Synthetic mimics of antimicrobial peptides from triaryl scaffolds

Article abstract of DOI:10.1021/jm101410t

In this report, we describe the synthesis of a new series of small amphiphilic aromatic compounds that mimic the essential properties of cationic antimicrobial peptides using Suzuki-Miyaura coupling. The new design allowed the easy tuning of the conformational restriction, controlled by introduction of intramolecular hydrogen bonds, and the overall hydrophobicity by modifications to the central ring and the side chains. This approach allowed us to better understand the influence of these features on the antimicrobial activity and selectivity. We found that the overall hydrophobicity had a more significant impact on antimicrobial and hemolytic activity than the conformational stiffness. A novel compound was discovered which has MICs of 0.78 μg/mL against S. Aureus and 6.25 μg/mL against E. Coli, similar to the well-known antimicrobial peptide, MSI-78.

Full text of DOI:10.1021/jm101410t

ARTICLE
pubs.acs.org/jmc
Synthetic Mimics of Antimicrobial Peptides from Triaryl Scaffolds
Hitesh D. Thaker,†,§ Federica Sgolastra,†,§ Dylan Clements,‡ Richard W. Scott,‡ and Gregory N. Tew*,†
†Department of Polymer Science and Engineering, University of Massachusetts, Amherst, Massachusetts 01003, United States
‡PolyMedix Inc., 170 North Radnor-Chester Road, Suite 300, Radnor, Pennsylvania 19087, United States
S Supporting Information
b
ABSTRACT: In this report, we describe the synthesis of a new
series of small amphiphilic aromatic compounds that mimic the
essential properties of cationic antimicrobial peptides using
Suzuki-Miyaura coupling. The new design allowed the easy
tuning of the conformational restriction, controlled by intro-
duction of intramolecular hydrogen bonds, and the overall
hydrophobicity by modifications to the central ring and the side chains. This approach allowed us to better understand the
influence of these features on the antimicrobial activity and selectivity. We found that the overall hydrophobicity had a more
significant impact on antimicrobial and hemolytic activity than the conformational stiffness. A novel compound was discovered
which has MICs of 0.78 μg/mL against S. Aureus and 6.25 μg/mL against E. Coli, similar to the well-known antimicrobial peptide,
MSI-78.
’ INTRODUCTION
bacterial cell membrane, leading to increased permeability and
eventually cell death. Because of the difference in membrane
phospholipid composition, bacterial membranes have been pro-
posed to be more negatively charged than mammalian ones,
and this enables AMPs to be selective toward bacteria. Several
models have been proposed to describe the mechanism of
interaction between AMPs and bacterial membranes, although
the exact mechanism is still not clear.4,8,9 In addition, some AMPs
are also known to kill bacteria by interacting with intracellular
macromolecules.3 Since AMPs target the fundamental feature of
bacteria, unlike conventional antibiotics which have very specific
binding sites, resistance development has proven to be more
difficult.10,11
The discovery and development of antibiotics and antibacter-
ial agents for treatment of bacterial infections were some of the
most profound medical advances of the 20th century. The use of
antibiotics has significantly reduced illness and death caused by
bacterial infection. However, over the past few decades, there has
been an alarming increase in bacterial resistance to even our best
antibiotics. The evolution and spread of these multidrug resistant
bacteria have become a major threat to global health care.1
Consequently, there has been increased interest in identifying
and developing novel compounds that can act as suitable
antibiotics.
Antimicrobial peptides (AMPs) have been investigated as
potential antibiotics because of their broad spectrum activity,
immunomodulatory response, and unique mode of action.2-5
AMPs are found in almost all multicellular organisms and form
the core of the innate immune system. Most AMPs show direct
antimicrobial activity against a variety of bacteria, fungi, protozoa,
and viruses.6 Hundreds of AMPs that exhibit a large variety of
sequences and structures have been isolated and identified.
AMPs can be broadly classified into R-helical and β-sheet
peptides, although other secondary structures like extended coils
or loops are also present.7 Despite their large sequence diversity,
AMPs do share some common structural characteristics. They
are generally short, composed of 12-50 amino acids, with a net
positive charge ranging from þ2 to þ9, mainly because of the
presence of lysine and/or arginine, and have hydrophobic
residues. They generally adopt an amphiphilic structure where
hydrophilic and hydrophobic residues segregate onto opposite
regions, either in the presence of a solvent or upon interaction
with the cell membrane.2,3,8 For many of these cationic AMPs,
the mechanism of action has been suggested to primarily involve
interaction with the negatively charged components of the
AMPs, with all their unique features, appear to be quite
promising as antibacterial drug candidates, but they do have
some disadvantages when considered for clinical use. AMPs
usually have high cytotoxicity, poor tissue distribution and are
susceptible to proteolysis and hydrolysis. The high cost involved
in the synthesis of AMPs is another factor hampering their use as
drug candidates.12-14 This has led several research groups to
focus on the design and synthesis of unnatural backbones that
mimic the structure and activity of AMPs.15,16 A number of
studies, based on this peptidomimetic approach, have reported
synthetic mimics of antimicrobial peptides (SMAMPs) including
peptoids,17 β-peptides,18-20 cyclic peptides,21 synthetic
polymers,22-26 oligo-acyl lysines,27,28 and aromatic oligo-
mers.29-32 The ability to recapitulate AMP activity in SMAMPs
has allowed many of the problems plaguing peptide based drug
development to be overcome such that a SMAMP is in phase I
clinical trials for pan-staph infections.33
Received: October 29, 2010
Published: March 09, 2011
r
2011 American Chemical Society
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Previously, our research group designed a series of aromatic
oligomers based on arylamide,22,30 urea,31 and phenylene-
ethynylene24,34 backbones with broad spectrum antimicrobial
activity and selectivity. The class of arylamide oligomers was
designed de novo using molecular dynamics and utilized hydrogen
bonding to add conformational rigidity to the backbones. Detailed
analysis of these oligomers revealed that replacing the central
benzene ring with pyrimidine further rigidified the conformation
due to intramolecular hydrogen bonding and led to a more
potent structure.30 These oligomers with hydrogen bonding also
displayed enhanced antibacterial activities toward S. aureus and
E. coli.30 The class of phenylene-ethynylene (PE) oligomers, with
strictly hydrocarbon backbone, demonstrated excellent antibac-
terial activity and some selectivity. The PE oligomers had no hydrogen
bonds but still could adopt facially amphiphilic conformations via
rotation around single bonds in the backbone.35 The study of all
these oligomers summarized above has shown that a formal
secondary structure, such as an R-helix, is not critical as long as
there is a correct balance and segregation of hydrophobic and
hydrophilic groups. It also demonstrated that oligomers with and
without restricted conformations could be potent SMAMPs.
When a rigid scaffold is used, the design must lead to the correct
conformer for maximum potency; if a flexible conformation is
employed, many conformers are available but an entropic penalty
is incurred when the SMAMP binds to the membrane.36
This report describes a novel series of aryl oligomers synthe-
sized by Suzuki-Miyaura coupling. The general design principle
of the molecule is shown in Figure 1. This new design is
advantageous because of its synthetic versatility that allows the
facile construction of a library of compounds with different
backbones and side chains. We have altered the central ring
providing a systematic study of intramolecular hydrogen bonding
and thus conformational restriction. We have also explored the
effect of hydrophobicity via modifications of both polar and
nonpolar side chains. The results demonstrated that antimicro-
bial and hemolytic activities of this particular class of SMAMPs
are more responsive to changes in hydrophobicity than con-
formational stiffness.
’ RESULTS
Design. Several amphiphilic aryl oligomers were synthesized
using Suzuki-Miyaura coupling and evaluated to develop a
structure-activity relationship (SAR) of antimicrobial potency.
Initially, the central ring of the backbone was varied to observe
the effect of different degrees of rotational restriction on the
antimicrobial efficiency of those compounds. With this aim, three
series of compounds carrying pyridazine, pyridine, or benzene as
the central ring and β-alanine as the polar side group were built
(Figure 2). We expected the molecule with pyridazine ring (1a-d)
to have the most rigid conformation because of its ability to
lock the conformation via two hydrogen bonds (H-bonds),
compared to the presence of only one H-bond in the pyridine
ring (2a-c) or none in the case of the benzene ring (3a-e).
To evaluate the effect of different nonpolar and polar groups
on both structural and biological properties, the side chains
were varied as well. For the nonpolar groups, two different
substituents were used, i.e., tert-butyl (t-butyl), which is hydrophobic
and electron-donating, and CF3, which is a smaller hydrophobic
group and electron-withdrawing. The molecule without a non-
polar side group 1c was also synthesized and compared to 1a and
1b to explore the effect of having nonpolar side groups in the
molecule.
To test the effect of the spacer length between the aromatic
backbone and the cationic amine, β-alanine and aminovaleric
acid polar side groups containing three and five carbons in the
side chain, respectively, were employed (Figure 3). Compound
3e was synthesized to test the effect of guanidine versus primary
amine, since the guanidinium group is present in many natural
AMPs and has been shown to improve antimicrobial activity of
SMAMPs.29
Synthesis. Scheme 1 shows a general example of the synthesis
of oligomers 1a-c. The biaryl carbon-carbon bond of the
backbone was constructed using modified Suzuki-Miyaura
coupling conditions37 between 3,6-dibromopyridazine and a
4-substituted aniline boronic ester (4a-c), which was prepared
via the borylation of the corresponding commercially available
bromoaniline.38,39 Polar side chains were added by EDC/HOBT
coupling to the oligomers where R is the electron-donating tert-
butyl or H, but this synthetic strategy was not effective for
oligomers with the electron-withdrawing CF3 group because of
its deactivating effect on the amine. For those compounds, the
amide coupling was carried out in moderate yield using POCl3/
pyridine conditions (see Experimental Section). The final pro-
duct of all oligomers was obtained as a salt by deprotection of the
terminal amine Boc functionality using DCM/trifluoroacetic
acid. Oligomers 2a-c and 3a-c were obtained in comparable
yields from 2,5-dibromopyridine and 1,6-dibromobenzene,
Figure 1. Representative scaffold showing design principles.
