Side-chain-to-tail thiolactone peptide inhibitors of the staphylococcal quorum-sensing system

Article abstract of DOI:10.1016/S0960-894X(03)00497-9

The expression of many staphylococcal virulence factors are regulated by the agr locus via a two-component signal transduction system (TCSTS), which is activated in response to a secreted autoinducer peptide (AIP). By exploiting the unique chemical archit

Full text of DOI:10.1016/S0960-894X(03)00497-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2449–2453
Side-Chain-to-Tail Thiolactone Peptide Inhibitors of the
Staphylococcal Quorum-Sensing System
R. John Scott,a Lu-Yun Lian,c S. Hanna Muharram,b Alan Cockayne,b Stewart J. Wood,a
Barrie W. Bycroft,a Paul Williamsa,b and Weng C. Chana,*
aSchool of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK
bInstitute of Infection, Immunity & Inflammation, University of Nottingham, University Park, Nottingham NG7 2RD, UK
cDepartment of Biomolecular Sciences, University of Manchester Institute of Science & Technology, The Mill, Sackville Street,
Manchester M60 1QD, UK
Received 7 April 2003; revised 14 May 2003; accepted 15 May 2003
Abstract—The expression ofmany staphylococcal virulence afctors are regulated by the agr locus via a two-component signal
transduction system (TCSTS), which is activated in response to a secreted autoinducer peptide (AIP). By exploiting the unique
chemical architecture ofthe naturally occurring AIP-1, several potent inhibitors ofstaphylococcal TCSTS were designed and syn-
thesized using either a linear or branched solid-phase approach. These inhibitors are competitive binders and contain the crucial 16-
membered side-chain-to-tail thiolactone peptide pharmacophore.
# 2003 Elsevier Ltd. All rights reserved.
Staphylococcus aureus is a versatile pathogen capable of
causing life-threatening infections that are mediated
primarily through the phenotypic expression ofviru-
lence factors.1,2 The agr locus in S. aureus, which con-
sists oftwo divergent operons expressed rfom the
tide ‘tail’. The unusual thiolactone structure is derived
from the C-terminal carboxyl group being bonded to
the sulfhydryl group of an N-terminally located cysteine
residue. Although four S. aureus AIPs have been repor-
ted to-date, they differ in their primary sequence but
retained the unique thiolactone structure. Interestingly,
these AIPs are capable ofcross-inhibiting the activities
promoters P2 and P3, is critically involved in the
1,3
expression ofvirulence determinants.
The P2 operon
3,5,6
encodes for elements of a quorum-sensing system, these
being proteins necessary for the biosynthesis of an
autoinducer molecule, AIP, and the two-component
sensor (AgrC): response regulator (AgrA) proteins.
Activation ofthe TCSTS is mediated by interaction of
the autoinducer with its cognate sensor protein resulting
in upregulation oftranscription both from promoter P2,
amplifying the response, and from promoter P3, initiat-
ing the production ofRNA III. Ultimately, the RNA
III upregulates the production ofseveral exotoxins and
enzymes, and represses the expression ofa range of
bacterial cell-surface proteins.3,4
ofnon-selfstaphylococcal TCSTS.
The staphylococcal autoinducer molecule, for example,
AIP-1 1, is peptidic, comprising ofa 16-membered
macrocyclic thiolactone attached to a tri- or tetra-pep-
The inhibition ofstaphylococcal quorum-sensing sys-
tems, specifically the TCSTS, have been shown to abol-
ish the production ofenterotoxin C3, lipase and toxic
shock syndrome toxin-1.3,6 Moreover, following inter-
nalization by epithelial cells, the ability ofthe S. aureus
to effect endosomal escape and intracellular replication
*Corresponding author. Tel.: +44-115-9515080; fax: +44-115-
9515078; e-mail: weng.chan@nottingham.ac.uk
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00497-9

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R. J. Scott et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2449–2453
appeared to be dependent on the agr-regulated produc-
7
noteworthy feature of the antagonists 4 and 5 is the
presence ofthe (4-substituted phenoxy)butyryl group
which functions as a Tyr-surrogate at the N-terminus of
the exocyclic ‘tail’.
tion ofexoproteins. Thus, the blockade ofstaphylo-
coccal TCSTS constitutes
a
unique therapeutic
approach,8 which is based on the attenuation ofbacter-
ial virulence and hence disrupting the capacity ofthe
bacteria to cause infection.
