Expedient synthesis of SMAMPs via click chemistry

Article abstract of DOI:10.1021/ml400155a

A novel series of synthetic mimics of antimicrobial peptides (SMAMPs) containing triazole linkers were assembled using click chemistry. While only moderately active in buffer alone, an increase in antimicrobial activity against Staphylococcus aureus and Escherichia coli was observed when these SMAMPs were administered in the presence of mouse serum. One compound had minimum inhibitory concentrations (MICs) of 0.39 μg/mL and 6.25 μg/mL, respectively, and an HC₅₀ of 693 μg/mL. These values compared favorably to peptide-based antimicrobials. A correlation between the net positive charge and SMAMP antimicrobial activity was observed. The triazole linker, an amide surrogate, was found to provide better antimicrobial activity against both S. aureus and E. coli when compared to other analogues.

Full text of DOI:10.1021/ml400155a

Letter
pubs.acs.org/acsmedchemlett
Expedient Synthesis of SMAMPs via Click Chemistry
Tsung-hao Fu,† Yan Li,† Hitesh D. Thaker,† Richard W. Scott,‡ and Gregory N. Tew*,†
†Polymer Science and Engineering Department, University of Massachusetts, 120 Governors Drive, Amherst, Massachusetts 01003,
United States
‡PolyMedix, Inc., 170 North Radnor-Chester Road, Suite 300, Radnor, Pennsylvania 19087, United States
S
* Supporting Information
ABSTRACT: A novel series of synthetic mimics of antimicrobial peptides (SMAMPs) containing triazole linkers were
assembled using click chemistry. While only moderately active in buffer alone, an increase in antimicrobial activity against
Staphylococcus aureus and Escherichia coli was observed when these SMAMPs were administered in the presence of mouse serum.
One compound had minimum inhibitory concentrations (MICs) of 0.39 μg/mL and 6.25 μg/mL, respectively, and an HC50 of
693 μg/mL. These values compared favorably to peptide-based antimicrobials. A correlation between the net positive charge and
SMAMP antimicrobial activity was observed. The triazole linker, an amide surrogate, was found to provide better antimicrobial
activity against both S. aureus and E. coli when compared to other analogues.
KEYWORDS: Synthetic mimics of antimicrobial peptides, click chemistry, antibiotics, Gram-positive and Gram-negative bacteria,
1,2,3-triazoles
nfections caused by bacteria are an increasing threat to
Ihuman safety and well-being. Every year a large number of
deaths are caused by bacterial infections.1 Traditional anti-
biotics used to treat these bacterial infections, such as β-lactams,
are becoming ineffective as these bacteria develop resistance to
them. While bacteria are developing resistance at an alarming
rate, the development of new antibiotics has slowed
considerably, with many pharmaceutical companies abandoning
their antibiotic research, resulting in fewer and fewer antibiotics
approved by the FDA (Food and Drug Administration).2
Antimicrobial peptides (AMPs) represent a class of antibiotics
currently being investigated as an alternative to traditional
antibiotics.3 They are peptides that show broad-spectrum
antimicrobial activity against various pathogens. Unlike tradi-
tional antibiotics, AMPs do not target bacterial enzymes, which
can mutate easily. Although the exact mechanisms of how
AMPs operate are not completely clear, their ability to
permeabilize the bacterial cell membrane4−7 has been identified
along with several other targets.8,9
Since their initial discovery, hundreds of AMPs have been
catalogued by scientists around the world, and web-based
databases have been established to keep track of the vast
number of AMPs.10,11 Despite this, there has been little success
of developing AMPs into FDA-approved drugs. One of the very
few examples is pexiganan, also known as MSI-78, which
reached phase III clinical trials but was not approved by the
FDA due to the fact that it was no more effective than existing
treatments.12 A major difficulty in AMP drug development is
production.13 AMPs are typically isolated from nature in
minute amounts. Peptide synthesis methodologies are too
expensive for manufacturing AMPs on the scale necessary for
clinical testing. Using genetic engineering to produce AMPs in
yeast or bacteria has proven to be ineffective due to the toxicity
of AMPs toward their expression host.
