Novel inhibitors of surface layer processing in Clostridium difficile

Article abstract of DOI:10.1016/j.bmc.2011.06.042

Clostridium difficile, a leading cause of hospital-acquired bacterial infection, is coated in a dense surface layer (S-layer) that is thought to provide both physicochemical protection and a scaffold for host-pathogen interactions. The key structural comp

Full text of DOI:10.1016/j.bmc.2011.06.042

Bioorganic & Medicinal Chemistry 20 (2012) 614–621
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel inhibitors of surface layer processing in Clostridium difficile
T. H. Tam Dang a, Robert P. Fagan b, Neil F. Fairweather b,c, Edward W. Tate a,c,
⇑
a Department of Chemistry, Imperial College London, London SW72AZ, United Kingdom
b Department of Life Sciences, Imperial College London, London SW72AZ, United Kingdom
c Institute of Chemical Biology, Imperial College London, London SW72AZ, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 21 June 2011
Clostridium difficile, a leading cause of hospital-acquired bacterial infection, is coated in a dense surface
layer (S-layer) that is thought to provide both physicochemical protection and a scaffold for host-patho-
gen interactions. The key structural components of the S-layer are two proteins derived from a polypep-
tide precursor, SlpA, via proteolytic cleavage by the protease Cwp84. Here, we report the design,
synthesis and in vivo characterization of a panel of protease inhibitors and activity-based probes (ABPs)
designed to target S-layer processing in live C. difficile cells. Inhibitors based on substrate-mimetic pep-
tides bearing a C-terminal Michael acceptor warhead were found to be promising candidates for further
development.
Keywords:
Activity based probe
Activity based protein profiling
Clostridium difficile
Surface layer protein
Protease inhibitor
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
molecular weight (LMW) SLP and high molecular weight (HMW)
SLP.12–14 Both the high and low molecular weight subunits are de-
Clostridium difficile is an anaerobic Gram-positive bacterium and
produces spores that can survive for extended periods in the envi-
ronment. First isolated by Hall and O’Toole in 1935 and named
Bacillius difficilis due to difficulties of isolation and culture,1 by
the late 1970s it was identified as the cause of diarrhoea and colitis
occurring mostly as a complication of antibiotic therapy. During
the past few years, there has been renewed interest in C. difficile
due to the recognition that the disease is more common, more se-
vere, and more resistant to standard treatment than previously
thought.2,3 C. difficile is the most frequent nosocomial (hospital ac-
quired) pathogen, with a mortality and morbidity rate greater that
due to better-known methicillin-resistant Staphylococcus aureus
(MRSA) in many parts of the United States and Europe. It causes
a wide range of gastrointestinal diseases ranging from mild diar-
rhoea to more life threatening pseudomembranous colitis.4
rived from a single gene, slpA.15,16
This gene is strongly transcribed during the entire growth
phase17 and up to 400 molecules of SlpA per cell per second are
produced, translocated, and cleaved during exponential phase.
The translated gene product SlpA is known to undergo at least
two rounds of post-translational cleavage: firstly near the N-termi-
nus to remove the signal sequence that targets the nascent chain
for translocation across the cytoplasmic membrane, and then
internally to release the two mature SLPs, LMW–SLP and HMW–
SLP (Fig. 1).18–20 It is presumed that removal of the signal peptide
occurs prior to or during translocation across the membrane, while
the second cleavage takes place either within the membrane or
within the cell wall. After cleavage, the mature proteins form a
complex and reassemble to build the S-layer. We recently reported
the structure of the resultant LMW/HMW–SLP complex using a
combination of genetic and structural techniques.21
As noted above, proteases are of interest as potential virulence
factors in C. difficile. The MEROPS database (Sanger Institute, UK)
lists 139 known and putative peptidases in C. difficile including
known and unknown functions, and in addition 63 non-peptidase
homologues. Given the difficulty of identifying bacterial proteases
by homology, this number may considerably underestimate the
real number of proteases. However, with the notable exception
of the self-processing toxins, only a few protease activities have
been reported to date in C. difficile, despite its importance as a
pathogen. Proteolytic activity has been observed in ten strains of
C. difficile, with gelatinase, collagenase, hyalurodinase and chon-
droitin sulfatase activity observed.22,23 However, the proteases
responsible are not characterized and their identities remain
The main virulence factors of C. difficile are toxins A and B,5–8
but other virulence factors have also been described, such as adhe-
sins and hydrolytic enzymes.9–11 Among these, the surface layer
proteins (SLPs) are proposed to aid the colonization of the host,
and induce both inflammatory and antibody responses in the host.