Two contrasting synthetic strategies were initially
investigated for the total synthesis of (Ala5)AIP-1 2.
Since the required peptide chains were assembled using
standard Fmoc/tBu solid-phase synthesis conditions9
and followed by regioselective unmasking of the reactive
groups at the macrocyclization sites, trityl-based pro-
tecting groups were utilized to mask these crucial reac-
tive functionalities.
During the course ofour structure–activity relationship
(SAR) studies ofAIP-1, we discovered a potent TCSTS
inhibitor, (Ala5)AIP-1 2.6 The dose–response (agr-blaZ
reporter1) curve, which was acquired in the presence of
0.25 mM AIP-1, was reminiscent ofa competitive
acceptor antagonist.6 Thus, functionally similar com-
pounds will now be referred to as AgrC antagonists.
The sensor-kinase AgrC is comprised oftwo large
domains, that is, an N-terminal sensor domain display-
ing transmembrane helices and extracellular loops, and
a cytoplasmic C-terminal histidine kinase domain.1,3 We
herein report our endeavors in the development ofa
practical and efficient methodology for the solid-phase
synthesis ofthese macrocyclic peptides. The overall syn-
thetic strategy is based on solid-phase assembly ofeither
a linear or branched protected peptide (see Fig. 1),
release from the solid support, and followed by poly-
mer-supported carbodiimide mediated cyclization in
solution. We have applied these synthetic methods for the
syntheses ofthree other potent AgrC antagonists 3–5. A
For the linear approach, in which the macrocyclization
was carried out through the formation of a thioester
bond between the Cys sulfhydryl and C-terminus car-
boxyl groups, the required protected peptide
6
(R1=Me, R2=(CH2)2SMe) was obtained via standard
Fmoc/tBu solid-phase peptide assembly9 on N-Fmoc-
Met-O-2-chlorotrityl polystyrene.10 Notably, the Cys
residue was installed using four-equivalent excess of the
S-4-methoxytrityl protected N-Fmoc-cysteine, which
was activated using N,N0-diisopropylcarbodiimide
(DIPCDI) in the presence of1-hydroxybenzotriazole
(HOBt).11 Subsequent treatment ofthe assembled pep-
tide-resin with TFA:Et3SiH:CH2Cl2 (1:1:98 v/v) for 30–
40 min resulted in the chemoselective acidolysis ofthe 2-
chlorotrityl ester resin linkage and S-4-methoxytrityl to
afford the partially protected octapeptide 6 (R1=Me,
R2=(CH2)2SMe) in typically quantitative yields. Partial
purification of 6 was accomplished by filtration ofthe
acidolytic resin suspension into pyridine:MeOH (1.5:75
v/v), evaporation ofthe filtrate to dryness in vacuo, tri-
turation ofthe residual material with ice-cold water,
and finally drying the precipitated peptide in vacuo. A
small fraction of the partially protected peptide 6 was
exposed to TFA:H2O:Et3SiH:EtSMe (90:5:3:2) for 1 h,
followed by RP-HPLC analysis,12 which revealed a
peptide purity >95%.
Macrocyclization was then achieved by exposing a
dilute solution of 6 in CHCl3 (1–2 mM) to N-poly-
styrene methyl-N0-cyclohexylcarbodiimide (5 equiv), in
13
the presence of7-aza-1-hydroxybenzotriazole
and 4-
dimethylaminopyridine (0.1 equiv) for 3–4 days at
ambient temperature. The polymer-supported carbodii-
mide (2% DVB, 200–400 mesh) is a commercially
available analogue of N-polystyrene methyl-N0-iso-
propylcarbodiimide.14 Following a standard workup,
90% TFA-mediated acidolytic treatment ofthe crude
protected macrocyclic peptide afforded the desired
Figure 1. The linear 6 and branched 7 retro-synthetic analyses to the
side-chain-to-tail thiolactone peptides.

