported value (74000 deg.cm-
2.dmol-1) reported for helical ATC-oligomers in water.
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FT-IR studies. We showed in a recent study that the amide I frequencies provide
unambiguous structural indicators of the H-bond network for helically folded
ATC-containing oligomers.37 Free ATC carbonyls were expected to give amide I
absorption at ν(CO)>1640 cm-1 (1645 cm-1 for ATC-CONH-R and around 1675 cm-
1
for ATC-CONH2) while the bounded ATC C=O should be highly redshifted
around 1620-1625 cm-1. Fig. 2B shows the spectrum of oligomer 3, which is
representative of the entire series (Fig S3 and Table S13). The band at 1618 cm-1
was attributed to the bound ATC C=O. No band was observed around 1645 cm-1,
which confirmed that most of the ATC carbonyls were involved in hydrogen-
bonds as expected for a C9-helical folding. Because oligomers were obtained as
trifluoroacetate salts, strong absorption bands at 1147 cm-1 and 1198 cm-1
corresponding to C-F stretching modes were also observed. The band at 1678 cm-
1
was attributed to the overlay of carbonyl vibrations corresponding to the
trifluoroacetate salts and to the C-terminus ATC-CONH2. Estimations of
contributions of discrete subcomponent absorptions in the amide I region have
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been achieved using curve-fitting approaches with Gaussian functions.
Quantitative analysis revealed that the absorption at 1619 cm-1 and 1678 cm-1
contributes to 55% and 45% of the C=O components respectively that perfectly
settled the C9-helical structure (eleven bound ATC C=O vs one free ATC C=O
and six TFA).
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NMR analyses and structure calculation: We finally explored the folding of the
oligomers by NMR. NMR analyses of 3 and 4 were conducted in H2O/D2O (9:1),
pH 6.5. In the case of oligomers 6-9, NMR analysis could not be conducted in
water because of poor 1H signal dispersion but nearly all backbone resonances of
6-9 could be assigned in CD3OH. As previously observed on ATCs-containing
oligomers,35, 36 all the amide protons signals were strongly downfield. Such a
strong NH deshielding (> 9 ppm) has been recognized as a structural marker
related to the formation of the C9 hydrogen-bonding pattern.37 In addition,
3J(NH,CH) values < 6 Hz (5.3 ± 0.3 Hz) were typical of  values around -60°
(Table S8) as expected for a C9-helix. Further evidences of C9-helical folding came
from the analysis of the 2D-ROESY spectra, which revealed characteristic

medium sequential NH(i)/CH(i-1) and weak CH(i-1)/ CH(i) correlations. In the

case of oligomer 3, many weak NH(i)/CH(i-1), NH(i)/CH(i-1) and CH(i-
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1)/CH(i) NOE connectivities documented as structural markers of the ATC helix
were visible (Fig. 2C and Tables S8).37 For the longer sequence 4, and for
oligomers 6-9 these canonic weak NOEs were also detectable but could not be
unequivocally assigned due to signal overlaps.
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Taking all together, the NMR, FT-IR and CD data were strong evidences of the
facially amphiphilic morphology of the designed -peptides. NOEs were finally
used as restraints for NMR solution structure calculations of 3 using a simulated
annealing protocol with AMBER 16.43 Unambiguous 99 distance restraints were
introduced to generate an ensemble of convergent 15-lowest energy structures in
water (Fig. 2D). The lowest energy structure has been further optimized with
Gaussian 0944 at the SMD M06-2X/6-31G(d) level of theory in water and its
electrostatic potential was calculated at the same level of theory (Supporting
Information). As expected, we obtained a well-defined C9-helix in which all the
cationic lateral chains were distributed along a single face (Fig. 2E, Table S12). By
contrast to the majority of cationic AMPs, which are intrinsically disordered in
solution because of electrostatic repulsion between the side chains, oligomer 3
was folded. The length of the oligomer is 34.7 Å with three helical turns, each
displaying a pitch of 11.5 Å as previously reported.35
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C
A/
D/
E/
80x103
60
1
2
40
ATC9
3
4
5
6
7
8
9
20
0
ATC8
-20
-40
-60
ATC7
200
220
240
260
/ nm
280
300

ATC6
ATC5
B/
12x10-3
1618 cm-1
ATC CO 1-8
55%
TFA C-F
1678 cm-1
TFA CO
ATC CO 9
45%
TFA C-F
10
8
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ATC4
ATC3
6
4
2
0
ATC2
ATC1
2000
1800
1600
1400
1200
1000
-1
Wavenumber (cm )
C/
N
+
NH3
90°
O
O
S
O
O
N
H
N
N
H
N
H
H
N
S
N
N
S
N
S
N
+
NH3
Figure 2. A/ Far-UV CD spectrum of 3 in water (pH 4.3) at 25 µM between 200
and 300 nm. B/ FT-IR spectrum (Black) and deconvolution (Red) of oligomer 3 in
the Amide I in water (pH 4.3) at 5 mM. C/ Typical Inter-residue NOEs pattern
along 3 in water pH 6.5. D/ Superimposition of the 15 lowest energy NMR
solution structures of the oligomer 3 in water. E/ Lowest-energy NMR structure
of compound 3 optimized at the M06-2X/6-31G(d) level of theory in water using
the solvation model based on density (SMD) method and its electrostatic
potential (color scale: blue +0.541e, green 0e, red -0.541e) calculated using the
Merz-Singh-Kollman scheme at the same level of theory in water.