These proteins may adhere directly to the host cell or enhance
competition with host flora. As in several other bacteria, the
surface layer proteins are the predominant surface proteins in C.
difficile. However, in contrast to most other species, where the
S-layer is composed of one main structural protein, the S-layer of
C. difficileis constructed from two distinct proteins:
a low
⇑
Corresponding author.
E-mail address: e.tate@imperial.ac.uk (E.W. Tate).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.042

T. H. Tam Dang et al. / Bioorg. Med. Chem. 20 (2012) 614–621
615
for inhibition of papain cysteine proteases by E-64 derivatives. In
this model, the warhead would sit in the Ser355 (P1) position
and the peptide residues would make interactions predominantly
via their side chains in the pocket that accommodatesthe P2 Lys
in the majority of strains, or P2 Tyr in some strains. In our previous
work, this hypothesis was explored through a structure–activity
study that matched this inhibitor binding mode to the P2 position,
whilst there was no clear preference shown for residues at P3 or
beyond.34 Inhibitors containing Lys, Argand Tyr were effective,
whilst most other residues were not tolerated with the exception
of Leu, which is present in the parent inhibitor E-64 (Scheme 1).
In the present study, we explored the influence of inhibitor reactiv-
ity on inhibition of SlpA processing across a selection of warheads
at the N-terminus of the specificity element, including halo- and
acyloxyacetamides. We also examined the influence of backbone
direction by placing a Michael acceptor motif at either the N- or
C-termini.
Figure 1. Post-translational processing of SlpA. The high- and low-molecular
weight (HMW and LMW) surface layer proteins (SLPs) are synthesized in C. difficile
as a ‘full-length’ precursor, SlpA. (A) During the process of translocation across the
membrane the signal peptide (SP) is removed, presumably by a signal peptidase; (B)
further processing by the papain class cysteine protease Cwp84 generates the
mature S-layer proteins; (C) LMW and HMW SLPs self-assemble to form the S-layer.
2.2. Probe synthesis
A flexible route was designed to provide ready access to N-ter-
minal warhead combinations (Scheme 1), based on our solid phase
route to E-64 analogues.34 Starting from Biotin NovaTag resin, the
specificity element was built up by standard Fmoc/tBu solid phase
peptide synthesis (SPPS), and the warhead moiety coupled directly
to the N-terminal amine prior to cleavage from the resin as a C-ter-
minal PEG-Biotin conjugate. N-Terminal chloroacetamide (CAA),
fluoroacetamide (FAA) and Michael acceptor (MA) probes were
readily synthesized in acceptable overall yields, with a range of
specificity elements derived from our previous work on N-terminal
epoxysuccinamides (EP).
unknown. This situation is exacerbated by limited genetic tools in
C. difficile; the generation of unconditional knockout mutants in C.
difficile is generally feasible,24 but it is currently extremely difficult
to construct either multiple or conditional knockouts.6 This has led
to notable problems, for example, the difficulty encountered in
establishing the essentiality of the major C. difficile toxins for viru-
lence.25 Alternative chemistry-based approaches such as activity-
based protein profiling (ABPP) thus have particular interest for
characterization of enzymes and post-translational protein pro-
cessing in such challenging and medically important organ-
isms,26–29 with a view to identifying novel drug targets for future
intervention.30–33
A solid phase synthesis route was also developed for the syn-
thesis of a prototype C-terminal Michael acceptor probe (Scheme
2). In this approach, the specificity element was anchored to a
polystyrene resin via a side chain, enabling the C-terminal acid
In the first reported application of ABPP in Clostridia, we devel-
oped activity-based probes (ABPs) against the protease responsible
for processing SlpA in C. difficile.34 Using these probes, the protease
was identified in vivo as Cwp84, a papain class cysteine protease,
and confirmed in a Cwp84 knockout strain.35 In parallel, the Shen,
Garcia and Bogyo labs have exploited a different class of ABPs to
characterize the proteolytic activation pathway of the C. difficile
toxins,36–39 focusing on toxin in isolation rather than identification
of proteases de novo in live bacteria. Here, we report the design
and synthesis of a broader range of potential Cwp84 inhibitors,
and characterize their ability to inhibit SlpA processing in live
bacteria.
to be replaced. For this purpose, ethyl-3-[Fmoc-L-Ser]-(E)-propeno-
ate, a protected serine analogue bearing a Michael acceptor in
place of the acid was synthesisedstarting from Fmoc-Ser(tBu)-OH.