R. J. Scott et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2449–2453
2451
peptide analogue 2 as a white amorphous solid in good
yield and purity >40%. Significantly, RP-HPLC ana-
lysis12 showed virtually absence ofthe linear peptide
residue was installed as the S-tbutylsulphenyl (StBu)16
protected amino acid, in which the thio-group was che-
moselectively and efficiently unmasked17 on the solid-
phase under the mild reducing conditions, 10 equiv
Bu3P in isopropanol-DMF (1:10) within 5–6 h at room
temperature—we found that the Bu3P-mediated depro-
tection was incomplete (ꢀ30% in 5 h) when THF was
used as the solvent, (ii) S-acylation was achieved by
repeated treatment with activated Trt-Met-OH18 to
afford the polymer-bound branched peptide 10, and (iii)
concomitant release from resin and N-deprotection by
mild acidolytic treatment of 10 to afford the partially
protected peptide 7 in good yield and purity (>95%).
Macrocyclization was then undertaken using the pre-
viously established conditions, that is, a CHCl3 solution
of 7 (2 mM) was added polymer-bound carbodiimide
and 7-aza-1-hydroxybenzotriazole (HOAt). Following
workup and 90% TFA-mediated acidolytic treatment of
the crude protected macrocyclic peptide, the peptide 2
was obtained in acceptable yield and purity. However,
following purification by RP-HPLC, 2 was obtained in
yield (10–15%) that is comparable to the linear syn-
thetic strategy. Nonetheless, we are confident that the
branched strategy will facilitate the combinatorial
synthesis ofAIPs.
and only a small amount (<5%) ofthe
d-Met8
diastereoisomer. Following purification by RP-HPLC,
the thiolactone octapeptide 2 was obtained as a lyophi-
lized powder (15–20% yield).15
We then focused our attention on the branched
approach for the synthesis of the cyclic peptide 2. In this
strategy, the thioester bond was formed on the solid-
phase and the final macrocyclization ofthe partially
protected peptide 7 was through an amide bond
between the Ile7-carboxyl and Met8-amine groups. We
envisaged that this branched strategy has a number of
merits, including (i) formation of the ‘difficult’ thioester
bond can be forced to completion by exploiting solid-
phase methods, and (ii) has a readily accessible site (on
a pre-assembled peptide sequence) for the installation of
unique C-terminal amino acids, since this position has
been shown to have profound effect on Agr-binding.6
Scheme 1 outlines the overall synthetic method for the
branched strategy. The key features are: (i) the Cys
Compared to our lead compound 2, we previously
observed that the Gly-analogue, (Gly5)AIP-1 displayed
ꢀ40-fold decreased in activity as an antagonist at the
AgrC-1 acceptor.6 We hypothesized that increasing the
side-chain hydrophobicity ofthe crucial amino acid-5
would enhance potency. Thus, the methyl homologue,
(Abu5)AIP-1 3 was synthesized using the solid-phase
strategies outlined above and purified by RP-HPLC to
afford 3 as a lyophilized powder (10–19% yield).19
The effect ofintroducing Tyr 1-surrogates on the activity
ofour lead compound 2 was investigated. Our approach
here was to replace the Tyr residue with aromatic-bear-
ing acyl groups with increased hydrophobicity. Thus,
we have selected 4-benzylphenoxyalkanoyl as the gen-
eric des-amino-Tyr-surrogate, which was installed at the
N-terminus using 4-benzoylphenoxyalkanoic acid
building blocks. The reasons for utilizing this synthon
approach are two-fold: (i) structural variants of ben-
zoylphenol are readily accessed by the Friedel–Crafts
acylation ofsubstituted benzene using
p-methoxy-
benzoyl chloride,20 and (ii) biaryl ketones are photo-
activatable21 and hence the 4-benzoylphenoxyalkanoyl-
derivatized AIP-1 could be used for photolabelling
studies ofAgrC. The ketone ufnctionality in the ben-
zoylphenoxy moiety was readily reduced by a TFA:Et3-
SiH mixture, which is also used for the global
deprotection ofsynthesized peptide.