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1
2
3
4
5
6
7
8
9
Relative hydrophobicity of 3-9. Since the hydrophobic/hydrophilic balance is
recognized as a crucial parameter influencing both selectivity and affinity for
bacteria, we examined the relative lipophilicity of the oligomers by RP-HPLC.
The percentage of acetonitrile required for elution of peptide 3-9 from C18
analytical column are listed Table S14. Variations in elution properties of
sequences 3, 4, 7 and 8 were small indicating very similar lipophilic properties.
However, as expected for molecules displaying a much larger hydrophobic
surface resulting from branched lateral chains, HPLC analyses showed that
oligomer 6 was more lipophilic than all the other sequences. Reducing the
overall charge as in compound 9 also led to a similar polarity decrease.
Nevertheless, all the peptides remained highly soluble in water (up to 10 mM,
pH 4.0 to 7.5).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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41
42
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59
60
Antimicrobial activity. Minimal inhibitory concentration (MIC) and Minimal
bactericidal concentration (MBC) values (Table 1) were determined against both
Gram-positive (Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis) and
Gram negative (Escherichia coli, Pseudomonas aeruginosa) bacteria as well as a yeast
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(Candida albicans). Three independent experiments were conducted using
Melittin, the main cytolytic component of the bee venom, as positive control.
Comparison of oligomers 1-4 shows a strong correlation between sequence
length and activity. While shorter oligomers 1 and 2 were little to no active, 9-
mer 3 showed significant bactericidal activity, which is even more marked with
the longest peptide 4. Since the sequences 3 and 4 span a length of 34.7 Å and
46.3 Å that match the membrane thickness ∼35-40 Å, it is likely that
antimicrobial activity results from perpendicular insertion of peptides into the
lipid bilayer as proposed in the barrel-stave and toroidal models.45, 46 The
scrambled 5 peptide remained active on B. subtilis which is the most sensitive
strain regardless of the compound. However it was three to almost twenty-five
times less potent on the other bacteria than the facing sequence 4 demonstrating
that controlling spatial segregation of the cationic chains is important for the
antimicrobial effect. Based on results obtained with 1-4, sequence lengths of 6-9
were fixed at nine and twelve monomer units. As shown in Table 1, the 12-mer 8
displaying guanidine groups was the most potent among the all series and
compared to its parent 4, showed an antimicrobial spectrum extended toward S.