Formation of the aldehyde Fmoc-Ser(tBu)-H via reduction of the
intermediate benzyl thioester was followed by Wittig olefination
and deprotection of the tert-butyl ether to render the required ana-
logue in good overall yield. After loading on chlorotrityl resin
probes could be constructed containing a C-terminal serine, as
found at Ser355 in the SlpA cleavage site. We postulated that this
would favor adoption of a backbone comparable to the natural sub-
strate, placing the Cwp84 active site cysteine thiolate in close prox-
imity to the Michael acceptor.
2. Results and discussion
2.1. Probe design
2.3. In vivo inhibition of S-layer processing
We next tested each of the probes synthesized above in an
in vivo assay for inhibition of S-layer processing described in
our previous work.34 In brief, bacteria were cultured for 16 h
in the presence of an inhibitor (or DMSO control), and surface
layer proteins extracted and analyzedby Western blot against
an antibody reactive for SlpA. In the presence of an effective
inhibitor of S-layer processing SlpA remains unprocessed, and
can be detected as a single band at 74 kDa. Amongst the N-ter-
minal warheads, the chloroacetamides (CAA) showed inhibition
activity against S-layer processing at 100 lM (overnight culture),
and this appears to be directed by the selectivity element
(Fig. 2A).
Arg and Leu-Arg sequences were found to have activity whilst
Ala and Ser did not, which correlates with the structure–activity
pattern in previous studies.34 The N-terminal FAA and MA
Previously-reported epoxysuccinyl inhibitors and probes for
S-layer processing are based on the structure of E-64 (Scheme 1),
and the design of our new probes took a similar approach of com-
bining a specificity element that binds at the same site as the nat-
ural substrate (SlpA), and a reactive electrophilic warhead to trap
irreversibly the nucleophilic active center of the protease. The SlpA
cleavage site is well-characterized in C. difficile reference strain
630, occurring at Ser355 in the sequence . . .LETKS-ANDT. . ., with
the LMW and HMW SLPs lying to the N-terminal and C-terminal
sides of this cleavage site, respectively.40 Interestingly, tyrosine is
present in place of lysine at P2 in several strains of SlpA. Peptide-
based irreversible (or ‘suicide’) Cwp84 inhibitors reported to date
have been proposed to bind the protease with the amide backbone
reversed relative to the natural substrate, the most common mode
probes showed no activity at 100 lM in this assay (data not

616
T. H. Tam Dang et al. / Bioorg. Med. Chem. 20 (2012) 614–621
O
H
N
O
O
O
Fmoc
O
N
H
N
O
PEG3-Biotin
= PEG3-Biotin
O
H
H
H
N
Biotin NovaTag resin
O
S
N
H
R
SPPS
HN
+
O
H2N
Peptide
N
FmocHN
PEG3-Biotin
OH
PEG3-Biotin
Biotin NovaTag
resin
O
1. NMM
O
H
N
H
N
2. TFA-H2O-TIPS
Peptide
PEG3-Biotin
Cl
H2N
Peptide
N
+
Cl
Cl
PEG3-Biotin
CAA-Peptide-PEG3-Biotin
1. HCTU, DiPEA
2. TFA-H2O-TIPS
O
O
F
H
N
H
N
H2N Peptide
N
Na+
O
+
Peptide
PEG3-Biotin
F
PEG3-Biotin
FAA-Peptide-PEG3-Biotin
1. HCTU, DiPEA
2. TFA-H2O-TIPS
O
H
N
O
H
N
Peptide
PEG3-Biotin
O
H2N Peptide
N
+
HO
EtOOC
O
PEG3-Biotin
MA-Peptide-PEG3-Biotin
O
O
O
O
O
H
N
PEG3-Biotin
O
O
N
H
N
H
HO
N
H
O
O
O
HN
NH
NH
N
H
NH2
EtEP-LR-PEG3-Biotin
E-64
H2N
NH
Scheme 1. Synthesis of N-terminal chloroacetamide (CAA), fluoroacetamide (FAA), and Michael acceptor (MA) probes. For each warhead, Peptide = -Arg-(R), -Leu-Arg-(LR), -
Ala-(A) or -Ser-(S).The specificity element was synthesized on PEG-Biotin-Novatag™ resin using standard SPPS with subsequent coupling of the desired warhead. CAA
warhead: ClCOCH2Cl (1.2 equiv), NMM (2.5 equiv), 1 h, rt, overall yield 4–37%. FAA warhead: NaCOOCH2F (5 equiv), HCTU (5 equiv) and DiPEA (6 equiv), 1 h, rt, overall yield
6–15%. MA warhead: HOOCCH@CHCOOEt (5 equiv), HCTU (5 equiv) and DiPEA (6 equiv), 1 h, rt, overall yield 10–39%.
shown), implying that their reactivity is insufficient, or that they
make poor contacts in the enzyme active site. The activity of the
structurally similar CAA and EtEP warheads suggests that the
lack of activity against Cwp84 is more likely the result of
reduced chemical reactivity in the warhead. In contrast to the
N-terminal Michael acceptors, the C-terminal MA inhibitor
Ac-LETKS-MA showed good activity at 100 lM against S-layer
processing, comparable to that of our previous E-64-based N-ter-
minal epoxides (Fig. 2A). This activity was maintained in two
ABPs, Ac-K(Biotin)YS-MA and Ac-K(Biotin)LS-MA, supporting the
hypothesis that these inhibitors bind with the backbone in the
‘native’ conformation, and tolerate significant divergence at P3.