Figure 2. Aryl oligomers with β-alanine polar side chain and different central rings.
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Figure 3. Aryl oligomers prepared for investigating the effect of different polar side groups.
Scheme 1. Synthetic Pathway for Pyridazine Oligomersa
a (i) Pinacolborane, PdCl2(dppf) CH2Cl2, Et3N, dioxane, 100°C, 3 h; (ii) 3,6-dibromopyridazine, PdCl2(dppf) CH2Cl2, Na2CO3 (aq), DMF, 90°C,
3
3
18 h; (iii) (a, c) Boc-β-Ala-OH, EDC/HOBT, CH2Cl2, room temp, overnight; (b) Boc-β-Ala-OH, POCl3, pyridine, 0°C, 1 h; (iv) TFA/CH2Cl2 (1:3),
room temp, 1 h.
Figure 4. Acetyl derivatives of oligomers used for H-bond investigation.
respectively, using the same synthetic pathway. The synthesis
and characterization of all compounds is reported in detail in the
Experimental Section.
Table 1. Spectroscopic Data of Model Compounds
IRd
1H NMRc
H-Bond Investigation. Conformational analysis of the com-
pounds was performed using acetyl derivatives as models
(Figure 4), assuming that variable polar side chains do not affect
the establishment or strength of the intramolecular H-bonding
and thus the related backbone rigidity. Acetylation was per-
formed via iodine catalysis according to the literature40 (see
Experimental Section for detailed synthesis.)
δN-H in CDCl3 δN-H in DMSO- Δδ(δDMSO)-(δCDCl3) νN-H
(ppm) d6 (ppm) (ppm)
(cm-1
compd
)
6a
11.33
10.40
-0.93
2924
6b 11.88
10.92
-0.96
-0.93
2924
6c 11.68
10.75
12.11,a 9.72b
2922
7b 12.24,a 8.78b
8b 8.52
-0.13,a 0.94b 2917,a 3302b
We evaluated the presence and strength of the intramolecular
H-bond between the nitrogen of the central ring and amide
group involved as the H-donor. It is well-known that H-bonding
9.39
0.87
3259
a N-H involved in H-bond with pyridine ring. b N-H not involved in
H-bond with pyridine ring. c Tetramethylsilane (TMS) was used as the
internal standard for H-bond studies. d In solid state.
1
is typically associated with a downfield shift of the H NMR
signals corresponding to the involved proton and with a shifting
of the IR stretching band of the donor group toward lower
frequencies.41,42 Linear correlations between the NMR and IR
data have been reported in the literature.43,44 Here we compared
information about the presence and strength of H-bonding in
solution. Table 1 summarizes the final shifts observed for the
1
the solvent effect on the H NMR amide signal in different
amide protons in pure solvents. In neat CDCl3, the pyridine
nitrogen acts as a better amide proton acceptor than pyrida-
zine, since the H-bonded amide proton is shifted further
backbones while gradually changing the solvent composition of
diluted samples (∼2.5 mM) from CDCl3 to DMSO-d6 using
tetramethylsilane (TMS) as standard. This analysis provides
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Figure 5. Influence of the solvent composition on the amide proton chemical shift: (a) 6b vs (b) 7b.
downfield (δ = 12.24 ppm in 7ba instead 11.88 ppm in 6b). Both
compounds, however, show the presence of intramolecular
H-bonding when compared to the negative control 8b with
benzene as the central ring (δ = 8.52 ppm). Similar chemical
shifts observed in the cases of compounds 6a-c indicate that the
ring substituent, either electron-withdrawing or electron-donat-
ing, has no significant impact on the H-bond.
The difference in H-bonding strength is more evident when
the ratio DMSO-d6/CDCl3 is increased (Figure 5). Because
DMSO is a H-bonding solvent with its oxygen atom as the
acceptor, its presence leads to competition between intramole-
cular and intermolecular H-bond formations, resulting in a
upfield displacement of the proton chemical shift. However,
compounds with stronger intramolecular H-bonding are known
to be less affected by these changes in solvent composition.45
Upon an increase of the DMSO-d6/CDCl3 ratio, a substantial
upfield shift of the amide proton’s resonance for compounds
H-bonding on the main conformation adopted in solution. No
NOE signal was observed between the amide proton of the side
chain and the aromatic protons of the pyrizadine central ring,
indicating that those intramolecular H-bonds are strong enough
to prevent rotation around the aryl carbon-carbon bonds of the
backbone and therefore lock the structure into the predicted facially
amphiphilic conformation (see Supporting Information Figure S1).
Figure 6 shows the NOESY spectra of compound 7b in DMSO-d6
solvent in which the asymmetry of the molecule allows the differ-
entiation between the two different amide protons (a and b). There
is a NOE signal between b and d protons, indicating that the ring is
involved in free rotation. However, the absence of signal between
a and c protons indicates that this side of the molecule is con-
formationally locked because of the presence of H-bond.
Antimicrobial Activity. All the compounds were tested
against three different pathogenic bacteria: two Gram-negative
(E. coli and K. pneumoniae) and one Gram-positive (S. aureus).
Their antimicrobial activity was quantified in terms of minimum
inhibitory concentration (MIC), i.e., the lowest concentration of
compound that inhibits bacterial growth by more than 90%.
These values were determined according to the Hancock method
for cationic antimicrobial peptides, which is a modification of the
classical microbroth dilution method recommended by the
Clinical and Laboratory Standards Institute (CLSI).47,48 The
results are shown in Tables 2 and 3.
6a-c (Δδ(δ
= -0.96 ppm) was observed, consis-
DMSO)-(δCDCl3
)
tent with a reduction in intramolecular H-bonding (Figure 5a),
whereas the amide proton in 7ba was only slightly sensitive to
the solvent composition (Δδ(δ
= -0.13 ppm). In
DMSO)-(δCDCl3
)
contrast, our negative controls (compound 8b and amide proton
in 7bb without H-bonds) showed downfield shifts of the proton
amide resonance (Figure 5b) due to the change in solvent
1
dielectric constant. The H NMR data are supported by the
differences in the IR values of the N-H stretching frequencies
for the various amide protons (see Supporting Information for IR
data). All these data showed that pyridine can establish a stronger
intramolecular H-bond than the pyridazine ring, in agreement
with studies reported in the literature.46
Two-dimensional NOESY (nuclear Overhauser effect spec-
troscopy) experiments were also performed on the same model
compounds to further investigate the effect of intramolecular
In general, all compounds shown in Table 2 have activity
comparable to that of magainin analogue compound 17 (MSI-78),49
an antimicrobial R-helical peptide with peptide sequence G-I-G-
K-F-L-K-K-A-K-K-F-G-K-A-F-V-K-I-L-K-K-NH2, and show bet-
ter activity against Gram-positive (S. aureus) than Gram-negative
bacteria (E. coli and K. pneumoniae). Among the tert-butyl
containing SMAMPs, 3a, with the benzene central ring, shows
the best activity with MIC values of 3.13 μg/mL for S. aureus and
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Figure 6. Partial NOESY spectra of compound 7b in DMSO-d6 solvent. Only NHb shows a NOE effect with ArHd. Meanwhile there is no signal
between NHa and ArHc, which proves the ability of the intramolecular H-bonding to restrict the rotation around the biaryl bond of the backbone.
Table 2. Biological Activity and Hydrophobicity
Table 3. Biological Activity and Hydrophobicity
MIC (μg/mL)
MIC (μg/mL)
HC50
RTa
b
compd S.aureus E.coli K.pneumoniae HC50 (μg/mL) RT (min) log Kow
compd S. aureus E. coli K. pneumoniae (μg/mL) (min) log Kow
1a
1d
3a
3d
3e
12.5 50
100
25
82.53
nda
39.3
39.9
43.3
44.2
46.3
1.89
3.86
3.95
5.91
4.86
1a
2a
3a
1b
2b
3b
1c
17
12.5
12.5
3.13
25
50
100
25
82.53
39.02
6.46
39.3
38.1
43.3
35.9
37.9
38.9
20.5
1.89
2.76
3.95
0.00
0.86
2.05
-1.93
12.5 25
12.5
6.25
50
3.13 6.25
3.13 6.25
0.78 6.25
12.5
12.5
12.5
6.46
12.5
50
34.46
12.78
190.7
356.3
36.46
>1000
120d
25
50
>100
25
a nd = not determined.
3.13
50
12.5
50
>50
8-16c
8-16c 16-32c
in activity. However, 1d is still not as potent as its benzene
counterpart 3d. Compound 3e, which replaced the primary amine
with guanidines, shows no increase in activity against E. coli but has
maximum potency against S. aureus with an MIC of 0.78 μg/mL.
Hydrophobicity. In order to further examine the impact of
hydrophobicity, we measured the retention time (RT) of the
compounds by HPLC using a C8 column and determined
log Kow by software calculations. ECOSAR software (by the
USA E.P.A.) was used to calculate the log Kow value according
the KOWWIN library. The results are listed in Tables 2 and 3.