aureus and E. faecalis. Reducing the overall charge (compound 9) increased the
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
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selectivity toward Gram + bacteria while decreasing the activity on Gram - germs
1
2
3
4
5
and C. albicans.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
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45
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Table 1. Antimicrobial and hemolytic activities of ATC-containing oligomers 1-9
Gram +
Gram -
Yeast
Sequenc
e
Lengt
h (n=)
Charg
e
B. subtilis
S. aureus
E. faecalis
E. coli
P. aeruginosa
C. albicans
[b]
HD50
ATCC6633[a ATCC6538[a ATCC29121[a ATCC8739[a ATCC9027[a]
ATCC10231[a
]
]
]
]
]
Melittin
0.8 (1.1)
>150 (>150)
30 (30)
0.9 (1.2)
1.6 (1.6)
3.4 (4.7)
3.1 (3.1)
1.6 (1.6)
>150 (>150)
62.5 (130)
10.4 (10.4)
3.7 (3.7)
50 (62.5)
25 (25)
3
1
2
3
4
5
6
7
8
9
1
2
3
4
4
4
3
4
4
+2
+4
+6
+8
+8
+8
+6
+8
+4
>150 (>150)
>150 (>150)
>150 (>150)
26 (57)
>150 (>150)
>150 (>150)
>150 (>150)
>150 (>150)
>150 (>150)
10.4 (12.5)
100 (>100)
20.8 (25)
>150 (>150)
>150 (>150)
5.2 (7.3)
>150 (>150)
>150 (>150)
42 (83)
> 100
> 100
> 100
> 100
> 100
>100
>100
>100
46
0.8 (0.8)
0.5 (0.5)
0.8 (0.8)
4.2 (6.2)
0.5 (0.5)
0.6 (0.8)
0.5 (0.5)
1.6 (1.6)
3.9 (5.2)
100 (>100)
16.7 (25)
5.2 (8.3)
100 (>100)
14.6 (19)
54 (100)
14.6 (16.5)
0.8 (0.8)
14.6 (16.5)
3.1 (7.8)
9.4 (12.5)
6.2 (6.2)
25 (50)
1.8 (2.3)
4.2 (4.7)
3.1 (8.3)
3.1 (11.5)
23 (30)
> 100 (nd)
[a] Minimal inhibitory concentration (MIC) values in µM for ATC-peptides 1-9. Minimal bactericidal concentration
(MBCs) values in µM are indicated in bracket [b] dose required to lyse 50% of the red blood cells (HD50) are in µM. nd not
determined
Hemolytic activities. We next investigated the susceptibility of human cells to
oligomers 3-9 by monitoring erythrocytes lysis. Interestingly, no hemotoxicity
was detected up to 100 µM for most of the γ-peptides, including the most active
one 8, compared to melittin (HD50=3 µM). Only the 12-mer 9 with an overall
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charge of +4 led to significant hemolysis. This result is consistent with other
reports demonstrating correlations between hydrophobicity and bacteria cell
selectivity.19, 47
1
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3
4
5
6
7
8
9
10
11
12
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SEM experiments: Based on these results, we chose to focus on the most potent
oligomer 8 and evaluated its impact on E. coli morphology by surface electron
microscopy (SEM). In order to define the best incubation period to observed
antimicrobial effects, we conducted time-kill study on E. coli over 2h incubation
at 37°C. The average log reduction of the viable cell count ranged between 0.95
and 4.73 log10 cfu/ml on incubating the bacteria for 2ꢀh at the MIC and 8xMIC.
Significant decrease of the growth rate was observed between 40 to 60 min at 4x
and 8xMIC (Fig. S4). Once again such a fast killing kinetic is consistent with a
mechanism involving membrane destabilization. Thus we decided to perform
TEM experiments after 60 min contact between bacteria cells and compound 8.
The untreated E. coli cells were about 2.0 µm long and displayed a smooth and
intact surface (Fig. 4A-B). After incubation with peptide 8, the bacteria shortened
to as little as 1.3 µm and appeared more compact indicating that the bacteria
were not able to grow to maximum length (Fig. 4C-E). As previously observed
with other helical AMPs,48 the cells exhibited increased region of cellular
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Journal of Medicinal Chemistry
aggregation. Beside these changes of morphology, the cells showed blisters and
protruding vesicles on their surface (Figure 3E). In addition, whereas there were
no fragments for control untreated bacteria, numerous spherical elements whose
diameters were lower than 0.1µm were observed around bacteria, the number of
which increasing with the peptide concentration. Despite some dispersion in
size, such fragments, probable microsomes, were classically reported with other
AMPs associated with membrane permeation and/or depolarization.48-50 Finally,
at supra-MIC numerous lysed cells were observed.
1
2
3
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5
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Vehicle
MIC
8 x MIC
A/
C/
E/
2.00 µm
2.00 µm
2.00 µm
B/
D/
F/
1.00 µm
1.00 µm
1.00 µm
Figure 4. A/, B/ SEM micrographs untreated E. coli on glass platelets. In isotonic
medium the cells are long, intact and evenly shaped. C/ and D/ After 60 min incubation
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with MIC of 8, the cells appear shorter and more compact and showed protruding
vesicles on their corrugated surface. E/ and F/ After 60 min incubation with 8xMIC of 8,
blisters were visible on cell surface leading to numerous spherical elements in the
medium and numerous lysed cells were observed.
1
2
3
4
5
6
7
8
9
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Mechanistic studies: To get more evidences of the mode-of-action of 8, we
performed membrane depolarization experiments on E. coli. The modifications in
ion permeability was studied using 3,3′-Dipropylthiadicarbocyanine iodide
[DiSC3(5)], a voltage-sensitive fluorescent probe. The carbocyanine accumulates
on polarized membranes resulting in self-quenching of fluorescence while upon
membrane depolarization, the dye is released and fluorescence de-quenched.51-53
Melittin was chosen as a positive control and cefotaxim and ampicillin as
negative control. As expected for membrane-active antimicrobial peptides, both
melittin and compound 8 cause a dose-dependent increase of membrane
depolarization at bactericidal concentrations (Figure S5).