Scheme 1. Reagents and conditions: (i) 20% piperidine-DMF; (ii)
Fmoc-amino acid:TBTU:HOBt:DIEA (1:1:1:2, 4–8 equiv); (iii) Boc2O
(4–8 equiv), 18 h; (iv) Bu3P (10 equiv), iPrOH-DMF (1:10), 6 h; (v) (a)
Trt-Met-OH (10 equiv)-DIPCDI (5 equiv), DMAP (0.01 equiv),
CH2Cl2-DMF (1:1), 3 h, (b) Trt-Met-OH (5 equiv)-DIPCDI (2.5
equiv), DMAP (0.01 equiv), 6 h, (c) repeat (b); (vi) TFA-Et3SiH-
CH2Cl2 (1:1:98), 40 min; (vii) polymer-bound carbodiimide:HOAt (5:1
equiv), CHCl3, 72 h; (viii) (a) TFA:H2O:Et3SiH:EtSMe (90:5:3:2), 1 h;
(b) purification by RP-HPLC using Kromasil C8 preparative column
(10ꢂ150 mm).
The synthesis of N-4-(4-benzylphenoxy)butyryl-(des-
Tyr1; Ala5)AIP-1 4 was accomplished using the linear
approach. Thus, following solid-phase assembly of the
linear peptide, the N-terminus was acylated using
DIPCDI-activated 4-benzoylphenoxybutyric acid (mp
94–95 ꢁC), which was readily prepared by O-alkylation
of4-hydroxybenzophenone with ethyl 4-bromobutyrate
followed by saponification using methanolic aqueous

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R. J. Scott et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2449–2453
NaOH. The partially protected peptide intermediate
was obtained by 1% TFA-mediated acidolysis, which
was then subjected to polymer-bound carbodiimide-
mediated macrocyclization. The cyclization reaction
was carried out in CHCl3:DMF (ꢀ40:1) due to solubi-
lity issues. Treatment ofthe crude protected cyclic pep-
tide 11 with TFA:H2O:Et3SiH:EtSMe (90:5:3:2) resulted
in global deprotection and reduction ofthe ketone
functionality to afford 4, which was purified by RP-
HPLC (yield 5–10%).22 The observed low HPLC-pur-
ified yield was partly due the unexpected poor solubility
ofthe peptide 4 in either water, MeCN or aqueous
MeCN.
nist 3. Our recent 1H NMR studies (temperature
coefficient and NOESY) established that the 16-mem-
bered macrocyclic domain in both the agonist 1 and the
antagonist 2 display similar and well-defined conforma-
tion that is stabilized by two intramolecular H-bonds —
the H-bond donors being the backbone amide NHs of
the Asp/Ala5 and Ile7 residues (data will be reported
elsewhere). We suspect that the side-chain-to-tail thio-
lactone pharmacophore is the ‘address’ region ofthe pep-
tide ligands, which is in-part responsible for molecular
recognition.
In addition, it was gratifying to observe that both analo-
gues 4 and 5, comprising the installed des-amino-Tyr-
surrogates, exhibited good antagonistic potency though
somewhat lower compared to the octapeptide 2. The
good binding capacity ofthe analogue 5, which con-
tained a photophore tag, will facilitate further work in
obtaining a detailed map ofthe AgrC ligand-binding site.
Ofparticular significance is the exceptionally high inhib-
itory potency ofthe AIP-1 analogues 2–5 towards the
AgrC-2 acceptor, to which the natural agonist is AIP-21.
We believe that these new antagonists will provide an
invaluable platform for the design and synthesis of the
next generation ofglobal AgrC antagonists, especially
those with structural variants of4-benzylphenoxyalk-
anoyl installed at the N-terminus.
In addition, the peptide analogue 523 was obtained by
treatment ofthe protected cyclic peptide 11 with TFA:
H2O:iPr3SiH:EtSMe (90:5:3:2) for 30 min. The sterically
hindered iPr3SiH and a shorter reaction time were used
in order to minimize undesired reduction ofthe benzo-
phenone moiety.