The data shows that the central ring influences the overall
hydrophobicity of the molecule, with oligomers containing the
central benzene ring being the most hydrophobic and those with
the pyridazine ring being the least hydrophobic. For instance,
compound 3b (RT = 38.9 min and log Kow = 2.05) is more
hydrophobic than 1b (RT = 35.9 min and log Kow = 0.00). The
major effect on hydrophobicity, however, is due to the presence
of nonpolar side groups. In all the cases, compounds with tert-
butyl side groups are more hydrophobic than their CF3 counter-
parts. However, the type of polar side group (β-alanine vs
aminovaleric acid) does not have a significant impact on the
overall hydrophobicity of the molecule. For example, com-
pounds 1a and 1d have very close retention times, 39.3 and
39.9 min, respectively.
a Measured by HPLC using C8 column with a gradient of 1% acetoni-
trile/min starting with 100% water. b According to KOWWIN’s estima-
tion method. c See ref 49. d See ref 32.
6.25 μg/mL for E. coli. Changing the central ring to pyridine and
pyridazine leads to a decrease in activity. In comparison to
compound 3a, compound 1a shows 8-fold and 4-fold decreases
in activity against E. coli and S. aureus, respectively. A similar trend
is observed with compounds having CF3 as the side group.
The nature of the nonpolar side group seems to impact the
antimicrobial activity of the compounds as well. Within the
pyridazine series, changing the side group from tert-butyl to
CF3 leads to a 2-fold decrease in activity against S. aureus but no
change against E. coli. Compared to compound 2a, compound 2b
with CF3 shows a 2-fold decrease in activity against S. aureus and
4-fold decrease against E. coli. Compound 1c, which does not
have any nonpolar side group, shows poor activity for both S.
aureus and E. coli. The better activity showed by the tert-butyl
series can be attributed to the higher hydrophobicity and
bulkiness of the tert-butyl group.
Since the compounds containing tert-butyl, in general, showed
better activity than their CF3 counterparts, we further expanded
this series by changing the polar side group of SMAMPs 1a and
3a from β-alanine to aminovaleric acid. This led to the two
corresponding new compounds, 1d and 3d, containing two
intramolecular hydrogen bonds and no intramolecular hydrogen
bonds, respectively. This allowed us to evaluate the effect of the
polar side chain’s flexibility alone or in combination with backbone
rigidity on antimicrobial activity (Table 3). The incorporation of
aminovaleric acid slightly improves the activity in the case of the
pyridazine oligomer 1d compared to 1a, but this was not true in
the case of benzene oligomer 3d where no change was observed
Hemolytic Activity. In order to evaluate the cytotoxicity of
these compounds toward mammalian cells, the ability to induce
lysis in human erythrocytes was measured as an HC50 value, i.e.
the lowest concentration that causes the hemolysis of 50% of red
blood cells. The SMAMPs showed hemolytic activity consistent
with the RTs obtained from HPLC, thereby indicating a correla-
tion between HC50 and hydrophobicity. Compound 3a, with the
benzene central ring and tert-butyl side groups (RT = 43.3 min),
is the most hemolytic in the series with HC50 of 6.46 μg/mL,
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whereas compound 1a (RT = 20.5 min) showed no measurable
hemolysis within the given concentration range.
The hydrophobicity of all the compounds was calculated as
the theoretical log Kow value and compared to RT values
measured by HPLC. Kow is the n-octanol/water partition coeffi-
cient which is a common measure of compound hydrophobicity,
and it has been used in various structure-activity studies for
correlating many solute properties.52 The theoretical log Kow
value was calculated to determine if a robust correlation between
the software calculations and experimental HPLC values existed.
This would enable the design of new molecules with optimum
hydrophobicity using only calculations. In general, a linear
correlation between the log Kow and the RT values for com-
pounds was observed in Table 2. However, some deviation was
observed when the polar side chains of the molecules became
more flexible (see Supporting Information Figure S2). For
example, 1a and 1d have similar RT values (39.3 and 39.9 min)
but have a considerable difference in their log Kow values (1.89
and 3.86). At this point, further studies are necessary to establish
the value of correlations between RT and log Kow for this class of
compounds. Therefore, for the present study, we chose to follow
the experimental RT values to associate hydrophobicity with the
activity of these SMAMPs. Plots were made with MIC vs RT for
both S. aureus and E. coli (see Supporting Information Figure S3).
A relatively linear trend between the activity and hydrophobicity
was noticed only in the case of S. aureus.
Previous studies on aryl oligomers have shown that the
antimicrobial and hemolytic activity is a result of a proper balance
of several parameters including charge, amphiphilicity, hydropho-
bicity, etc. The molecules discussed in this paper have antimicrobial
activities comparable to the magainin analogue compound 17.
Improving the selectivity of these compounds would require fine-
tuning one or more of the parameters described above. Additional
investigations are ongoing to evaluate the influence of increasing
the size and number of charges on the trends observed for the
present compounds. This class of molecules provides an easy
synthetic tool to make new antimicrobial agents with all the
advantages of abiotic structures over peptides in terms of stability
and scale-up production cost for drug development.
’ DISCUSSION
The design and synthesis of new peptidomimetics with potential
therapeutic applications have attracted attention in recent years.15,36
Although the exact conformational aspects responsible for the
activity of SMAMPs are not known, all these compounds
resemble the AMPs in terms of their charge and amphiphilicity.
In our current study, we designed a novel scaffold to investigate a
structure-activity relationship between the various structural
and physicochemical parameters (hydrophobicity, conforma-
tional restriction) and antimicrobial activity. The difference
between our scaffold and the previously studied aryl oligomers
is the use of Suzuki-Miyaura coupling for the formation of a
direct carbon-carbon bond between the aryl groups, instead of
amides (in the case of arylamide oligomers),22,29 ureas (in the
case of urea oligomers),31 or triple bonds (in the case of
phenylene-ethynylene oligomers)24,34 between the aryls. In this
series of molecules, three aryl groups were used and the charge
was kept constant at þ2, which has been suggested to be the
minimum requirement for antimicrobial activity.50,51 The effect
of conformational restriction and hydrophobicity was studied by
varying the central ring and side chains, respectively.
Previous studies on the arylamide and arylurea oligomers showed
that conformationally restricted molecules, as a result of intramo-
lecular H-bonding, improved activity and selectivity.30 Additionally,
increased conformational stiffness of the compound led to better
activity in vivo.29 With this background, we decided to evaluate the
effect of changing the number of H-bonds by varying the central
ring from pyridazine to pyridine and then to benzene. 1H NMR and
NOESY studies confirmed that the pyridazine central ring locks the
conformation of the molecule by two intramolecular H-bonds with
the amide hydrogens, whereas the benzene central ring allows free
rotation around the C-C aryl bonds. However, in contrast to our
expectations and previous studies, pyridazine-based oligomers were
less active than the benzene oligomers, while compounds with
pyridine had an intermediate activity. This anomaly can be attrib-
uted to the increase in hydrophobicity of benzene-based com-
pounds compared to pyridazine ones. Also it can be assumed that
the benzene compounds, being more flexible, orient themselves
better at the bacterial membrane leading to increased antibacterial
activity. The comparison between compounds 1a and 3b in Table 2
seems to support the latter hypothesis. These two compounds in
fact have similar hydrophobicity (expressed as both RT and
log Kow), but 1a, with two intramolecular H-bonds, is less active
than 3b (MIC of 12.5 μg/mL vs 3.13 μg/mL against S. aureus). On
the other hand, the increased potency displayed by compounds with
tert-butyl and the fact that the benzene-based oligomers are the
most hydrophobic indicate that in this series of SMAMPs, hydro-
phobicity is more important than conformational stiffness.
’ CONCLUSION
A new series of SMAMPs have been synthesized using
Suzuki-Miyaura coupling in which the backbone and the side
chains were systematically varied to evaluate the impact of
conformational stiffness and overall hydrophobicity on the
antimicrobial and hemolytic activity. The presence of intramo-
lecular H-bonding and its stabilization of the oligomer confor-
mation were demonstrated by NMR and IR spectroscopy, while
hydrophobicity was evaluated by HPLC and software calcula-
tions. The data set obtained for the complete series was
compared with the corresponding antimicrobial and hemolytic
activity trend, expressed as MIC and HC50, respectively, in order
to establish a correlation between all these parameters. Analysis
of the data leads to the conclusion that, for this class of molecules,
the overall hydrophobicity has a more significant impact on the
antimicrobial and hemolytic activity than the conformational
stiffness. This is observed in particular with Gram-positive
bacteria, which are more sensitive to these molecular alterations
than Gram-negative bacteria. However, further investigations are
ongoing to evaluate the importance of molecular size and
number of positive charges, considering that a proper balance
of all these features is essential for the biological activity of these
SMAMPs.
The series of compounds was further extended by modifying
the polar side chains. However, the modification of the polar side
chain from β-alanine to aminovaleric acid did not have a
significant impact on the hydrophobicity of the compound.
Compounds 1a and 1d, as well as 3a and 3d, with similar RT
values, showed the same antimicrobial activity, confirming that
overall hydrophobicity plays a fundamental role in controlling
the activity for this class of compounds. Compound 3e showed
the best antimicrobial activity against S. aureus (MIC of 0.78 μg/
mL), as expected because of the presence of a guanidinium group.
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’ EXPERIMENTAL SECTION
= 2.1 Hz, 1H, ArH), 7.42 (dd, J = 2.1, 8.7 Hz, 1H, ArH), 6.72 (d, J = 8.7
Hz, 1H, ArH), 6.14 (br s, 2H, NH), 1.30 (s, 12H, Me). 13C NMR (75
MHz, CDCl3) δ: 156.25, 134.34 (d, JCF = 3.5 Hz), 129.64 (d, JCF = 3.5
Hz), 125.12 (q, JCF = 268.5 Hz), 118.52 (q, JCF = 32.3 Hz), 114.26,
84.12, 24.99. m/z = 287.1 (calcd), 287.3 (obtained).
Materials. All the chemicals (reagent grade) were purchased from
Aldrich, VWR, Acros, or Fisher and used as received unless otherwise
indicated. Dichloromethane (DCM), pyridine, and triethylamine
(TEA) were distilled over CaH2 under nitrogen prior to use. 1,4-
Dioxane was distilled from sodium/benzophenone. Column chroma-
tography was carried out using a Combiflash-ISCO column machine.