Evaluation of resistance development: we finally conducted antibiotic
resistance experiments. Experiments were performed to measure the
susceptibility of E. coli to develop resistance against one conventional antibiotic,
cefotaxim and melittin as well as 8. These experiments were performed by a
serial passage of E. coli to sub-lethal concentrations of the three compounds,
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Journal of Medicinal Chemistry
followed by determination of the MIC values every 24 h period. For cefotaxim
the MIC increased in as little as five exposures, and raised from <0.05 µg/ml to
0.8 μg/ml after the 8th passage. In contrast, no significant change in the MIC was
observed for 8 and melittin over the entire time course of the experiment in
sharp contrast to the conventional antibiotic.
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CONCLUSION
This work illustrates the potential of ATC-based -peptide oligomers as scaffold
for the development of poly-cationic AMPs mimetics. Knowing the side chain
distribution along the backbone, we rationally designed facially amphiphilic
sequences with three to twelve ATC units. By contrast to the majority of cationic
AMPs, which are intrinsically disordered in solution because of electrostatic
repulsion between the side chains, ATC oligomers retain a well-defined
secondary structure without the need for membrane binding or helix promoting
organic solvents. We first explored the relationship existing between the
oligomer size and the antimicrobial activities and identified that a minimum of
nine residues is necessary to gain antibacterial effects. In addition, we
demonstrated that a spatial segregation of the cationic and hydrophobic side
chains is important for the antimicrobial effect. Among the nine peptides
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described, oligomer 8 was the most potent in term of in vitro antimicrobial
activity and cell specificity. Much important is that we didn't detect any
hemolytic activity even at 100 µM, which is crucial for the development of i.v.
antibiotics. While detailed mechanisms of action for 8 cannot be extracted solely
from the data reported here, SEM images were consistent with membrane
alteration mechanisms as expected for poly-cationic amphiphilic molecules.
1
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EXPERIMENTAL SECTION
General procedures. All reagents and solvents were obtained from commercial
sources and used without further purification. Analytical HPLC analyses were
run on an Agilent Technology 1220 Infinity LC equipped with a Chromolith
Speed Rod RP-C18 185 Pm column (50 x 4.6 mm, 5 µm) with a gradient from
100% (H2O/TFA 0.1%) to 100% (CH3CN/TFA 0.1%) in 5 min; flow rate 4 mL/min;
UV detection at 214 nm (conditions B). Retention times are reported as follows:
Rt = (min). LC/MS analyses were recorded on a Quattro micro ESI triple
quadrupole mass spectrometer (Micromass, Manchester, UK) equipped with a
Chromolith Speed Rod RP-C18 185 Pm column (50 x 4.6 mm, 5 µm) and an
Alliance HPLC System (Waters, Milford, USA); gradient from 100%
(H2O/HCO2H 0.1%) to 100% (CH3CN/HCO2H 0.1%) in 3 min; flowrate 3 mL/min;
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Journal of Medicinal Chemistry
UV detection at 214 nm. High-Resolution Mass Spectrometric analyses were
performed with a time-of-flight (TOF) mass spectrometer fitted with an
Electrospray Ionisation source (ESI) in positive ion mode. Intermediates 11 and
N-Fmoc-ATC-OH 13a-d have been synthesized as previously reported.42 All
oligomers 1-9 tested for biological activity showed > 95% purity as assessed by
RP-HPLC (Chromolith Speed Rod RP-C18 185 Pm column 50x4.6 mm, 5 µm;
flow rate: 5.0 mL/min; gradients from 100/0 to 0/100 eluents A/B over 5 min, in
which eluents A=H2O / TFA 0.1% and B=CH3CN / TFA 0.1%; Detection was done
at 214 nm).
