Design and synthesis of macrocyclic peptomers as mimics of a quorum sensing signal from Staphylococcus aureus

Article abstract of DOI:10.1021/ol800908h

(Chemical Equation Presented) We report the design and synthesis of macrocyclic peptide-peptoid hybrids (peptomers) as analogs of autoinducing peptide I (AIP-I) from Staphylococcus aureus. Our solid-phase synthetic route includes efficient microwave-assisted reactions and a tandem macrocyclization-cleavage step, and we demonstrate its compatibility with parallel synthesis through the generation of a focused peptomer library. One of the peptomers was capable of stimulating biofilm formation in S. aureus, a phenotype linked to AIP-I receptor (AgrC-I) inhibition.

Full text of DOI:10.1021/ol800908h

ORGANIC
LETTERS
2008
Vol. 10, No. 12
2329-2332
Design and Synthesis of Macrocyclic
Peptomers as Mimics of a Quorum
Sensing Signal from Staphylococcus
aureus
Sarah A. Fowler, Danielle M. Stacy, and Helen E. Blackwell*
Department of Chemistry, UniVersity of Wisconsin-Madison, 1101 UniVersity AVenue,
Madison, Wisconsin 53706-1322
blackwell@chem.wisc.edu
Received December 22, 2007
ABSTRACT
We report the design and synthesis of macrocyclic peptide-peptoid hybrids (peptomers) as analogs of autoinducing peptide I (AIP-I) from
Staphylococcus aureus. Our solid-phase synthetic route includes efficient microwave-assisted reactions and a tandem macrocyclization-
cleavage step, and we demonstrate its compatibility with parallel synthesis through the generation of a focused peptomer library. One of the
peptomers was capable of stimulating biofilm formation in S. aureus, a phenotype linked to AIP-I receptor (AgrC-I) inhibition.
There is an urgent, global need for new antibacterial
therapies, and quorum sensing (QS) has emerged as an
attractive target for intervention.1 Many bacterial pathogens
have evolved to overcome traditional antibiotics that target
growth.2 However, as inhibition of QS pathways should
block virulence, not bacterial growth, resistant mutants are
anticipated to arise at considerably slower rates (if at all).3
Gram-positive bacteria use small peptides as QS signals,1
and the macrocyclic autoinducing peptides (AIPs) utilized
by Staphylococcus aureus for QS have been well studied
due to the enormous clinical relevance of this pathogen.4
Non-native ligands that intercept QS pathways in S. aureus
would hold significant value as chemical tools to study
the fundamental mechanisms of QS and their role in
pathogenesis.1b,c,5,6 In this context, peptidomimetics that
(4) (a) George, E. A.; Muir, T. W. ChemBioChem 2007, 8, 847–855.
(b) Lyon, G. J.; Novick, R. P. Peptides 2004, 25, 1389–1403. (c) Chan,
W. C.; Coyle, B. J.; Williams, P. J. Med. Chem. 2004, 47, 4633–4641. (d)
Novick, R. P.; Muir, T. W. Curr. Opin. Microbiol. 1999, 2, 40–45.
(5) Gorske, B. C.; Blackwell, H. E. Org. Biomol. Chem. 2006, 4, 1441–
1445
.
(1) (a) Waters, C. M.; Bassler, B. L. Ann. ReV. Cell DeV. Biol. 2005,
21, 319–346. (b) Greenberg, E. P. J. Clin. InVest. 2003, 112, 1288–1290.
(c) Lyon, G. J.; Muir, T. W. Chem. Biol. 2003, 10, 1007–1021.
(2) (a) Walsh, C. T. Antibiotics: Actions, Origins, Resistance; ASM
Press: Washington, DC, 2003. (b) Walsh, C. Nat. ReV. Microbiol. 2003, 1,
65–70. (c) Wright, G. D. Curr. Opin. Chem. Biol. 2003, 7, 563–569.
(3) (a) Clatworthy, A. E.; Pierson, E.; Hung, D. T. Nat. Chem. Biol.