These disadvantages of AMPs thus motivated the develop-
ment of synthetic mimics of antimicrobial peptides (SMAMPs),
synthetic molecules designed to capture the active structural
features of AMPs, such as amphiphilicity. SMAMPs can exhibit
improved antimicrobial activity and are easier to synthesize
than AMPs due to their simplified structure. Examples of
SMAMPs include peptoids,14 β-peptides,15−17 short linear18,19
and cyclic peptides,20 synthetic polymers,21−24 oligo-acyl
lysines,25,26 ceragenins,27 and aromatic oligomers.28−33 In our
research group, we previously synthesized SMAMPs possessing
triaryl scaffolds linked with urea,29 amide,30 and acetylene34
moieties. Of the three, SMAMPs with amide linkers proved to
be the most active, and one of these has completed a phase II
clinical trial for pan-Staph infections in humans. 1,2,3-Triazoles
are commonly used as bioisosteres of amides35,36 in medicinal
chemistry, and replacing the amide functionality with a triazole
can lead to improved activities.37−39 Therefore, it seemed
reasonable to determine if SMAMPs containing triazole linker
would be more active as well. Additionally, 1,2,3-triazoles are
formed via click chemistry, which is highly reliable,
regiospecific, and functional group tolerant. To the best of
our knowledge, the use of click chemistry to create a SMAMP
scaffold has not been demonstrated before. Hence, we set out
Received: April 23, 2013
Accepted: July 22, 2013
© XXXX American Chemical Society
A
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to synthesize SMAMPs of the general structure 1 (Scheme 1)
Scheme 2. Synthesis of SMAMP 7 via Click Chemistry
and characterize their antimicrobial properties.
Scheme 1. General Synthetic Design of SMAMPs Containing
Triazoles Utilizing Click Chemistry
Previous studies showed that the central aromatic core and
charge density were important parameters in optimizing
SMAMP activity.33 As a result, three groups of SMAMPs
were targeted in this endeavor. The first group (SMAMPs 4−5)
consists of SMAMPs with a central phenyl ring flanked by two
phenyl groups and a net positive charge of two or four (Figure
1). The second group (SMAMPs 6−10) consists of SMAMPs
and Enterococcus faecalis (ATCC 29212), and Gram-negative
Escherichia coli (ATCC 25922), Pseudomonas aeruginosa
(ATCC 10145), and Klebsiella pneumoniae (ATCC 13883).
Their antimicrobial activity was expressed in terms of minimum
inhibitory concentration (MIC). These values were determined
according to Hancock’s method for cationic antimicrobial
peptides, which is a slight modification of the classical
microbroth dilution method recommended by the Clinical
and Laboratory Standards Institute (CLSI), and they were
measured either in the absence or presence of 40% mouse
serum.43,44 The upper limit of mouse serum was 40%, as the
bacteria had difficulty growing when the percentage was
increased further. The results are shown in Table 1. SMAMPs
4−11 possessed a variable spectrum of activity, generally
showing lower MIC values toward S. aureus and E. coli than E.
faecalis, P. aeruginosa, and K. pneumoniae. Interestingly,
SMAMP 10 showed modest MIC values against P. aeruginosa,
which is a difficult bacteria to kill.
The hemolytic activity of SMAMPs 4−11 toward mamma-
lian cells was also evaluated, where the ability to induce lysis in
human erythrocytes was measured as an HC50 value, the lowest
concentration that causes 50% lysis of the red blood cells.
Surprisingly, SMAMPs 4−11 all displayed higher antimicrobial
activity (lower MIC values) toward S. aureus in the presence of
40% mouse serum. This serum effect was less pronounced
toward E. coli but was still observed for the majority of
SMAMPs. This result was unexpected because it is well-known
that antibiotics are generally less effective when administered in
the presence of serum, as the binding of antibiotics to serum
proteins usually deactivates the antibiotic.45 Svenson et al. had
previously studied the binding of cationic peptides to albumin,
and the results indicated that the binding of peptides to
albumin lowers the effective concentration of peptides needed
to combat bacteria.46 Hence, the MIC values of the peptides
Figure 1. Three groups of SMAMPs investigated for their
antimicrobial activity.
with a central naphthyl ring flanked by two phenyl groups and a
net positive charge ranging from two to six. The third group
consists of SMAMP 11, with a central phenanthryl ring flanked
by two phenyl groups and a net positive charge of four.