1
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isovaleramide: Isovaleryl chloride (1.0 equiv., 16.6 mmol, 2.0 mL) was diluted
with 20 mL of dry THF under an argon atmosphere. Then ammoniac was
bubbled into the solution for 30 min at 0°C and the white turbid mixture was
evaporated to quantitatively yield isovaleramide as a white powder which was
used without further purification. 1H NMR (400 MHz, DMSO-d6) δ 0.83 (d, J = 6.4
Hz, 6H); 1.90-1.93 (m, 3H); 6.72 (s, 1H); 7.21 (s, 1H).
isothiovaleramide: Isovaleramide (1.0 equiv., 16.6 mmol) was dissolved in
dimethoxyethane (DME, 120 mL) and Lawesson’s reagent (0.55 equiv., 9.1 mmol,
3.7 g) was then added in one portion. The mixture was refluxed at 100°C for 2 h
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(HPLC monitoring). Solvent was evaporated and the yellowish residue was
dissolved in a mixture of EtOAc (100 mL) and aqueous saturated NaHCO3
solution (100 mL) which was stirred for 15 min. After transfer in a separating
funnel, the organic layer was washed with brine, dried over MgSO4, filtered and
concentrated under reduced pressure to yield crude product (yellow oil) which
was purified by flash chromatography on silica gel (DCM 100% to DCM/MeOH:
94/6) to yield pure isovaleric thioamide as slight yellow crystals. Yield 43 % (841
1
2
3
4
5
6
7
8
9
10
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60
1
mg). H NMR (400 MHz, DMSO-d6) δ 0.98 (d, J = 6.6 Hz, 6H, H1); 2.23 (m, 1H,
H2); 2.50 (d, 2H, J = 6.9 Hz, H3); 6.90 (br, 1H, NH5); 7.76 (br, 1H, NH5). LC Rt =
1.00 min. LC-MS: (ESI+): m/z 118.2 ([M+H]+) 100%.
1,1-dimethylprop-2-en-1-yl
4-[(1S)-1-(N-Fmoc)-aminoethyl]-2-isobutyl-1,3-
thiazole-5-carboxylate (12e): A solution of 11a (1.0 equiv., 4.5 mmol, 2.25 g) and
isovaleric thioamide (1.6 equiv.) in EtOH was stirred 4 h at 40°C. After
evaporation of the solvent, the crude product was purified by flash
chromatography (Cyclohexane 100% to Cyclohexane/EtOAc: 80/20) to yield pure
1
Fmoc-ATC-Odimethylallyle 12e as a clear oil. Yield 41% (952 mg). H NMR (400
MHz, CDCl3) δ 1.0 (d, J = 7.5 Hz, 6H, iBu CH3); 1.48 (d, J = 6.7 Hz, 3H, CH3); 1.65
(s, 6H, dimethylallyl CH3); 2.11 (m, 1H, iBu CH); 2.82 (d, J = 7.1 Hz, 2H, iBu CH);
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Journal of Medicinal Chemistry
4.23 (t, J = 7.00 Hz, 1H, Fmoc CH); 4.35 (m, 2H, Fmoc CH2); 5.15 (d, J = 10.9 Hz,
1H, CH2=CH); 5.27 (d, J = 17.3 Hz, 1H, CH2=CH) 5.68 (m, 1H, CH-NH); 6.02 (d, J
= 8.7 Hz, 1H, NH); 6.18 (dd, J = 10.9, 17.3 Hz, 1H, CH2=CH); 7.29 (d, J = 7.6 Hz,
1H, Ar); 7.31 (d, J = 7.3 Hz, 1H, Ar); 7.39 (d, J = 7.6 Hz, 1H, Ar); 7.41 (d, J = 7.3 Hz,
1H, Ar); 7.59 (d, J = 4.9 Hz, 1H, Ar); 7.61 (d, J = 7.3 Hz, 1H, Ar); 7.76 (d, J = 7.6 Hz,
1
2
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13
2H, Ar). C NMR (100 MHz, CDCl3) δ 22.3 (CH3); 22.4 (2C, iBu CH3); 26.7 (2C,
dimethylallyl CH3); 29.9 (iBu CH); 42.6 (iBu CH2); 46.7 (NH-CH); 47.4 (Fmoc
CH); 66.8 (Fmoc CH2); 83.1 (dimethylallyl C(CH3)2); 113.5 (CH2=CH); 120.1 (2C,
Ar); 122.7 (Ar); 125.3 (2C, Ar); 127.1 (2C, Ar); 127.7 (2C, Ar); 141.4 (2C, Ar); 142.0
(CH2=CH); 144.2 (Ar); 144.3 (Ar); 155.7 (O-CO-N); 160.5 (COO); 162.5 (Thiaz C4);
174.0 (Thaz C2). LC Rt = 2.85 min. LC-MS: (ESI+): m/z 541.1 ([M+Na]+) 40%, 519.1
([M+H]+) 85%, 451.1 ([M-C5H9+2H]+) 100%. HRMS (ESI+) m/z calcd. for
[C30H35N2O4S]+: [M+H]+ 519.2318, found 519.2318.