Measurements. 2D-NOESY and 1H and 13C NMR spectra were
obtained at 300 MHz or 75 MHz, using a Bruker DPX-300 NMR
spectrometer. Chemical shifts (δ) are reported in ppm and coupling
constants (J) in Hz. The abbreviations for splitting patterns are the
following: s, singlet; br s, broad singlet; d, doublet; dd, doublet of
doublets; t, triplet; q, quartet; m, multiplet. Mass spectral data including
results from high resolution mass spectrometry (HRMS) were obtained
at the University of Massachusetts, Mass Spectrometry Facility. IR values
were measured using a Perkin-Elmer Spectrum 100. Analytical HPLC
B. General Procedure for Oligomerization (Suzuki Coupling). In a
Schlenk tube, Na2CO3 (8.4 mmol, 10 equiv) dissolved in water
(∼8 mL) was added to a solution of aniline boronic ester (2.1 mmol,
2.5 equiv), dibromoaryl (0.84 mmol,
1 equiv), and PdCl2-
(dppf) CH2Cl2 catalyst (0.04 mmol, 0.05 equiv) in DMF (HPLC
3
grade, 10 mL) at room temperature. The Schlenk tube was degassed
by three freeze-pump-thaw cycles, then purged with nitrogen. The
mixture was stirred at 90 °C for 18 h. The reaction mixture, cooled to
room temperature, was then quenched with water (50 mL) and
extracted with ethyl acetate (50 mL ꢀ 3). The combined organic layers
were washed with a saturated aqueous solution of NaHCO3 (50 mL)
and brine (50 mL), dried over anhydrous Na2SO4, and evaporated under
reduced pressure. The crude product was purified by flash column
chromatography using hexanes/ethyl acetate (80:20) eluent to give pure
compound as a solid. According to this procedure, the following
compounds were synthesized.
ꢀ
was carried out on a Waters system using an Agilent Zorbax SB-C8, 80 Å,
4.6 mm ꢀ 150 mm i.d. (5 μm) column, eluted by water and acetonitrile,
both containing 0.1% of TFA. Detection was by UV detector at 254 nm
wavelength. The elution was performed by gradually increasing the ratio
of acetonitrile in water by 1%/min, starting with 100% water, with a flow
rate of 1 mL/min. The purity of the final compounds as determined by
analytical HPLC was, in general, greater than 95%.
Synthesis and Compound Data. A. General Procedure for
4-Substituted 2-(Pinacolboronic ester)aniline (Borylation). In a
flame-dried Schlenk tube, to a mixture of 4-substituted 2-bromoaniline
(5.81 mmol, 1 equiv) and 1,10-bis(diphenylphosphino)ferrocenepalla-
dium(II) dichloride dichloromethane complex (0.3 mmol, 0.05 equiv)
in dry 1,4-dioxane (10 mL), TEA (23.28 mmol, 4 equiv) and pinacolborane
(17.4 mmol, 3 equiv) were added dropwise under nitrogen atmosphere.
The mixture was heated to 100 °C and stirred at that temperature for 3 h.
The mixture, cooled to room temperature, was then quenched with
saturated NH4Cl solution (10 mL) and extracted with ethyl acetate
(30 mL ꢀ 3). The combined organic layers were washed with brine
(30 mL) and dried over anhydrous Na2SO4. Solvent was evaporated under
reduced pressure and the crude product was purified by flash column
chromatography (hexanes/ethyl acetate, 90:10) to give a white powder.
According to this procedure, the following compounds were synthesized.
Synthesis of 2-[6-(2-Amino-5-tert-butylphenyl)pyridazin-3-yl]-4-
tert-butylaniline (5a). Starting from compound 4a and 3,6-dibromo-
pyridazine, compound 5a was obtained with a yield of 86%. H NMR
(300 MHz, DMSO-d6) δ: 8.15 (s, 2H, ArH), 7.52 (d, J = 2.1 Hz, 2H,
ArH), 7.24 (dd, J = 2.1, 8.5 Hz, 2H, ArH), 6.81 (d, J = 8.5 Hz, 2H, ArH),
6.47 (br s, 4H, NH), 1.29 (s, 18H, t-Bu). 13C NMR (75 MHz, DMSO-
d6) δ: 158.65, 145.20, 138.12, 127.61, 126.04, 125.16, 116.65, 116.46,
33.53, 31.22. m/z = 374.3 (calcd), 375.2 (obtained).
1
Synthesis of 2-[6-(2-Amino-5-tert-butylphenyl)pyridin-3-yl]-4-
tert-butylaniline (9a). Starting from compound 4a and 2,5-dibromo-
pyridine, compound 9a was obtained with a yield of 78%. 1H NMR (300
MHz, DMSO-d6) δ: 8.64 (d, J = 3.0 Hz, 1H, ArH), 7.92 (dd, J = 3.0,
9.0 Hz, 1H, ArH), 7.81 (d, J = 9.0 Hz, 1H, ArH), 7.48 (d, J = 3.0 Hz,
1H, ArH), 7.14 (m, 2H, ArH), 7.05 (d, J = 3.0 Hz, 1H, ArH), 6.73
(dd, J = 3.0, 6.0 Hz, 2H, ArH), 6.29 (br s, 2H, NH), 4.80 (br s, 2H,
NH), 1.28 (s, 9H, t-Bu), 1.25 (s, 9H, t-Bu). 13C NMR (75 MHz,
DMSO-d6) δ: 157.13, 147.34, 144.93, 143.01, 139.00, 137.86, 137.01,
132.69, 126.54, 126.47, 125.53, 125.20, 121.47, 119.91, 116.18, 115.30,
33.43, 33.38, 31.27. m/z = 373.3 (calcd), 374.3 (obtained).
Synthesis of 4-tert-Butyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)aniline (4a). Pure compound was obtained as a white solid with
53% yield. 1H NMR (300 MHz, DMSO-d6) δ: 7.33 (d, J = 2.4 Hz, 1H,
ArH), 7.19 (dd, J = 2.4, 8.6 Hz, 1H, ArH), 6.53 (d, J = 8.6 Hz, 1H, ArH),
5.34 (br s, 2H, NH), 1.28 (s, 12H, Me), 1.19 (s, 9H, t-Bu). 13C NMR (75
MHz, DMSO-d6) δ: 152.43, 136.78, 131.59, 129.97, 114.34, 83.06,
33.33, 31.40, 24.65. m/z = 275.2 (calcd), 275.3 (obtained).
Synthesis of 2-[4-(2-Amino-5-tert-butylphenyl)phenyl]-4-tert-bu-
tylaniline (10a). Starting from compound 4a and 1,6-dibromobenzene,
compound 10a was obtained with a yield of 88%. 1H NMR (300 MHz,
DMSO-d6) δ: 7.47 (s, 4H, ArH), 7.09 (dd, J = 2.1, 8.4 Hz, 2H, ArH),
7.02 (d, J = 2.1 Hz, 2H, ArH), 6.70 (d, J = 8.4 Hz, 2H, ArH), 4.68 (br s,
Synthesis of 2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-
4-(trifluoromethyl)aniline (4b). Pure compound was obtained as a
white solid with 49% yield. 1H NMR (300 MHz, DMSO-d6) δ: 7.58 (d, J
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4H, NH), 1.24 (s, 18H, t-Bu). 13C NMR (75 MHz, DMSO-d6) δ:
142.48, 138.77, 138.30, 128.86, 126.36, 124.89, 124.86, 114.99, 33.39,
31.36. m/z = 372.3 (calcd), 372.3 (obtained).
The combined organic layer was washed with saturated aqueous solution
of NaHCO3 and brine, dried over anhydrous Na2SO4, and concentrated.
The crude product was purified by flash column chromatography and
eluted with hexanes/ethyl acetate (60:40) to give pure compound as a solid.
According to this procedure, the following compounds were synthesized.
Synthesis of 2-{6-[2-Amino-5-(trifluoromethyl)phenyl]pyridazin-
3-yl}-4-(trifluoromethyl)aniline (5b). Starting from compound 4b and
3,6-dibromopyridazine, compound 5b was obtained with a yield of 50%.
1H NMR (300 MHz, DMSO-d6) δ: 8.35 (s, 2H, ArH), 7.97 (br s, 2H,
ArH), 7.47 (br s, 6H, ArH þ NH), 7.00 (d, J = 8.7 Hz, 2H, ArH). 13C
NMR (75 MHz, DMSO-d6) δ: 157.92, 151.01, 127.22 (d, JCF = 3.5 Hz),
126.61 (d, JCF = 3.5 Hz), 126.38, 125.14 (q, JCF = 268.6 Hz), 116.74, 115.78
(q, JCF = 32.1 Hz), 115.79. m/z = 398.1 (calcd), 399.1 (obtained).
Synthesis of N-{4-tert-Butyl-2-[6-(5-tert-butyl-2-acetamidophe-
nyl)pyridazin-3-yl]phenyl}acetamide (6a). Starting from compound
5a, compound 6a was obtained with 55% yield. H NMR (300 MHz,
1
DMSO-d6) δ: 10.40 (br s, 2H, NH), 8.08 (s, 2H, ArH), 7.82 (d, J = 8.5
Hz, 2H, ArH), 7.69 (d, J = 2.2 Hz, 2H, ArH), 7.54 (dd, J = 2.2, 8.5 Hz,
2H, ArH), 1.97 (s, 6H, Me), 1.33 (s, 18H, t-Bu). 13C NMR (75 MHz,
DMSO-d6) δ: 168.39, 158.45, 147.29, 133.85, 127.77, 127.13, 126.58,
124.41, 34.32, 31.09, 23.77. m/z = 458.3 (calcd), 459.3 (obtained).