T. H. Tam Dang et al. / Bioorg. Med. Chem. 20 (2012) 614–621
617
OtBu
OtBu
OH
OtBu
H
DCC/DMAP
Et3SiH
S
FmocHN
FmocHN
BnSH
FmocHN
Pd/C
O
RT/ o/n
RT/1h
O
O
Fmoc-L-Ser(tBu)-H
Fm oc-L-Ser(tBu)-SBn (91%)
RT/24 h
OtBu
COOEt
Ph3P
Chlorotrityl resin
OH
O
ClTrt
20% TFA/DCM
RT/2h
Tr t
FmocHN
COOEt
FmocHN
COOEt
FmocHN
COOEt
600C/6h
Ethyl-3-[Fmoc-L-Ser(tBu)]-(E)-
propenoate (37%, 2 steps)
Ethyl-3-[Fmoc-L-Ser]-(E)-
propenoate (62%)
SPPS
95% TFA
2.5% H2O
O
O
OH
O
Trt
2.5%TIS
HN
Peptide
N
H
COOEt
N
H
N
H
COOEt
Peptide
(1.5%-11% overall yield)
-Biotin)-Leu-(K(Biotin)L), or -Lys(e-Biotin)-Tyr-(K(Biotin)Y).
Scheme 2. Synthesis of C-terminal Michael acceptor (MA) probes. Peptide = -Leu-Thr-Glu-Lys-(LETK), -Lys(
e
2.4. In vivo labeling of Cwp84
Finally, we examined the ability of selected active ABPs to label
proteins in the S-layer of C. difficile. Following treatment of live
cells with the ABPs under the same inhibition conditions as above,
the cell lysates were subjected to Western blotting against Neutra-
vidin-horseradish peroxidase (HRP). Interestingly, the chloroacet-
amide CAA-LR-PEG3-Biotin provided the same labeling pattern as
our previously-reported epoxide ABP EtEP-LR-PEG3-Biotin, giving
a strong band at 74 kDa and a weaker band at slightly higher
apparent molecular weight (Fig. 2B). Previous studies strongly
suggest that Cwp84 is processed from an inactive zymogen to an
active form in the S-layer,34,41 and we have previously demon-
strated that these bands correspond to Cwp84, likely in pre-pro-
cessed and processed forms. The two C-terminal Michael
acceptor probes Ac-K(Biotin)YS-MA and Ac-K(Biotin)LS-MA both
strongly label the lower band, but do not appear to label the higher
molecular weight form of Cwp84. Both classes of new probe are
remarkably selective, with only minor biotinylated bands seen in
addition to Cwp84.
3. Conclusion
In summary, we report the discovery of two new classes of inhib-
itor for S-layer processing in C. difficile based on N-terminally linked
chloroacetamide (CAA) or C-terminal Michael acceptors (MA). The
structure–activity relationships for the CAA class follow those of
our previously reported N-terminal epoxy succinimidyl esters, and
show similarly highly selective irreversible labeling of Cwp84, the
protease responsible for SlpA processing. Taken together, these data
suggest acommon binding mode for the CAAs, and a mode of action
that encompasses aspects of the processing pathway for Cwp84.
These experiments also demonstrate that the warhead chemotype
Figure 2. (A) Testing N-terminal chloroacetamide (CAA) and C-terminal Michael
acceptor (MA)warheads in vivo against S-layer processing; K(Bt) = -biotinyl lysine;
structures of compounds are shown in Schemes 1 and 2. C. difficile strain 630 was
cultured overnight under anaerobic conditions in brain-heart infusion (BHI)
medium, and in the presence of 100 lM of each compound or DMSO vehicle, after
which surface layer proteins were isolated, run on SDS–PAGE, and Western blotted
against primary anti-LMW SLP antibody followed by horseradish peroxidase (HRP)
conjugated secondary antibody, with visualization by chemiluminescence. (B)
Labeling activity of selected N-terminal CAA and C-terminal MA activity-based
probes in vivo. Inhibited cell lysates were Western blotted against Neutravidin-
HRP, and visualized by chemiluminescence.
e

618
T. H. Tam Dang et al. / Bioorg. Med. Chem. 20 (2012) 614–621
is an important determinant for activity, supporting the hypothesis
that inhibition is partly dependent on warhead electrophilicity.