4-[(1S)-1-(N-Fmoc)-aminoethyl]-2-isobutyl-1,3-thiazole-5-carboxylatic
acid
(13e): The O-dimethylallylester 12e (1.8 mmol) was dissolved in dry THF (10
mL). Tetrakis (triphenylphosphine)palladium(0) (3 mol %) was then added to the
solution and the orange mixture was stirred for 5 min at r.t.. Then PhSiH3 (1.2
equiv.) was added and the solution was stirred for 30 min (HPLC monitoring).
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The solvent was then evaporated under vacuum to yield Fmoc-ATC-OH 13e as a
brown foam (1.25 g). ). LC Rt = 2.23 min. LC-MS: (ESI+): m/z 451.2 ([M+H]+)
100%.
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N-Boc-aminovaleramide (15): To a solution of 5-(Boc-amino)pentanoic acid 14
(1.0 equiv., 36.8 mmol, 8.0 g) in anhydrous THF at 0°C and under argon
atmosphere were sequentially added IBCF (1.2 equiv.) and triethylamine (1.2
equiv.). The white suspension was stirred for 10 min until complete formation of
the mixed anhydride (HPLC monitoring). Then gaseous ammonia was bubbled
for 30 min at r.t. to yield N-Boc-aminovaleramide 15 as a white powder.
1
Quantitative yield (8.02 g). H NMR (400 MHz, DMSO-d6) δ 1.37 (s, 9H, tBu);
1.42-1.46 (m, 4H, CH2-CH2-CH2-CH2); 2.01 (t, J = 7.2 Hz, 2H, CH2-CO); 2.88 (m,
2H, NH-CH2); 6.68 (s, 1H, CO-NH2); 6.76 (t, 1H, J = 5.4 Hz, NH); 7.21 (s, 1H, CO-
NH2).
N-Boc-aminopentanethioamide (16): 15 (1.0 equiv., 36.8 mmol, 8.02 g) was
dissolved in dry DCM (180 mL). Then Lawesson’s reagent was added in one
portion (0.55 equiv., 20.3 mmol, 8.19 g) and the white turbid mixture was heated
at 32°C overnight. After evaporation under reduced pressure, the yellow oil was
dissolved in a mixture of DCM (200 mL) and aqueous saturated NaHCO3
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Journal of Medicinal Chemistry
solution (200 mL) which was stirred for 15 min. After transfer in a separating
funnel, the organic layer was washed with brine, dried over MgSO4, filtered and
concentrated under reduced pressure to yield crude product (orange oil) which
was purified by chromatography on silica gel (Et2O/Petroleum ether: 5/5 to 9/1)
to yield pure N-Boc-aminopenthanethioamide 16 as white solid. Yield 50 % (4.28
g). 1H NMR (400 MHz, DMSO-d6) δ 1.37 (s, 11H, tBu and CH2); 1.60 (quin, J = 7.5
Hz, 2H, H4 or CH2); 2.44 (t, J = 7.5 Hz, 2H, CH2-CS); 2.89 (m, 2H, NH-CH2); 6.78
(t, J = 5.4 Hz, 1H, NH); 9.11 (s, 1H, CS-NH2); 9.31 (s, 1H, CS-NH2). LC Rt = 1.36
min. LC-MS: (ESI+): m/z 255.1 ([M+Na]+) 90%, 233.1 ([M+H]+) 25%, 133.1 ([M-
Boc+H]+) 100%.