These SMAMPs were synthesized by reacting two
equivalents of azide with one equivalent of bis-alkyne. A
representative procedure for the synthesis of SMAMP 7 is
depicted in Scheme 2. Starting with dibromide 12, a
Sonogashira reaction40 with trimethylsilylacetylene gave the
bis-trimethylsilylalkyne intermediate in excellent yields. Re-
moval of the trimethylsilyl groups with KF furnished bis-alkyne
13. The azide fragment was prepared by the alkylation of
diphenol 14 with Br(CH2)3NHBoc, followed by the conversion
of the intermediate bromide to azide 15 via procedures
developed by Zhu and Ma.41 Standard 1,3-dipolar cycloaddition
conditions developed by Sharpless et al.,42 using CuSO4 as the
copper source and sodium ascorbate as the reductant, gave 16
in 43% yield. The remainder of the mass balance consisted of
the product in which bis-alkyne 13 reacted with only one
equivalent of azide 15 and additional heating to 50 °C was
required for 13 to react completely with two equivalents of 15.
This difference in reactivity of the two alkyne moieties enabled
the synthesis of asymmetrical SMAMPs such as 9. Finally,
deprotection of 16 with trifluoroacetic acid (TFA) provided
SMAMP 7 as its TFA salt. Further details for the synthesis of all
SMAMPs can be found in the Supporting Information.
SMAMPs 4−11 were tested against five different bacteria
strains: Gram-positive Staphylococcus aureus (ATCC 27660)
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Table 1. Antimicrobial Activities with and without Mouse Serum and Hemolysis of the SMAMPs
MIC (μg/mL)
a
b
c
d
e
f
g
SMAMP
SA
SA+40%MS
EC
EC+40%MS
EF
25
PA
KP
HC50 (μg/mL)
h
4
50
12.5
0.39
12.5
0.39
0.19
0.39
0.19
0.39
>256
25
>50
>50
>50
>50
50
25
6.25
>50
6.25
3.13
25
25
50
>50
>50
>50
50
Nd
5
12.5
125.5
50
>1000
h
6
>50
>50
>50
50
Nd
7
12.5
25
25
693
81
8
25
50
9
12.5
12.5
25
25
50
>50
>50
>50
94
10
25
25
50
12.5
25
828
109
120
11
>50
8
12.5
128
50
i
pexiganan
4
a
b
c
d
S. aureus (ATCC 27660). S. aureus (ATCC 27660) in the presence of 40% mouse serum. E. coli (ATCC 25922). E. coli (ATCC 25922) in the
e
f
g
h
presence of 40% mouse serum. E. faecalis (ATCC 29212). P. aeruginosa (ATCC 10145). K. pneumoniae (ATCC 13883). Not determined due to
low antimicrobial activity. MIC and HC50 of pexiganan were reported previously: Ge et al. Antimicrob. Agents Chemother. 1999, 43, 782−788.
i
increased in the presence of albumin. There are only a limited
number of examples47−49 in which an increase in antimicrobial
activity was observed when an antibiotic was administered in
the presence of serum. The exact reasons for this phenomena
are not completely clear. It has been previously suggested that
changes in the pH has lowered the MIC.50 In addition, the
presence of other antibacterial peptides51 in serum or
unidentified synergistic host factors52 could contribute to the
observed lower MIC. Another explanation is that serum
increases the solubility or internalization of the antibiotic.
Several trends were observed in the antimicrobial activity in
the presence of mouse serum and hemolysis of SMAMPs 4−11.
Regardless of the size of the central ring in SMAMPs 4−11, the
antimicrobial activity increased dramatically when the net
positive charge was increased from two (SMAMPs 4 and 6) to
four (SMAMPs 5 and 7). Further increases in net positive
charge to five or six did not seem to impact the antimicrobial
activity significantly. The size of the aromatic core appeared to
have a proportional effect on hemolytic activity but not on MIC
values. When comparing SMAMPs 5 and 8, both with a net
positive of four, there was approximately a 12-fold increase in
hemolytic activity with the addition of one phenyl moiety in the
aromatic core. The hemolytic activity also varied with the
substitution pattern as the ortho-substituted SMAMP 8 had a
much higher hemolytic activity than its meta-substituted
counterpart, SMAMP 7. The effect of charge and hydro-
phobicity had been previously described by Strøm et al. in the
context of short cationic peptides.53 These peptides required a
minimal charge of +2 for antistaphylococcal activity, whereas
our SMAMPs 4−11 required a minimal charge of +4. The
presence of a larger hydrophobic group gave lower MIC values
in Strøm’s peptides; however in this report increasing the size
of the central aromatic ring did not have a significant effect on
MIC values.