2007, 3, 541–548. (b) Hentzer, M.; Givskov, M. J. Clin. InVest. 2003, 112,
1300–1307.
(6) For a related approach using antibodies, see: (a) Park, J.; Jagasia,
R.; Kaufmann, G. F.; Mathison, J. C.; Ruiz, D. I.; Moss, J. A.; Meijler,
M. M.; Ulevitch, R. J.; Janda, K. D. Chem. Biol. 2007, 14, 1119–1127
(7) George, E. A.; Novick, R. P.; Muir, T. W. J. Am. Chem. Soc. 2008,
130, 4914–4924
.
.
(8) For a review of peptoids, see: (a) Patch, J. A.; Kirshenbaum, K.;
Seurynck, S. L.; Zuckermann, R. N.; Barron, A. E. In Pseudopeptides in
Drug DeVelopment; Nielsen, P. E., Ed. Wiley-VCH: Weinheim, Germany,
2004
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10.1021/ol800908h CCC: $40.75
Published on Web 05/14/2008
2008 American Chemical Society

display enhanced biostability relative to native QS peptides
represent an important ligand class; such mimetics remain
largely unexplored as QS modulators in Gram-positive
bacteria, however.7 We hypothesized that N-substituted
glycine oligomers, or peptoids, would be a versatile structure
class from which to design such ligands due to their high
biostability and ease of synthesis.8,9 Furthermore, peptoids
have been successfully developed as mimics of peptides and
proteins to probe numerous other biological phenomena.10
Here, we report our initial investigations toward the design
and solid-phase synthesis of macrocyclic peptide-peptoid
hybrids (or peptomers)11 as analogs of AIP-I from S. aureus
(Figure 1). We demonstrate the utility of our synthetic route
in the present study for the design of peptoid analogs of AIP-I
(1). Acyclic AIP-I fails to activate AgrC receptors, indicating
that the macrocycle enforces an active conformation.4 We
reasoned that replacing the thioester with a more hydrolyti-
cally stable linkage could provide AIP-I analogs with
enhanced agonistic, or antagonistic, activity. We note,
however, that thioester replacement with lactones or lactams
in certain R-peptide AIP analogs reduced their agonistic
activities but had virtually no effect on antagonistic activi-
ties.4,7 These prior findings suggested that such substitutions
should be made with care in the design of new analogs.
In AIP-I (1), the presence of Phe-6 and Ile-7 are essential
for agonistic activity (e.g., replacement with Ala decreases
its EC50 by ∼500-fold).12 Additionally, truncated analogs
of AIP-I that lack the exocyclic Tyr-1-Ser-2-Thr-3 tail also
display low agonistic activity; interestingly, these peptides
exhibit strong antagonistic activity against AgrC receptors
instead. Recently, Williams and co-workers found that
replacement of Asp-5 with Ala in these truncated cyclic
peptides yielded one of the most potent AgrC-I antagonists
reported (2, IC50 ) 5 nM; Figure 2).4c Together, these SAR
Figure 1. S. aureus group I autoinducing peptide (AIP-I, 1).
through the construction of a focused library of peptomers
and report the initial biological testing of these compounds.
One of the peptomers was found to stimulate a QS response
in S. aureus (i.e., biofilm formation) and represents to our
knowledge the first peptomer modulator of a QS phenotype
in bacteria.
Figure 2. Truncated AIP-I analog (2) with antagonistic activity
against AgrC-I.
S. aureus uses AIP ligands and their cognate trans-
membrane receptors, AgrC proteins, for QS.1,4 AIP-AgrC
binding activates a two-component intracellular signaling
system that ultimately triggers the virulence response. S.
aureus has evolutionarily diverged into four distinct groups
(I-IV), each with its own unique AIP ligand and AgrC
receptor. Researchers have seized the opportunity to delineate
a set of structure-activity relationships (SARs) in these four
peptidic systems to better understand AIP-mediated QS.4 The
majority of these SAR data were derived for AIP-I (1) from
group I S. aureus (Figure 1). We built on these general SARs
data suggested that peptoid mimics of peptide 2 that lack a
thioester linkage could be modulators (most likely antago-
nists) of AgrC-I.