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4-[(1S)-1-(N-Fmoc)-aminoethyl]-2-(4-(Boc-
amino)butyl)-1,3-thiazole-5-carboxylate (12f): A solution of 11a (1.0 equiv., 1.95
mmol, 976 mg) and 16 (2.0 equiv.) in EtOH was stirred 90 min at r.t. then 30 min
at 40°C. After evaporation of the solvent under reduced pressure, the crude was
purified by flash chromatography (Cyclohexane/EtOAc: 90/10 to 60/40) to yield
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pure 12f as a clear oil. Yield 56% (697 mg). H NMR (400 MHz, CDCl3) δ 1.44 (s,
9H, tBu); 1.47 (d, J = 6.7 Hz, 3H, CH3-CHNH); 1.59 (m, 2H, CH2-CH2-NHBoc);
1.63 (s, 3H, dimethylallyl CH3); 1.65 (s, 3H, dimethylallyle CH3); 1.82 (quin, J = 7.6
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Hz, 2H, CH2-CH2-CH2-NHBoc), 2.98 (t, J = 7.6 Hz, 2H, CH2-(CH2)3-NHBoc); 3.16
(brm, 2H, CH2-NHBoc); 4.22 (t, J = 7.1 Hz, 1H, Fmoc CH); 4.35 (d, J = 7.8 Hz 2H,
Fmoc CH2); 4.66 (br, 1H, NH-Boc); 5.15 (d, J = 11.1 Hz, 1H, CH2=CH); 5.26 (d, J =
17.8 Hz, 1H, CH2=CH); 5.67 (m, 1H, CH-NHFmoc); 6.02 (brm, 1H, NH-Fmoc);
6.16 (dd, J = 11.1, 17.8 Hz, 1H, CH2=CH); 7.30 (m, 2H, Ar); 7.39 (t, J = 7.4 Hz, 2H,
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Ar); 7.61 (t, J = 7.7 Hz, 2H, Ar) ; 7.76 (d, J = 7.4 Hz, 2H, Ar). C NMR (100 MHz,
CDCl3) δ 22.1 (CH3-CHNH); 26.7 (2C, dimethylallyl CH3); 27.9 (CH2-CH2-CH2-
NHBoc); 28.6 (3C, tBu); 29.5 (C23); 33.2 (CH2-(CH2)3-NHBoc); 40.2 (CH2-NHBoc);
46.7 (CH2-NHBoc); 47.4 (CH Fmoc); 66.9 (CH2 Fmoc); 79.4 (tBu); 83.2 (CH2=CH-
C); 113.6 (CH2=CH); 120.1 (2C, Ar); 122.8 (S-C-CO); 125.3 (2C, Ar); 127.1 (2C, Ar);
127.8 (2C, Ar); 141.4 (2C, Ar); 142.0 (CH2=CH); 144.1 (Ar); 144.3 (Ar); 155.7
(FmocNH-CO); 156.1 (BocNH-CO); 160.4 (S-C-CO); 162.6 (C-CH(CH3)-NH);
174.5 (S-C-N). LC Rt = 2.62 min. LC-MS: (ESI+): m/z 656.1 ([M+Na]+) 25%, 634.1
([M+H]+) 100%, 578.0 ([M-tBu+2H]+) 30%, 534.1 ([M-Boc+H]+) 80%, 466.0 ([M-Boc-
C5H9+H]+) 55%. HRMS (ESI+) m/z calcd. for [C35H44N3O6S]+: [M+H]+ 634.2949,
found 634.2951.
4-[(1S)-1-(N-Fmoc)-aminoethyl]-2-(4-(Boc-amino)butyl)-1,3-thiazole-5-
carboxylic acid (13f): The O-dimethylallylester 12f (1.10 mmol) was dissolved in
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Journal of Medicinal Chemistry
dry THF (5 mL). Tetrakis (triphenylphosphine)palladium(0) (3 mol %) was then
added to the solution and the orange mixture was stirred for 5 min at r.t.. Then
PhSiH3 (1.2 equiv.) was added and the solution was stirred for 30 min (HPLC
monitoring). The solvent was then evaporated under vacuum to yield Fmoc-
ATC-OH 13f as a brown foam (630 mg). ). LC Rt = 2.12 min. LC-MS: (ESI+): m/z
588.2 ([M+Na]+ 20%, 566.2 ([M+H]+) 30%, 466.2 ([M-Boc+H]+) 100%.
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1,1-dimethylprop-2-en-1-yl 4-[(1S)-1-(N-Fmoc)-amino-3-methylbutyl]-2-(4-(Boc-
amino)butyl)-4-hydroxy-4,5-dihydrothiazole-5-carboxylate (17): 16 (1.1 equiv.,
5.41 mmol, 1.26 g) was dissolved in DME (35 mL) and then KHCO3 (8.0 equiv.,
39.3 mmol, 3.93 g) was added in one portion. The mixture was vigorously stirred
for 30 min at r.t.. 11b was dissolved in 40 mL of DME and added to the mixture
with a dropping funnel at 0°C. The reaction was stirred overnight at r.t.. The
crude was filtered to remove undissolved KHCO3 and the clear filtrate was
evaporated under reduced pressure. The resulting yellow oil was dissolved in
EtOAc (100 mL) then washed with water (1 × 100 mL) and brine (1 × 50 mL),
dried over MgSO4 and filtered. The solvent was evaporated to yield the
corresponding thiazoline 17 as an orange foam. Quantitative yield (3.44 g). LC Rt
= 2.57 min. LC-MS: (ESI+): m/z 694.3 ([M+H]+) 100%.