Acknowledgements
The synthetic AIP-1 peptide derivatives were tested for
their ability to inhibit staphylococcal TCSTS, specifi-
cally their capacity to antagonize AgrC-1 and AgrC-2
acceptors. The bacterial cell assay is based on the ability
ofthese compounds to competitively block the activity
ofAIP-1 and AIP-2 within their specific S. aureus
strains. The biological activity was monitored by the
robust agr-blaZ reporter1,3,6 that entailed the P3-driven
We are grateful for funding from BBSRC, UK (a
studentship to S.J.W.) and MRC, UK.
References and Notes
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Unexpectedly, the (Abu5)-analogue 3 showed lower
inhibitory potency compared to the lead peptide 2 as an
AgrC-1 antagonist. The hydrophobicity and/or steric
bulk ofthe amino acid-5 side-chain are likely to con-
tribute to the decreased binding capacity ofthe antago-
Table 1. Inhibition (IC50 nM)a ofstaphylococcal AgrC determined
using competitive assays and S. aureus Group I and II engineered with
the agr-blaZ reporter,1,3,6 RN6390B:pRN6683 and SA564:pRN6683,
respectively
4. (a) Morfeldt, E.; Tegmark, K.; Arvidson, S. Mol. Micro-
biol. 1996, 21, 1227. (b) Benito, Y.; Kolb, F. A.; Romby, P.;
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Novick, R. P.; Muir, T. W. Proc. Natl. Acad. Sci. U.S.A. 1999,
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AIP-1 derivatives
IC50 (nM)
AgrC-1
AgrC-2
(Ala5)AIP-1, 2
3
4
5
21ꢃ9
4ꢃ4
2.8ꢃ0.4
19ꢃ14
18ꢃ5
137ꢃ65
295ꢃ22
303ꢃ164
aAssays were carried out at least in triplicate, and sigmoidal dose-
response curves were generated using PRISM3 program.

R. J. Scott et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2449–2453
2453
6. McDowell, P.; Affas, Z.; Reynolds, C.; Holden, M. T. G.;
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min at 1.1 mL minꢄ1. Column dimension: 4.6ꢂ150 mm; efflu-
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TFA in MeCN-H2O (9:1).
1987, 43, 2675. (b) Beekman, N. J. C. M.; Schaaper, W. M. M.;
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19. 1H NMR (600 MHz, DMSO-d6, 303 K) dH 9.10 (Met8
NH), 8.63 (br s, Ser2 NH), 8.46 (Abu5 NH), 8.31 (Phe6 NH),
8.25 (Cys4 NH), 8.20 (br s, Ile7 NH), 7.92 (Thr3 NH), 7.86
(Tyr1 NH2), 7.28, 7.20 (2ꢂm, Phe6 ArHs), 7.08, 6.70 (Tyr1
ArHs), 5.22 (Ser2 CbOH), 4.90 (Thr3 CbOH), 4.54 (Ser2 CaH),
4.50 (Phe6 CaH), 4.30 (Abu5 CaH), 4.28 (Thr3 CaH), 4.25
(Cys4 CaH), 4.22 (Met8 CaH), 4.08 (Thr3 CbH), 3.92 (Tyr1
CaH), 3.80 (Ile7 CaH), 3.69, 3.54 (2ꢂm, Ser2 CbH2), 3.20 (Cys4
CbH), 3.02 (Tyr1 CbH), 2.90 (Cys4 CbH), 2.78 (Tyr1 CbH), 2.65
(Phe6 CbH), 2.55 (Met8 CgH), 2.50 (Phe6 CbH), 2.50 (s, Met8
CH3), 2.45 (Met8 CgH), 2.20, 1.95 (Met8 CbH2), 1.94 (Ile7
CbH), 1.69, 1.52 (Abu5 CbH2), 1.32 (Abu5 CH3), (1.18 (Ile7
CgH2), 1.06 (Thr3 CH3), 0.84 (Ile7 Cg’H3), 0.76 (Ile7 CdH3);
ES–MS+ m/z 931.35 (MH+), calcd 931.41; RP-HPLC Kro-
masil C8, 20–60% B in 25 min, tR 20.7 min.
13. Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
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J. Y. Org. Synth. Coll. Vol. 1988, 6, 951.