Synthesis of 2-{6-[2-Amino-5-(trifluoromethyl)phenyl]pyri-
din-3-yl}-4-(trifluoromethyl)aniline (9b). Starting from com-
pound 4b and 2,5-dibromopyridine, compound 9b was obtained
with a yield of 76%.1H NMR (300 MHz, DMSO-d6) δ: 8.68 (br s,
1H, ArH), 7.97 (br s, 2H, NH), 7.86 (br s, 1H, ArH), 7.42 (d, J = 8.2
Hz, 2H, ArH), 7.31 (m, 3H, ArH), 6.90 (t, J = 8.2 Hz, 2H, ArH), 5.77
(br s, 2H, NH). 13C NMR (75 MHz, CDCl3) δ: 157.62, 149.76,
147.94, 147.00, 137.72, 131.70, 127.80 (m), 127.05 (m), 126.66 (m),
122.81, 122.08, 120.38 (q, JCF = 32.7 Hz), 120.10, 119.21 (q, JCF = 32.6
Hz), 117.04, 115.41. m/z = 397.1 (calcd), 398.1 (obtained).
Synthesis of N-(2-{6-[2-Acetamido-5-(trifluoromethyl)phen-
yl]pyridazin-3-yl}-4-(trifluoromethyl)phenyl)acetamide (6b). Start-
ing from compound 5b, compound 6b was obtained with 80% yield. 1H
NMR (300 MHz, CDCl3) δ: 11.86 (br s, 2H, NH), 8.8 (d, J = 8.7 Hz,
2H, ArH), 8.15 (s, 2H, ArH), 7.90 (br s, 2H, ArH), 7.76 (d, J = 8.7 Hz,
2H, ArH), 2.25 (s, 6H, Me). 13C NMR (75 MHz, DMSO-d6) δ: 168.96,
157.74, 140.18, 128.49, 127.19 (m), 126.96, 125.91, 124.70 (q, JCF
=
32.4 Hz), 123.86, 122.31, 24.19. m/z = 482.1 (calcd), 482.2 (obtained).
Synthesis of 2-{4-[2-Amino-5-(trifluoromethyl)phenyl]phenyl}-
4-(trifluoromethyl)aniline (10b). Starting from compound 4b and
1,6-dibromobenzene, compound 10b was obtained as a white solid with a
yield of 90%. 1HNMR(300MHz, DMSO-d6) δ: 7.50 (s, 4H, ArH), 7.38 (dd,
J = 1.8, 8.5 Hz, 2H, ArH), 7.27 (d, J = 1.8 Hz, 2H, ArH), 6.88 (d, J = 8.5 Hz,
2H, ArH), 5.61 (br s, 4H, NH). 13C NMR (75 MHz, DMSO-d6) δ: 149.07,
137.21, 129.36, 127.10, 126.75 (d, JCF = 3.8 Hz), 125.39, 124.81, 123.52,
116.32 (q, JCF = 31.7 Hz), 114.64. m/z = 396.1 (calcd), 396.1 (obtained).
Synthesis of N-{2-[6-(2-Acetamidophenyl)pyridazin-3-yl]phe-
nyl}acetamide (6c). Starting from compound 5c, compound 6c was
obtained with 65% yield. 1H NMR (300 MHz, DMSO-d6) δ: 10.75 (br s,
2H, NH), 8.15 (s, 2H, ArH), 8.03 (d, J = 7.7 Hz, 2H, ArH), 7.81 (dd, J =
1.3, 7.7 Hz, 2H, ArH), 7.52 (t, J = 7.1 Hz, 2H, ArH), 7.35 (t, J = 7.1 Hz,
2H, ArH), 2.02 (s, 6H, Me). 13C NMR (75 MHz. DMSO-d6) δ: 168.41,
158.29, 136.56, 130.28, 130.03, 127.84, 127.20, 124.72, 123.94, 23.99.
m/z = 364.1 (calcd), 346.2 (obtained).
Synthesis of 2-[6-(2-Aminophenyl)pyridazin-3-yl]aniline (5c).
Starting from compound 4c (commercially available) and 3,6-dibromopyr-
idazine, compound 5c was obtained with a yield of 92%. 1H NMR (300
MHz, DMSO-d6) δ: 8.17 (s, 2H, ArH), 7.66 (d, J = 7.9 Hz, 2H, ArH), 7.16
(t, J = 7.1 Hz, 2H, ArH), 6.85 (m, 6H, ArH þ NH), 6.67 (t, J = 7.1 Hz, 2H,
ArH). 13C NMR (75 MHz, CDCl3) δ: 159.11, 147.23, 131.03, 129.12,
126.00, 118.37, 117.72, 117.56. m/z = 262.1 (calcd), 263.1 (obtained).
C. General Procedure for N-Acetylation. A large excess of acetyl
chloride was added to a mixture of oligomer and 20% iodine. The
mixture was stirred at room temperature, and the reaction time was
monitored by thin-layer chromatography (TLC). After the completion
of the reaction, iodine was quenched by a saturated aqueous solution of
Na2S2O3 and the product extracted using ethyl acetate (30 mL ꢀ 3).
Synthesis of N-(2-{6-[2-Acetamido-5-(trifluoromethyl)phenyl]-
pyridin-3-yl}-4-(trifluoromethyl)phenyl)acetamide (7b). Starting
from compound 9b, compound 7b was obtained with 51% yield. 1H
NMR (300 MHz, DMSO-d6) δ: 12.11 (br s, 1H, NH), 9.72 (br s, 1H,
NH), 8.86 (d, J = 2.1 Hz, 1H, ArH), 8.57 (d, J = 8.7 Hz, 1H, ArH), 8.18
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(m, 2H, ArH), 8.09 (dd, J = 2.1, 8.4 Hz, 1H, ArH), 7.93 (d, J = 8.7 Hz,
1H, ArH), 7.81 (d þ s, J = 8.4 Hz, 3H, ArH), 2.16 (s, 3H, Me), 1.98 (s,
3H, Me). 13C NMR (75 MHz, DMSO-d6) δ: 169.43, 169.22, 154.99,
148.33, 141.02, 139.74, 138.82, 132.98, 132.16, 127.73, 126.45(m),
123.76, 122.46, 25.22, 23.73. m/z = 481.1 (calcd), 481.2 (obtained).
Synthesis of tert-Butyl N-{2-[(2-{6-[2-(3-{[(tert-Butoxy)carbo-
nyl]amino}propanamido)-5-tert-butylphenyl]pyridin-3-yl}-4-tert-
butylphenyl)carbamoyl]ethyl}carbamate (12a). According to the
procedure described above, using compound 9a as starting oligomer,
1
compound 12a was obtained with 60% yield. H NMR (300 MHz,
DMSO-d6) δ: 11.58 (br s, 1H, NH), 9.46 (br s, 1H, NH), 8.71 (s, 1H,
ArH), 8.16 (d, J = 8.7 Hz, 1H, ArH), 7.95 (s, 2H, ArH), 7.74 (s, 1H, ArH),
7.46 (m, 3H, ArH), 7.40 (s, 1H, ArH), 6.85 (m, 1H, NH), 6.79 (m, 1H,
NH), 3.23 (m, 2H, CH2), 3.12 (m, 2H, CH2), 2.47 (t, J = 7.2 Hz, 2H,
CH2), 2.33 (t, J = 7.2 Hz, 2H, CH2), 1.34 (s, 18H, t-Boc), 1.32 (s, 18H, t-
Bu). 13C NMR (75 MHz, DMSO-d6) δ: 169.76, 168.95, 155.63, 155.36,
148.53, 147.45, 146.03, 137.66, 134.28, 133.41, 132.52, 132.19, 126.89,
126.64, 126.29, 125.71, 125.44, 122.49, 122.08, 77.47, 37.35, 36.51, 36.06,
34.19, 34.10, 31.01, 28.07. m/z = 715.4 (calcd), 716.9 (obtained).
Synthesis of N-(2-{4-[2-Acetamido-5-(trifluoromethyl)pheny-
l]phenyl-4-(trifluoromethyl)phenyl)acetamide (8b). Starting from
compound 10b, compound 8b was obtained with 40% yield. 1H NMR
(300 MHz, DMSO-d6) δ: 9.39 (br s, 2H, NH), 7.93 (d, J = 8.8 Hz, 2H,
ArH), 7.74 (d, J = 8.8 Hz, 2H, ArH), 7.64 (br s, 2H, ArH), 7.57 (s, 4H,
ArH), 1.98 (s, 6H, Me). 13C NMR (75 MHz, DMSO-d6) δ: 169.03,
138.95, 136.83, 135.04, 129.25, 126.75, 126.30, 125.95, 125.36, 124.86,
122.35, 23.32. m/z = 480,1 (calcd), 480.2 (obtained).
D. General Procedure for EDC/HOBT Coupling. In a round-bottom
flask under nitrogen atmosphere, the proper diaminearyl oligomer (0.5
mmol, 1 equiv), Boc-β-alanine (1.5 mmol, 3 equiv), and HOBT (1.5
mmol, 3 equiv) were dissolved in dry DCM (10 mL). The mixture was
cooled to 0 °C, and EDC (1.5 mmol, 3 equiv) was added. The reaction
mixture was stirred at room temperature overnight, then quenched with
water (10 mL) and extracted with ethyl acetate (20 mL ꢀ 3). The
combined organic layer was washed with a saturated aqueous solution of
NaHCO3 (20 mL) and brine (20 mL), dried over anhydrous Na2SO4,
and concentrated. The crude product was purified by flash column
chromatography and eluted with hexanes/ethyl acetate (60:40). Accord-
ing to this procedure, the following compounds were synthesized as solids.