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1,1-dimethylprop-2-en-1-yl 4-[(1S)-1-(N-Fmoc)-amino-3-methylbutyl]-2-(4-(Boc-
2,2,2-trifluoroacetamido)butyl)-1,3-thiazole-5-carboxylate (18): The thiazoline
17 was dissolved in 50 mL of dry THF at -15°C. Diisoproylethylamine (8.0 equiv.,
39.3 mmol, 6.80 mL) was added and the mixture was stirred for 15 min. Then
trifluoroacetic anhydride (4.0 equiv., 19.6 mmol, 2.74 mL) was added dropwise
(4-5 min). After 10 min of stirring at -15°C the mixture was diluted with DCM
(100 mL) and washed with aqueous saturated NaHCO3 solution (2 × 100 mL),
then with aqueous saturated KHSO4 solution (1 × 100 mL) and brine (1 × 50 mL).
The organic layer was dried over MgSO4, filtered and concentrated under
reduced pressure to yield 4.19 g of -15°C 18 as a red foam.
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1,1-dimethylprop-2-en-1-yl 4-[(1S)-1-(N-Fmoc)-amino-3-methylbutyl]-2-(4-(Boc-
amino)butyl)-1,3-thiazole-5-carboxylate (12g): KHCO3 (2.0 equiv., 9.82 mmol,
983 mg) was added to a solution of 18 dissolved in dry MeOH (50 mL). The
suspension was stirred overnight at r.t. then diluted with DCM (80 mL) and
washed with aqueous saturated NaHCO3 solution (1 × 50 mL) and brine (2 × 70
mL). The organic layer was dried over MgSO4, filtered, concentrated under
reduced pressure and filtered through a Celite pad to yield crude 12g as an
orange foam which was used for the next step without further purification. Yield
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93% (3.09 g). 1H NMR (400 MHz, CDCl3) δ 0.95 (d, J = 6.4 Hz, 3H, CH3-CH-CH3);
0.97 (d, J = 6.4 Hz, 3H, CH3-CH-CH3); 1.44 (s, 9H, tBu); 1.55-1.62 (m, 3H, CH2(iPr)
and CH2-CH2-NHBoc); 1.66 (s, 7H, dimethylallyl CH3 and CH3-CH-CH3); 1.70-
1.83 (m, 3H, CH2(iPr) and CH2-CH2-CH2-NHBoc); 2.96 (t, J = 7.6 Hz, 2H, CH2-
(CH2)3-NHBoc); 3.15 (brm, 2H, CH2-NHBoc); 4.21 (t, J = 7.0 Hz, 1H, CH Fmoc);
4.30 (dd, J = 7.5, 10.5 Hz, 1H, CH2 Fmoc); 4.39 (dd, J = 7.5, 10.5 Hz 1H, CH2 Fmoc);
4.67 (brm, 1H, NHBoc); 5.14 (d, J = 11.0 Hz, 1H, CH2=CH); 5.26 (d, J = 17.5 Hz,
1H, CH2=CH); 5.70 (m, 1H, Fmoc-NH-CH); 5.84 (brm, 1H, Fmoc-NH); 6.19 (dd, J
= 11.0, 17.5 Hz, 1H, CH2=CH); 7.29 (m, 2H, Ar); 7.38 (t, J = 7.3 Hz, 2H, Ar); 7.58 (d,
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J = 7.1 Hz, 1H, Ar); 7.59 (d, J = 5.3 Hz, 1H, Ar) 7.75 (d, J = 7.3 Hz, 2H, Ar). C
NMR (100 MHz, CDCl3) δ 22.3 (CH3-CH-CH3); 23.3 (CH3-CH-CH3); 25.1 (CH3-
CH-CH3); 26.7 (dimethylallyl CH3); 26.8 (dimethylallyl CH3); 27.1 (CH2-CH2-
CH2-NHBoc); 28.5 (3C, tBu); 29.5 (CH2-CH2-NHBoc); 33.2 (CH2-(CH2)3-NHBoc);
40.2 (CH2-NHBoc); 45.2 (CH2(iPr)); 47.4 (CH Fmoc); 49.0 (FmocNH-CH); 66.7
(CH2 Fmoc); 79.4 (tBu); 83.3 (CH2=CH-C); 113.5 (CH2=CH); 120.1 (2C, Ar); 123.4
(S-C-CO); 125.3 (2C, Ar); 127.1 (2C, Ar); 127.7 (2C, Ar); 141.4 (2C, Ar); 142.1
(CH2=CH); 144.1 (Ar); 144.2 (Ar); 155.9 (NH-CO Fmoc); 156.1 (NH-CO Boc); 160.5
(S-C-CO); 162.3 (S-C-C-N); 174.4 (S-C-N). LC Rt = 2.92 min. LC-MS: (ESI+): m/z
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