As an effort to showcase the importance of the 1,2,3-triazole
linker used in this study, the activity of SMAMP 7 was
compared to analogues previously synthesized in our laboratory
(Table 2, SMAMPs 17 and 18). When the 1,2,3-triazole linker
Table 2. Effect of the Linker Moiety on the Antimicrobial
and Hemolytic Activities of SMAMPs
a
b
S. aureus (ATCC 27660) in the presence of 40% mouse serum. E.
coli (ATCC 25922) in the presence of 40% mouse serum.
was removed from the scaffold (SMAMP 1733), there was a 4−
8-fold decrease in antimicrobial activity toward S. aureus and a
4-fold decrease toward E. coli in the presence of mouse serum.
Additionally, the hemolytic activity was approximately four
times higher. When the 1,2,3-triazole linker was replaced with
an acetylene moiety (SMAMP 18), again a significant decrease
in antimicrobial activity toward S. aureus in the presence of
mouse serum was observed as well as a lower HC50 value.
Overall, the presence of the 1,2,3-triazole linker enabled
SMAMP 7 to outperform analogues with other linkers in
terms of both antimicrobial and hemolytic activities.
In conclusion, the first example of utilizing click chemistry to
generate a SMAMP scaffold was reported. The size of the
aromatic core and the number and substitution pattern of the
ammonium groups were varied; the effects of these variables on
SMAMP antimicrobial and hemolytic activities were measured.
All SMAMPs had higher antimicrobial activities when
Out of all of these SMAMPs, 5, 7, and 10 were among the
most potent and showed the highest selectivity even compared
with pexiganan, the most thoroughly studied AMP to date. For
example, SMAMP 5 is much more potent than pexiganin and is
approximately 10 times less hemolytic giving selectivity values
(HC50/MIC) of >2500 and >160 for S. aureus and E. coli,
respectively. When 5, 7, and 10 were compared to various
SMAMPs in the literature, it was found that 5, 7, and 10
possessed better antimicrobial activity against S. aureus than
most peptoids, β-peptides, short linear and cyclic peptides,
synthetic polymers, and oligo-acyl lysines, which typically
possessed MIC values of 2−15 μg/mL.
C
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Letter
(10) AMSDb database. http://www.bbcm.univ.trieste.it/∼tossi/pag1.
htm (accessed February 1, 2013).
administered in the presence of mouse serum and exhibited
higher antimicrobial activity toward S. aureus over E. coli.
SMAMPs 5, 7, and 10 possessed comparable antimicrobial
activities toward S. aureus as the most potent SMAMPs
reported in the literature while maintaining lower hemolytic
activity. The importance of the linker was also investigated by
comparing 7 to 17−18, and a pronounced difference in
antimicrobial and hemolytic activities was observed, thus
validating the use of the 1,2,3-triazole moiety. As a result of
this study, we have identified three SMAMPs, 5, 7, and 10, that
outperformed pexiganan, and they are currently being
investigated for use as new antibiotics.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Procedure for the synthesis of 7, characterization data for 4−
11, and procedures for determining antimicrobial and
hemolytic activity. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Fax: (413) 545-2873. E-mail: tew@mail.pse.umass.edu.
■
(18) Haug, B. E.; Stensen, W.; Kalaaji, M.; Rekdal, Y.; Svendsen, J. S.
Synthetic Antimicrobial Peptidomimetics with Therapeutic Potential.
J. Med. Chem. 2008, 51, 4306−4314.
(19) Makovitzki, A.; Avrahami, D.; Shai, Y. Ultrashort antibacterial
and antifungal lipopeptides. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
15997−16002.
Author Contributions
T-h.F. and Y.L. contributed equally.
Notes
The authors declare no competing financial interest.
(20) 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. Systemic Antibacterial Activity of Novel
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ACKNOWLEDGMENTS
■
This work was supported by the NIH (AI-074866 and U01 AI-
082192). Mr. Michael Lis and Ms. Katie Gibney are
acknowledged for helpful and challenging discussions. Mass
spectral data were obtained at the University of Massachusetts
Mass Spectrometry Facility which is supported, in part, by the
National Science Foundation.
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