Our initial ligand design involved several incremental
changes to the structure of peptide 2; each step was guided
by computational studies to gauge the impact of the
perturbations on the overall conformation of 2.13 First, we
sought to determine whether the two key hydrophobic
residues in 2, Phe-6 and Ile-7, could be converted into their
analogous peptoid residues (Npm and Nssb, respectively).8
For ease of synthesis in this first generation ligand design,
we selected to convert only these two residues into peptoid
units. Thus, we would generate peptide-peptoid hybrid
products (or peptomers).11,14 Second, we chose to replace
the Cys-4-derived thioester with a more hydrolytically stable
amide linkage. We selected L-2,3-diaminopropionic acid as
a Cys-4 replacement at the outset. However, modeling studies
revealed that the 16-membered ring of this peptomer analog
failed to overlay well with the Williams inhibitor 2; in fact,
the N-substituted side chains were ∼60° out of plane relative
to the peptidic side chains (data not shown). Further
computational work revealed that by expanding the ring by
one atom through the replacement of Ala-5 with (S)-3-
aminobutanoic acid (to give peptomer 3; Figure 3), the
(9) (a) Gorske, B. C.; Jewell, S. A.; Guerard, E. J.; Blackwell, H. E.
Org. Lett. 2005, 7, 1521–1524. (b) Zuckermann, R. N.; Kerr, J. M.; Kent,
S. B. H.; Moos, W. H. J. Am. Chem. Soc. 1992, 114, 10646–10647
.
(10) For selected recent examples, see: (a) Xiao, X. S.; Yu, P.; Lim,
H. S.; Sikder, D.; Kodadek, T. Angew. Chem., Int. Ed. 2007, 46, 2865–
2868. (b) Hara, T.; Durell, S. R.; Myers, M. C.; Appella, D. H. J. Am.
Chem. Soc. 2006, 128, 1995–2004. (c) Seurynck, S. L.; Patch, J. A.; Barron,
A. E. Chem. Biol. 2005, 12, 77–88.
(11) Ostergaard, S.; Holm, A. Mol. DiVers. 1997, 3, 17–27.
(12) McDowell, P.; Affas, Z.; Reynolds, C.; Holden, M. T. G.; Wood,
S. J.; Saint, S.; Cockayne, A.; Hill, P. J.; Dodd, C. E. R.; Bycroft, B. W.;
Chan, W. C.; Williams, P. Mol. Microbiol. 2001, 41, 503–512.
(13) Using molecular mechanics in MOE (v. 2006. 08). See Supporting
Information for details.
(14) Peptomers were modeled with Nssb units (derived from S-sec-butyl
amine) but replaced with racemic (Nsb) units for synthesis.
(15) (a) Blackwell, H. E. Org. Biomol. Chem. 2003, 1, 1251–1255. (b)
Kappe, C. O. Angew. Chem., Int. Ed. 2004, 43, 6250–6284.
2330
Org. Lett., Vol. 10, No. 12, 2008

Scheme 1. Solid-Phase Synthesis of Peptomer Mimic 4
Figure 3. (Left) Initial cyclic peptomer (3) designed to mimic
peptide 2. (Right) Streamlined cyclic peptomer 4. Npm and Nssb
peptoid units replace residues 6 and 7 in peptide 2, respectively.