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15. 1H NMR (600 MHz, DMSO-d6, 303 K) dH 9.08 (Met8
NH), 8.70 (Ser2 NH), 8.26 (Ile7 NH), 8.25 (Cys4 NH), 8.00
(Ala5 NH), 7.95 (Thr3 NH), 7.80 (Phe6 NH), 7.28, 7.20 (2ꢂm,
Phe6 ArHs), 7.08, 6.70 (Tyr1 ArHs), 5.24 (Ser2 CbOH), 4.95
(Thr3 CbOH), 4.54 (Ser2 CaH), 4.50 (Phe6 CaH), 4.28 (Cys4
CaH), 4.27 (Thr3 CaH), 4.25 (Met8 CaH), 4.10 (Thr3 CbH),
4.03 (Ala5 CaH), 4.00 (Tyr1 CaH), 3.77 (Ile7 CaH), 3.69, 3.56
(2ꢂm, Ser2 CbH2), 3.20 (Cys4 CbH), 3.03 (Tyr1 CbH), 2.96
(Phe6 CbH2), 2.90 (Cys4 CbH), 2.78 (Tyr1 CbH), 2.54 (Met8
CgH), 2.51 (s, Met8 CH3), 2.45 (Met8 CgH), 2.19 (Met8 CbH),
1.94 (Met8 CbH and Ile7 CbH), 1.18 (Ile7 CgH2), 1.06 (Thr3
CH3 and Ala5 CH3), 0.82 (Ile7 Cg’H3), 0.78 (Ile7 CdH3); ES–
MS+ m/z 917.27 (MH+), calcd 917.39; RP-HPLC Kromasil
C8, 20-60% B in 25 min, tR 19.6 min.
22. 1H NMR (600 MHz, DMSO-d6, 303 K) dH 9.10 (Met8
NH), 8.26 (Ile7 NH), 8.20 (Cys4 NH), 8.04 (Ser2 NH), 7.95
(Ala5 NH), 7.90 (BnPh ArH), 7.82 (Phe6 NH), 7.75 (BnPh
ArH), 7.70 (Thr3 NH), 7.45, 7.30 (BnPh ArHs), 7.28, 7.20
(2ꢂm, Phe6 ArHs), 7.14, 7.08, 6.85 (BnPh ArHs), 5.05 (Ser2
CbOH), 4.90 (Thr3 CbOH), 4.50 (Phe6 CaH), 4.40 (Ser2 CaH),
4.28 (Cys4 CaH), 4.24 (Met8 CaH), 4.21 (Thr3 CaH), 4.05
(Thr3 CbH), 4.02 (Ala5 CaH), 3.95 (PhOCH2CH2), 3.87
(PhCH2Ph), 3.78 (Ile7 CaH), 3.62, 3.55 (2ꢂm, Ser2 CbH2), 3.20
(Cys4 CbH), 2.98 (Phe6 CbH2), 2.91 (Cys4 CbH), 2.55 (Met8
CgH), 2.52 (s, Met8 CH3), 2.45 (Met8 CgH), 2.34
(CH2CH2CO), 2.20 (Met8 CbH), 1.95 (Met8 CbH and Ile7
CbH), 1.94 (PhOCH2CH2CH2CO), 1.19 (Ile7 CgH2), 1.05
7
(Ala5 CH3), 1.02 (Thr3 CH3), 0.80 (Ile7 Cg H3), 0.76 (Ile
0
16. Weber, U.; Hartter, P.; Hoppe-Seyler’s, Z. Physiol. Chem.
1970, 351, 1384.
17. (a) Eritja, R.; Ziehler-Martin, J. P.; Walker, P. A.; Lee,
T. D.; Legesse, K.; Albericio, F.; Kaplan, B. E. Tetrahedron
CdH3); ES–MS+ m/z 1006.37 (MH+), calcd 1006.44; RP-
HPLC Kromasil C8, 50–100% B in 20 min, tR 10.8 min.
23. ES–MS+ m/z 1020.76 (MH+), calcd 1020.42; RP-HPLC
Kromasil C8, 50–100% B in 20 min, tR 8.9 min.