Synthesis of tert-Butyl N-{2-[(2-{4-[2-(3-{[(tert-Butoxy)carbo-
nyl]amino}propanamido)-5-tert-butylphenyl]phenyl}-4-tert-butyl-
phenyl)carbamoyl]ethyl}carbamate (13a). According to the proce-
dure described above, using compound 10a as starting oligomer, compound
13a was obtained with 60% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.25
(br s, 2H, NH), 7.44 (s, 4H, ArH), 7.39 (s, 4H, ArH), 7.30 (s, 2H, ArH),
6.77(m, 2H, NH),3.15(m, 4H, CH2),2.33(t, J= 7.5 Hz, 4H, CH2), 1.34(s,
18H, t-Boc), 1.32 (s, 18H, t-Bu). 13C NMR (75 MHz, DMSO-d6) δ: 170.01,
155.53, 148.40, 138.19, 135.78, 132.28, 128.81, 127.22, 126.75, 124.70, 77.63,
36.71, 36.27, 34.29, 31.22, 28.26. m/z = 714.4 (calcd), 715.9 (obtained).
Synthesis of tert-Butyl N-{2-[(2-{6-[2-(3-{[(tert-Butoxy)carbo-
nyl]amino}propanamido)-5-tert-butylphenyl]pyridazin-3-yl}-4-
tert-butylphenyl)carbamoyl]ethyl}carbamate (11a). According to
the procedure described above, using compound 5a as starting oligomer,
Synthesis of tert-Butyl N-{2-[(2-{6-[2-(3-{[(tert-Butoxy)car-
bonyl]amino}propanamido)phenyl]pyridazin-3-yl}phenyl)carbamo-
yl]ethyl}carbamate (11c). According to the procedure described above,
using compound 5c as starting oligomer, compound 11c was obtained with
40% yield. 1H NMR (300 MHz, DMSO-d6) δ:10.82(brs, 2H, NH), 8.14(s,
2H, ArH), 8.04 (d, J = 7.2 Hz, 2H, ArH), 7.82 (d, J = 6.8 Hz, 2H, ArH), 7.52
(m, 2H, ArH), 7.35 (m, 2H, ArH), 6.83 (br s, 2H, NH), 3.19 (m, 4H, CH2),
2.45 (m, 4H, CH2), 1.32(s, 18H, t-Boc). 13C NMR (75 MHz, DMSO-d6) δ:
169.59, 158.29, 155.52, 136.49, 130.29, 130.04, 127.82, 127.19, 124.79,
124.03, 77.64, 36.99, 36.51, 28.19. m/z = 604.3 (calcd), 605.2 (obtained).
1
compound 11a was obtained with 60% yield. H NMR (300 MHz,
DMSO-d6) δ: 10.46 (br s, 2H, NH), 8.06 (s, 2H, ArH), 7.84 (d, J = 8.4
Hz, 2H, ArH), 7.70 (br s, 2H, ArH), 7.55 (d, J = 8.4 Hz, 2H, ArH), 6.81
(m, 2H, NH), 3.16 (m, 4H, CH2), 2.40 (t, J = 6.9 Hz, 4H, CH2), 1.34 (s,
18H, t-Boc), 1.31 (s, 18 h, t-Bu). 13C NMR (75 MHz, CDCl3) δ: 170.31,
159.34, 156.03, 147.22, 134.69, 128.57, 127.87, 125.78, 122.87, 79.18,
37.75, 36.63, 34.63, 31.38, 28.42. m/z = 716.4 (calcd), 717.9 (obtained).
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Synthesis of tert-Butyl N-{4-[(2-{4-[2-(5-{[(tert-Butoxy)carbo-
nyl]amino}pentanamido)-5-tert-butylphenyl]phenyl}-4-tert-bu-
tylphenyl)carbamoyl]butyl}carbamate (14). By use of the same
procedure, but using N-Boc-5-aminovaleric acid instead of alanine,
compound 14 was synthesized with 60% yield. 1H NMR (300 MHz,
DMSO-d6) δ: 9.15 (s, 2H, NH), 7.44 (s, 4H, ArH), 7.39 (s, 4H, ArH),
7.30 (s, 2H, ArH), 6.80 (br s, 2H, NH), 2.90 (m, 4H, CH2), 2.18 (m,
4H, CH2), 1.50 (m, 4H, CH2), 1.35 (s, 22H, CH2 þ t-Boc), 1.32 (s, 18H, t-
Bu). 13C NMR (75 MHz, DMSO-d6) δ: 171.43, 155.31, 148.00, 137.90,
135.49, 132.14, 128.52, 126.92, 126.43, 124.44, 77.05, 54.67, 34.99, 33.96,
30.90, 28.86, 27.99, 22.19. m/z = 770.5 (calcd), 771.8 (obtained).
140.66, 139.21, 138.43, 132.52, 131.73, 127.13, 126.59, 126.47, 126.01,
125.90, 125.77, 125.68, 124.04, 123.61, 123.10, 122.41, 122.29, 122.07, 77.65,
37.93, 36.57, 36.47, 28.18, 28.13. m/z = 739.3 (calcd), 739.3 (obtained).
E. General Procedure for POCl3 Coupling. In a round-bottom flask
under nitrogen atmosphere, a specific diamine aryl oligomer (0.5 mmol,
1 equiv) and Boc-β-alanine (1.25 mmol, 2.5 equiv) were dissolved in dry
pyridine (5 mL). Once the temperature was cooled to 0 °C, POCl3 (1.25
mmol, 2.5 equiv) was added dropwise. The reaction mixture was stirred
at that temperature for 1 h. Ethyl acetate was added and the organic layer
washed with brine. Pyridine was removed by washing quickly with 1 M
HCl. The organic phase was then washed with a saturated solution of
NaHCO3 and dried over anhydrous Na2SO4. The solvent was removed
under reduced pressure, and the product was purified by flash column
chromatography with hexanes/ethyl acetate (60:40) eluent. According
to this procedure, the following compounds were obtained as solids.
Synthesis of tert-Butyl N-{2-[(2-{4-[2-(3-{[(tert-Butoxy)carbo-
nyl]amino}propanamido)-5-(trifluoromethyl)phenyl]phenyl}-
4-(trifluoromethyl)phenyl)carbamoyl]ethyl}carbamate (13b). Ac-
cording to the procedure described above, using compound 10b as
1
starting oligomer, compound 13b was obtained with 50% yield. H
NMR (300 MHz, DMSO-d6) δ: 9.45 (br s, 2H, NH), 7.93 (d, J = 8.6 Hz,
2H, ArH), 7.74 (d, J = 8.6 Hz, 2H, ArH), 7.63 (br s, 2H, ArH), 7.57 (s,
4H, ArH), 6.83 (m, 2H, NH), 3.17 (m, 4H, CH2), 2.40 (t, J = 6.9 Hz, 4H,
CH2), 1.35 (s, 18H, t-Boc). 13C NMR (75 MHz, DMSO-d6) δ: 170.19,
155.57, 138.79, 136.89, 135.19, 129.29, 126.82, 126.57, 125.99, 124.84,
122.34, 77.65, 36.50, 28.21. m/z = 738.3 (calcd), 739.3 (obtained).
F. General Procedure for Boc Deprotection. t-Boc protected oligomer
(0.16 mmol) was dissolved in dry DCM (1.5 mL), and trifluoroacetic
acid (TFA) was added (0.5 mL). After 1 h the product was precipitated
by a mixture of cold hexane and ethyl ether and filtrated. The pure
product, achieved as a salt with TFA, was dried under vacuum overnight.
The following compounds were obtained in a quantitative yield.
Synthesis of tert-Butyl N-{2-[(2-{6-[2-(3-{[(tert-Butoxy)carbo-
nyl]amino}propanamido)-5-(trifluoromethyl)phenyl]pyridazin-
3-yl}-4-(trifluoromethyl)phenyl)carbamoyl]ethyl}carbamate (11b).
According to the procedure described above, using compound 5b as starting
oligomer, compound 11b was obtained with a purity of about 90%. 1HNMR
(300 MHz, CDCl3) δ: 11.99 (br s, 2H, NH), 8.81 (d, J = 8.7 Hz, 2H, ArH),
8.16 (s, 2H, ArH), 7.92 (s, 2H, ArH), 7.78 (d, J = 8.7 Hz, 2H, ArH), 5.30 (br
s, 2H, NH), 3.52 (m, 4H, CH2), 2.74 (m, 4H, CH2), 1.35 (s, 18H, t-Boc).
Synthesis of Oligomer 1a. 1H NMR (300 MHz, DMSO-d6) δ: 10.58
(br s, 2H, NH), 8.09 (s, 2H, ArH), 7.78 (m, 10H, ArH þ NH), 7.59 (d, J
= 8.4 Hz, 2H, ArH), 3.02 (m, 4H, CH2), 2.63 (t, J = 6.5 Hz, 4H, CH2),
1.35 (s, 18H, t-Bu). 13C NMR (75 MHz, DMSO-d6) δ: 168.50, 158.45,
147.83, 133.29, 128.41, 127.77, 127.21, 126.72, 124.72, 35.02, 34.42,
33.22, 31.14. HRMS m/z = 517.3291 (calcd), 517.3293 (obtained).