peptide (2) and peptomer structures occupied virtually the
same space (95.4% fit value; Figure 4 left).13 Third, in this
ester loading (∼70%) was achieved in a short reaction time
(20 min) using µW heating (as determined by UV Fmoc
quantitation).17 DBU (1,8-diaza-bicyclo[5.4.0] undec-7-ene)
was used for subsequent Fmoc deprotection.18 We next
coupled the two peptoid residues (Nsb and Npm) to resin 5
using our recently reported, µW-assisted peptoid synthesis
method.9a,14 Thereafter, we performed DIC coupling reac-
tions under µW heating and a basic Fmoc deprotection to
couple two units of Fmoc-ꢀAla-OH to the resin-bound
peptomer. The resin was acetylated after each peptide
coupling reaction to block any unreacted amines from
subsequent reactions. This procedure generated resin-bound,
acyclic peptomer 7 with a loading of 0.41 mmol/g.17
We reasoned that subjecting resin 7 to base would facilitate
both Fmoc-deprotection and a tandem macrocyclization-
cleavage to release peptomer 4. We were pleased to observe
clean formation of the 17-membered macrocycle 4 upon
treatment of 7 with 2 equiv of piperidine at room temperature
(rt; 25% conversion from 7). The crude peptomer was
separated from the Fmoc piperidine-fulvene adduct by
preparative reverse-phase HPLC to give peptomer 4 in >95%
purity and 13% overall yield (calculated from Ala-resin 5).
Longer reaction times, either at room temperature or elevated
temperatures (e.g., 40-80 °C in a µW reactor), failed to
increase the macrocyclization-cleavage yield (data not shown).
We therefore utilized the room temperature, macrocycliza-
tion-cleavage conditions for the rest of this study.
Figure 4. Overlaid computed models. (Left) AIP-I analog 2
(magenta) with peptomer 3 (colored by atom type). (Right) AIP-I
analog 2 (magenta) with peptomer 4 (colored by atom type).
initial study of AIP-I mimics, we sought a highly streamlined
peptomer target to expedite synthesis. Additional modeling
of 3 with further structural replacements revealed that
peptomer 4, containing an L-Ala-(ꢀ-Ala)2 linkage (Figure 3),
was an excellent mimic of peptide 2 (97.3% fit value; Figure
4 right).13 We therefore selected peptomer 4 as our target
scaffold for this study.
We designed a solid-phase synthetic route to macrocyclic
peptomers based on scaffold 4, as solid-phase methods for
both R-peptide and peptoid syntheses are robust and well
established (Scheme 1).9 For ease of synthesis, this route
made use of only commercially available N-Fmoc-amino
acids, amines, and resin. In addition, we incorporated
microwave (µW)-assisted reactions throughout the route,15
as these transformations can dramatically expedite the rates
of solid-phase syntheses.9a We selected an ester-linker
derived resin (Wang),16 as we sought to explore a tandem
cyclization-cleavage procedure to generate the macrocyclic
peptomers in the final step.
Our synthesis began with the loading of Fmoc-L-Ala-OH
onto Wang-linker derived polystyrene beads using a carbo-
diimide (DIC) coupling reaction (Scheme 1). Acceptable
We further examined our peptomer synthesis method
through the construction of a small (11-member) peptomer
library. Each library member differed in the composition of
its two peptoid units (Figure 5). Eleven amine building blocks
(16) Wang, S. S. J. Am. Chem. Soc. 1973, 95, 1328–1333.
(17) Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 37, 3404–3409.
(18) We used DBU, as opposed to piperidine, for Fmoc deprotection
throughout this synthesis to eliminate premature compound cleavage via
amide formation.
(19) Vuong, C.; Saenz, H. L.; Gotz, F.; Otto, M. J. Infect. Dis. 2000,
182, 1688–1693.
Org. Lett., Vol. 10, No. 12, 2008
2331

deactivated amines into the macrocyclic peptomers (e.g., spe
and 4fb, respectively; Figure 5).
We sought to determine if the peptomers were capable of
modulating the activity of AgrC-I in S. aureus. A previous
report by Otto and co-workers showed that inhibition of
AgrC-I (either by genetic knockout or with a known AgrC-I
inhibitor, the S. epidermidis AIP) can stimulate S. aureus
biofilm formation in a static biofilm assay.19 The precise role
of AgrC proteins in S. aureus biofilm formation is still being
elucidated and appears to be environmentally sensitive;20
however, we reasoned that this biofilm assay would allow
us to quickly assess if the peptomers could modulate AgrC-
I. We examined peptomers 4 and 8-17 and the S. epider-
midis AIP at 10 µM in static biofilm assays (using S. aureus
RN6390b)19 and observed that one peptomer (14) was
capable of stimulating biofilm formation by 2-fold versus
the untreated control and to 34% the level of the S.
epidermidis AIP (see Figure S-3; Supporting Information).
Additional experiments are required to elucidate the mech-
anism by which peptomer 14 promotes biofilm formation in
S. aureus. Nevertheless, as 14 was identified from a relatively
small group of compounds (11), this result serves to
underscore the potential value of peptidomimetic tools to
study QS in Gram-positive bacteria.
Figure 5. Generic structure of peptomer library (left corner) and
amines (with abbreviations) used in construction of the library.14
were selected for library synthesis that differed in their
overall steric bulk, hydrophobicity, and electronics. These
macrocyclic peptomers were designed not only to study the
scope of our method but also to eventually probe if differing
peptoid structure in the key Phe-6-Ile-7 positions affected
peptomer activity against AgrC-I.
We prepared the peptomer library (4 and 8-17) in parallel
using our optimized synthetic method (Table 1), and isolated
In summary, we have designed peptomer analogs of AIP-I
from S. aureus and developed a new solid-phase synthetic
route that provides access to these compounds. This straight-
forward route incorporates a series of µW-assisted reactions
and a tandem macrocyclization-cleavage step. We applied
this method for the parallel synthesis of a collection of
macrocyclic peptomer mimics of a truncated AIP-I analog
(2). One of the peptomers (14) was found to stimulate biofilm
formation in S. aureus, a phenotype linked to AgrC-I
inhibition. Ongoing studies are focused on applying our
synthetic route to peptomers of heightened structural com-
plexity (i.e., peptomer 3) and further evaluating the activities
of peptomer mimetics in QS assays in S. aureus.
Table 1. Structures and Yields for Peptomer Library Members
cyclization yield overall yield
peptomera
R1
R2
(%)b,c
(%)c,d
4
8
9
Nsb
Npm Nsb
Npm
22
31
33
25
15
23
36
32
16
36
21
13
15
20
13
10
11
20
15
11
22
13
Nip
Nch
Nsb
Nsb
Nsb
Npm
Npm
Nchm
N4mb
Nspe
10
11
12
13
14
15
16
17
Nmp Npm
Ntfe
Nsb
Nsb
Npm
N4mob
N4fb
Acknowledgment. We thank the NSF (CHE-0449959),
Burroughs Wellcome Foundation, Research Corporation,
Johnson & Johnson, and 3M for financial support of this
work. H.E.B. is an Alfred P. Sloan Foundation fellow. S.A.F.
acknowledges the ACS Division of Medicinal Chemistry for
a predoctoral fellowship and Pfizer for a Diversity in Organic
Chemistry Fellowship. We thank Dr. Michael Otto (NIH)
for a sample of the S. epidermidis AIP, Dr. Grant Geske
(UW-Madison) for assistance with computational modeling,
and Dr. Benjamin Gorske (UW-Madison) for contributive
discussions.
a See Figure 5 for full peptomer structures. b Isolated yield of cyclization-
cleavage step after purification to g94% purity by HPLC. c Peptomers
containing Nsb were isolated as mixtures of diastereomers; yields report
both diastereomers. d Overall yield of macrocyclic peptomer based on
loading of Ala-resin 5 (0.65 mmol/g).
each compound in g94% purity by preparative reverse-phase
HPLC. Overall yields were 11-22% for this multiple-step
solid-phase synthesis, and the yields of the tandem macro-
cyclization-cleavage step ranged from 15-36%. Consistent
with our previous report,9a we found that the µW-assisted
peptoid synthesis procedure allowed for the efficient incor-
poration of both sterically hindered and electronically
Supporting Information Available: Full details of pep-
tomer synthesis and characterization, modeling studies, and
biological testing and data. This material is available free of
charge via the Internet at http://pubs.acs.org.
(20) Yarwood, J. M.; Bartels, D. J.; Volper, E. M.; Greenberg, E. P J.
Bacteriol. 2004, 186, 1838–1850.
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