Synthesis of tert-Butyl N-{2-[(2-{6-[2-(3-{[(tert-Butoxy)carbo-
nyl]amino}propanamido)-5-(trifluoromethyl)phenyl]pyridin-3-yl}-
4-(trifluoromethyl)phenyl)carbamoyl]ethyl}carbamate (12b). Ac-
cording to the procedure described above, using compound 9b as
1
starting oligomer, compound 12b was obtained with 50% yield. H
Synthesis of Oligomer 2a. 1H NMR (300 MHz, DMSO-d6) δ: 11.44
(br s, 1H, NH), 9.71 (br s, 1H, NH), 8.72 (s, 1H, ArH), 7.99 (m, 3H,
ArH), 7.74 (br s, 7H, ArH þ NH), 7.49 (m, 3H, ArH), 7.41 (s, 1H,
ArH), 3.06 (m, 2H, CH2), 2.98 (m, 2H, CH2), 2.69 (t, J = 6.6 Hz, 2H,
CH2), 2.55 (t, J = 6.9 Hz, 2H, CH2), 1.33 (s, 18H, t-Bu). 13C NMR (75
MHz, DMSO-d6) δ: 168.93, 168.2, 155.72, 148.99, 147.78, 146.76,
137.79, 133.95, 133.57, 132.32, 127.49, 127.07, 126.92, 126.51, 126.06,
NMR (300 MHz, DMSO-d6) δ: 12.30 (br s, 1H, NH), 9.74 (br s, 1H,
NH), 8.85 (br s, 1H, ArH), 8.59 (d, J = 8.7 Hz, 1H, ArH), 8.17 (d, J = 8.3
Hz, 2H, ArH), 8.09 (dd, J = 2.0, 8.3 Hz, 1H, ArH), 7.95 (d, J = 8.3 Hz,
1H, ArH), 7.80 (m, 3H, ArH), 6.91 (m, 1H, NH), 6.84 (m, 1H, NH),
3.27 (m, 2H, CH2), 3.14 (m, 2H, CH2), 2.55 (t, J = 6.8 Hz, 2H, CH2),
2.39 (t, J = 6.8 Hz, 2H, CH2), 1.33 (s, 9H, t-Boc), 1.29 (s, 9H, t-Boc).
13C NMR (75 MHz, DMSO-d6) δ: 170.11, 169.91, 155.54, 154.58, 147.81,
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125.70, 122.86, 119.22, 115.25, 35.15, 34.42, 34.34, 33.87, 32.63, 31.18.
HRMS m/z = 516.3339 (calcd), 516.3327 (obtained).
Synthesis of Oligomer 3b. 1H NMR (300 MHz, DMSO-d6) δ: 9.83
(br s, 2H, NH), 7.85 (m, 10H, ArH þ NH), 7.61 (m, 6H, ArH), 3.05 (m,
4H, CH2), 2.65 (m, 4H, CH2). 13C NMR (75 MHz, DMSO-d6) δ: 169.10,
138.36, 136.83, 135.28, 129.21, 126.87, 126.22, 125.91, 124.89, 122.3, 34.87,
32.78. HRMS m/z = 539.1882 (calcd), 539.1900 (obtained).
Synthesis of Oligomer 3a. 1H NMR (300 MHz, DMSO-d6) δ: 9.53
(br s, 2H, NH), 7.76 (br s, 6H, NH), 7.43 (m, 8H, ArH), 7.32 (s, 2H,
ArH), 3.01 (m, 4H, CH2), 2.56 (t, J = 6.6 Hz, 4H, CH2), 1.33 (s, 18H, t-
Bu). 13C NMR (75 MHz, DMSO-d6) δ: 168.81, 148.59, 137.96, 135.49,
131.66, 128.64, 127.15, 126.58, 124.67, 34.99, 34.16, 32.37, 31.03.
HRMS m/z = 515.3308 (calcd), 515.3289 (obtained).
Synthesis of Oligomer 1c. 1H NMR (300 MHz, DMSO-d6) δ: 10.84
(br s, 2H, NH), 8.13 (s, 2H, ArH), 7.97 (d, J = 7.9 Hz, 2H, ArH), 7.81 (d,
J=6.6Hz,8H,ArHþ NH), 7.55 (dd, J= 6.6, 7.5 Hz, 2H, ArH), 7.38 (t, J=7.5
Hz, 2H, ArH), 3.04 (br s, 4H, CH2),2.66(m, 4H,CH2).13CNMR(75MHz,
DMSO-d6) δ: 168.49, 158.25, 135.94, 130.3, 130.15, 128.1, 127.76, 125.28,
124.47, 34.97, 33.39. HRMS m/z = 405.2039 (calcd), 405.2012 (obtained).
Synthesis of Oligomer 1b. 1H NMR (300 MHz, DMSO-d6) δ: 11.02
(br s, 2H, NH), 8.32 (s, 2H, ArH), 8.29 (d, J = 8.6 Hz, 2H, ArH), 8.11 (br
s, 2H, ArH), 7.96 (d, J = 8.6 Hz, 2H, ArH), 7.77 (br s, 6H, NH), 3.07 (m,
4H, CH2), 2.71 (t, J = 6.6 Hz, 4H, CH2). 13C NMR (75 MHz, DMSO-
d6) δ: 168.98, 157.63, 139.67, 128.33, 127.82, 127.23 (m), 125.87,
125.16 (q, JCF = 32.3 Hz), 124.44, 122.27, 34.79, 33.58. HRMS m/z =
541.1787 (calcd), 541.1768 (obtained).
Synthesis of Oligomer 3d. 1H NMR (300 MHz, DMSO-d6) δ: 9.26
(s, 2H, NH), 7.75 (br s, 6H, NH), 7.46 (s, 4H, ArH), 7.39 (br s, 4H,
ArH), 7.31 (s, 2H, ArH), 2.76 (br s, 4H, CH2), 2.22 (br s, 4H, CH2),
1.56 (br s, 8H, CH2), 1.33 (s, 18H, t-Bu). 13C NMR (75 MHz, DMSO-
d6) δ: 171.24, 148.23, 137.92, 135.61, 132.06, 128.51, 127.07, 126.49,
124.51, 34.59, 34.02, 30.93, 26.40, 21.72. HRMS m/z = 571.4012
(calcd), 571.3987 (obtained).
G. Synthesis of Compound 1d. Compound 1d was obtained using
Fmoc chemistry because of an unexpected instability toward the
common t-Boc deprotection conditions.
Synthesis of Oligomer 2b. 1H NMR (300 MHz, DMSO-d6) δ: 12.11
(s, 1H, NH), 10.02 (s, 1H, NH), 8.83 (s, 1H, ArH), 8.52 (d, J = 8.6 Hz,
1H, ArH), 8.15 (m, 3H, ArH), 7.96 (d, J = 8.6 Hz, 1H, ArH), 7.83 (m,
9H, ArH þ NH), 3.10 (m, 2H, CH2), 3.03 (m, 2H, CH2), 2.80 (t, J = 6.6
Hz, 2H, CH2), 2.62 (t, J = 6.5 Hz, 2H, CH2). 13C NMR (75 MHz,
DMSO-d6) δ: 169.02, 168.84, 154.48, 147.88, 140,08, 138.80, 138.35,
132.49, 131.91, 127.23 (br s), 126.86, 126.61 (br s), 126.25 (br s),
125.74 (br s), 125.48, 125.38, 124.51, 124.19, 123.24, 122.74, 34.89,
34.84, 32.81. HRMS m/z = 540.1834 (calcd), 540.1843 (obtained).
Synthesis of 9H-Fluoren-9-ylmethyl N-{4-[(4-tert-Butyl-2-{6-[5-
tert-butyl-2-(5-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}penta-
namido)phenyl]pyridazin-3-yl}phenyl)carbamoyl]butyl}carbamate
(15). In a round-bottom flask under nitrogen atmosphere, compound 5a
(0.5 g, 1.34 mmol), Fmoc-5-aminopentanoic acid (1.36 g, 4 mmol),
and HOBT (0.54 g, 4 mmol) were dissolved in dry THF (20 mL).
The mixture was cooled to 0 °C, and EDC (0.77 g, 4 mmol) was
added. The reaction mixture was stirred at room temperature over-
night, then quenched with water (10 mL) and extracted with ethyl
acetate (20 mL ꢀ 3). The combined organic layer was washed with a
saturated aqueous solution of NaHCO3 (20 mL) and brine (20 mL),
dried over anhydrous Na2SO4, and concentrated. The crude product
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was filtered over a pad of basic alumina to remove the amino acid in
excess and then purified by flash column chromatography using
hexanes/ethyl acetate (60:40) eluent to obtain compound 15 in 50%
yield. 1H NMR (300 MHz, CDCl3) δ: 11.39 (br s, 2H, NH), 8.45 (d,
J = 8.4 Hz, 2H, ArH), 7.99 (s, 2H, ArH), 7.71 (d, J = 7.5 Hz, 4H,
ArH), 7.54 (m, 8H, FmocH), 7.34 (t, J = 7.5 Hz, 4H, FmocH), 7.25
(m, 4H, FmocH), 5.15 (br s, 2H, NH), 4.31 (d, J = 6.9 Hz, 4H,
FmocH), 4.12 (t, J = 6.9 Hz, 2H, FmocH), 3.19 (m, 4H, CH2), 2.44
(m, 4H, CH2), 1.77 (m, 4H, CH2), 1.57 (m, 4H, CH2), 1.37 (s, 18H,
t-Bu). 13C NMR (75 MHz, CDCl3) δ: 171.43, 159.51, 156.62,
147.12, 144.08, 141.37, 134.99, 128.69, 127.95, 127.73, 127.12,
125.79, 125.18, 122.93, 122.83, 120.03, 66.63, 47.32, 40.74, 37.68,
34.66, 31.32, 29.56, 22.56. m/z = 1016.5 (calcd), 1018.4 (obtained).
The Fmoc group was then removed using literature procedure53 to
obtain 1d.
Synthesis of tert-Butyl N-[(1E)-({4-[(2-{4-[2-(5-{[(1E)-{[(tert-
Butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]-
amino}pentanamido)-5-tert-butylphenyl]phenyl}-4-tert-butylphe-
nyl)carbamoyl]butyl}amino)({[(tert-butoxy)carbonyl]imino})-
methyl]carbamate (16). Compound 3d (1 g, 1.25 mmol) was dissolved
in dry CH2Cl2 under nitrogen atmosphere and N,N-diisopropy-
lethylamine (i.e., DIEA) (1 mL, 5.6 mmol) was added. After few
minutes, N,N0-bis-Boc-1-guanylpyrazole (0.85 g, 2.75 mmol) was
added and the mixture stirred overnight at room temperature. After
addition of ethyl acetate, the solution was washed with an aqueous
solution of 10% KHSO4 and extracted three more times with ethyl
acetate. The organic layers were washed with a saturated aqueous
solution of NaHCO3 and brine, dried over anhydrous Na2SO4, and
concentrated. Compound 16 was purified by flash column chroma-
tography (hexanes/ethyl acetate 60:40) and obtained as a solid with
90% yield. 1H NMR (300 MHz, CDCl3) δ: 11.47 (s, 2H, NH), 8.35
(br s, 2H, NH), 8.03 (d, J = 8.5 Hz, 2H, ArH), 7.49 (s, 4H, ArH), 7.41
(dd, J = 2.1, 8.5 Hz, 2H, ArH), 7.33 (m, 4H, ArHþNH), 3.39 (d, J =
6.9 Hz, 4H, CH2), 2.29 (t, J = 6.9 Hz, 4H, CH2), 1.71 (d, J = 7.0 Hz,
4H, CH2), 1.63 (d, J = 7.0 Hz, 4H, CH2), 1.45 (s, 18H, t-Boc), 1.44
(s, 18H, t-Boc), 1.35 (s, 18H, t-Bu). 13C NMR (75 MHz, DMSO-d6)
δ: 171.58, 171.55, 170.36, 163.15, 155.25, 152.13, 138.17, 132.42,
128.84, 126.68, 124.71, 82.88, 78.07, 66.95, 35.22, 34.19, 31.15,
28.28, 27.97, 27.59. m/z = 1054.7 (calcd), 1055.6 (obtained).
Then the t-Boc group was removed according to procedure F to give
compound 3e as a salt in a quantitative yield.
Synthesis of 5-Amino-N-(2-{6-[2-(5-aminopentanamido)-5-tert-
butylphenyl]pyridazin-3-yl}-4-tert-butylphenyl)pentanamide (1d).
Compound 15 (0.6 g, 0.6 mmol) was dissolved in THF (10 mL) with
1-hexanethiol (0.8 mL, 6 mmol). Then DBU (4.5 μL, 0.03 mmol)
was added dropwise and the mixture allowed to stir for 24 h. After
removal of solvents under reduced pressure, compound 1d was
precipitated from ethyl ether as a white solid (60% yield). 1H NMR
(300 MHz, CD3OD) δ: 8.11 (s, 2H, ArH), 7.87 (d, J = 8.5 Hz, 2H,
ArH), 7.78 (d, J = 2.1 Hz, 2H, ArH), 7.62 (dd, J = 2.1, 8.5 Hz, 2H,
ArH), 2.65 (t, J = 7.1 Hz, 4H, CH2), 2.38 (t, J = 7.2 Hz, 4H, CH2),
1.66 (m, 4H, CH2), 1.51 (m, 4H, CH2), 1.42 (s, 18H, t-Bu). 13C
NMR (75 MHz, MeOD) δ: 174.27, 160.73, 150.09, 134.68, 129.57,
129.48, 128.83, 127.91, 125.80, 41.71, 37.55, 35.55, 32.10, 31.68,
23.82. HRMS m/z = 573.3917 (calcd), 573.3892 (obtained).
H. Synthesis of Compound 3e. Compound 3e was directly derived
from 3d by addition of guanidinium group to the terminal amines,
followed by t-Boc deprotection.
Synthesis of Oligomer 3e. 1H NMR (300 MHz, DMSO-d6) δ: 9.23
(s, 2H, NH), 7.54 (br s, 2H, NH), 7.42 (s, 4H, ArH), 7.37 (d, J = 6.5 Hz,
4H, ArH), 7.28 (s, 2H, ArH), 6.5-7.5 (br s, 6H, NH), 3.06 (d, J = 5.9
Hz, 4H, CH2), 2.19 (m, 4H, CH2), 1.51 (m, 4H, CH2), 1.43 (m, 4H,
CH2), 1.30 (s, 18H, t-Bu). 13C NMR (75 MHz, DMSO-d6) δ: 156.91,
148.69, 138.30, 136.16, 132.40, 128.82, 127.51, 126.84, 124.90, 40.57,
35.06, 34.35, 31.26, 28.13. HRMS m/z = 655.4448 (calcd), 655.4460
(obtained).
Antimicrobial Activity. All biological testing was conducted by
Polymedix, Inc. (Philadelphia, PA) using a modified microbroth dilution
assay recommended by the Clinical and Laboratory Standards Institute
(CLSI) which has been developed for determining in vitro antimicrobial
activities of cationic agents. Modifications were made to minimize loss of
the antimicrobial agent due both to adsorption onto glass or plastic
surfaces and to the precipitation at high concentrations. Bacteria were
grown in Mueller-Hinton broth (MH broth) at 37 °C overnight, and
the bacterial growth was measured by turbidity as optical density at λ =
600 (OD600) using an Eppendorf BioPhotometer. Compounds were
first dissolved in DMSO and Hancock solution (0.01% acetic acid, 0.2%
bovine serum albumin) to make 2-fold dilution stock series and then
diluted 10-fold to cell culture in 96-well plates to be tested in duplicate at
100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.2, 0.1, and 0.05 μg/mL.
Minimal inhibitory concentrations (MICs) were obtained by measuring
cell growth at OD600 after incubation with compounds for 18 h at 37 °C.
Each compound was tested as a di-TFA salt, except for compound 1d
which was tested as diamine, against ATCC bacterial strains (E. coli
25922, S. aureus 27660, and K. pneumonia 13883).
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Hemolytic Activity. HC50 was determined by measuring the
quantity of hemoglobin released from red blood cells (RBCs) after their
lysis. RBCs collected by centrifugation from human whole blood were
diluted in a TBS solution to obtain a 0.22% RBC stock suspension. In a
96-well plate, serial 1:2 dilutions of each compound in water were added
to the RBC solution (final concentrations tested: from 1000 μg/mL to
lower) and the plate was incubated in a shaker at 37 °C for 1 h. After
centrifugation at 3000 rpm for 5 min, 30 μL of supernatant was removed
and added to 100 μL of H2O in a sterile polystyrene 96-well flat bottom
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plate. Hemoglobin concentration in the supernatant was read at OD405
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’ ASSOCIATED CONTENT
S
Supporting Information. Correlation between log Kow
b
and retention time (RT), activity relation between antimicrobial
activity and hydrophobicity, and partial NOESY data of com-
pound 6b. This material is available free of charge via the Internet
at http://pubs.acs.org.
(16) Som, A.; Vemparala, S.; Ivanov, I.; Tew, G. N. Synthetic mimics
of antimicrobial peptides. Biopolymers 2008, 90, 83–93.
(17) Chongsiriwatana, N. P.; Patch, J. A.; Czyzewski, A. M.; Dohm,
M. T.; Ivankin, A.; Gidalevitz, D.; Zuckermann, R. N.; Barron, A. E. Peptoids
that mimic the structure, function, and mechanism of helical antimicrobial
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: 413-577-1612. Fax: 413-545-0082. E-mail: tew@mail.
pse.umass.edu.
Author Contributions
§These authors contributed equally.
(19) Porter, E. A.; Weisblum, B.; Gellman, S. H. Mimicry of host-
defense peptides by unnatural oligomers: antimicrobial beta-peptides.
J. Am. Chem. Soc. 2002, 124, 7324–7330.
’ ACKNOWLEDGMENT
(20) Porter, E. A.; Wang, X.; Lee, H. S.; Weisblum, B.; Gellman, S. H.
Non-haemolytic beta-amino-acid oligomers. Nature 2000, 404, 565.
(21) Dartois, V.; Sanchez-Quesada, J.; Cabezas, E.; Chi, E.; Dubbelde,
C.; Dunn, C.; Granja, J.; Gritzen, C.; Weinberger, D.; Ghadiri, M. R.; Parr,
T. R., Jr. Systemic antibacterial activity of novel synthetic cyclic peptides.
Antimicrob. Agents Chemother. 2005, 49, 3302–3310.
(22) Tew, G. N.; Liu, D.; Chen, B.; Doerksen, R. J.; Kaplan, J.;
Carroll, P. J.; Klein, M. L.; DeGrado, W. F. De novo design of biomimetic
antimicrobial polymers. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5110
5114.
This work was funding with support from NIH (Grants AI-
074866, AI-O82192) and NSF (Grants DMR-0820506, CMMI-
0531171). Dr. Jing Jiang is greatly acknowledged for his helpful
discussions regarding compound synthesis. Semra Colak, Katie
Gibney, and Dr. Abhigyan Som are also acknowledged for their
invaluable comments on early drafts.
’ ABBREVIATIONS USED
(23) Mowery, B. P.; Lee, S. E.; Kissounko, D. A.; Epand, R. F.;
Epand, R. M.; Weisblum, B.; Stahl, S. S.; Gellman, S. H. Mimicry of
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Chem. 2004, 42, 3860–3864.
AMPs, antimicrobial peptides; SMAMPS, synthetic mimics of
antimicrobial peptides; PE, phenylene ethynylene; SAR, struc-
ture-activity relationship; TMS, tetramethylsilane; NOESY, nu-
clear Overhauser effect spectroscopy; MIC, minimum inhibitory
concentration; RT, retention time; TEA, triethylamine; DCM,
dichloromethane; HRMS, high resolution mass spectrometry
(25) Lienkamp, K.; Madkour, A. E.; Musante, A.; Nelson, C. F.;
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with unprecedented selectivity: a molecular construction kit approach.
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