The C-terminal electrophile MA class shows activity comparable
to both the epoxide and CAA inhibitors, despite a markedly differ-
ent configuration. In contrast to previously reported inhibitors, this
class has the potential to mimic a well-conserved serine at P1 in
SlpA and furthermore presents the remaining upstream residues
(P2–P5) without the need to invoke inversion of the main chain.
It is interesting to note that analogous inhibitors with an MA war-
head at the N-terminus areinactive, suggesting that correct orien-
tation of the backbone and/or the presentation of a serine in P1
contributes significantly to binding and activity. The lack of label-
ing of the higher molecular weight form of Cwp84 may have its ori-
gins in greater selectivity for a putative activated form of the
enzyme over an inactive form. An alternative explanation is that
the other classes of inhibitor hit an off-target protease that can
process and activate Cwp84 in trans, and thus the unprocessed
form is present only when this putative off-target is also inhibited.
Our parallel experiments exploring Cwp84 processing are reported
elsewhere,42 however, both scenarios are consistent with the C-
terminal MA possessing greater selectivity, perhaps as a result of
a more substrate-mimetic design.
Amino acids: N-a-9-Fluorenylmethoxycarbonyl (N-a-Fmoc)
protected amino acids were obtained from Novabiochem UK with
the following side chain protecting groups: Arg(Pbf), Lys(Boc),
Tyr(tBu), Thr(tBu), Glu(tBu), Ser(tBu).
Other reagents: Peptide synthesis grade dimethylformamide
(DMF) was purchased from National Diagnostics, UK. Benzotria-
zole-1-yloxy-tris-pyrrolidino-phosphonium hexafluorophosphate
(PyBop), N-hydroxybenzotriazole (HOBt), diisopropylethylamine
(DIPEA) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylamini-
um hexafluorophosphate (HBTU) were purchased from Novabio-
chem UK and Sigma Aldrich. Dry tetrahydrofuran (THF) and DMF,
chloroform, dimethylsulfoxide (DMSO), dichloromethane (DCM),
methanol, piperidine, thioanisole, tert-butylmethylether (TBME),
trifluoroacetic acid (TFA), ethanedithiol (EDT), iodoacetonitrile,
3-mercaptoethyl propionate, sodium thiophenolate, monobasic
and dibasic sodium phosphate, trimethylsilylisopropane (TIPS)
were regent grade from Sigma Aldrich. A ‘deprotection mixture’
of TFA–H2O–TIPS (97:2.5:1) was used for cleavage and deprotec-
tion reactions.
4.1. General procedures
In future it will be interesting to further explore the structure–
activity relationships within these new inhibitor classes. For
example, the apparent selectivity of the Michael acceptors for the
processed form of Cwp84 is significant, as it suggests a chemical
approach for probing these processes in vivo. Further screening
of C-terminal electrophilic peptide inhibitors against C. difficile
strains with diverse SlpA cleavage sites could probe the determi-
nants for binding in a systematic fashion, and thus provide a sound
structural basis for the design of improved or strain-selective
Cwp84 inhibitors. In the present study, we have focused on the
surface layer proteins, but it will also be of interest to explore po-
tential membrane and intracellular targets of these and other ABPs
in C. difficile. More generally, the activity-based labeling provided
by these classes of inhibitor adds to a growing range of powerful
chemical tools for investigating biological systems.43–46 They also
suggest potential applications in imaging the process of S-layer
biogenesis at the level of catalytic activity of the enzymes that
mediate post-translational modification of SlpA.
4.1.1. Solid phase peptide synthesis (SPPS)
Automated solid phase synthesis was performed using an Ad-
vanced ChemTech Apex 396 multiple peptide synthesizer (Ad-
vanced ChemTech Europe, Cambridge, UK). Peptide synthesis was
carried out in peptide synthesis grade DMF. Resin (25
well) was swelled in DMF for 60 min before coupling each cycle,
N- -amino Fmoc group was treated with 20% (v/v) piperidine in
DMF for 15 min, with two further repetitions. A fivefold excess of
amino acid over resin reactive groups (125 mol, 250 L of 0.5 M
lmol per
a
l
l
solution) was used for each coupling. In situ activation and cou-
pling was carried out for 45 min with a mixture of HBTU/HOBt
(125
250
l
mol, 250
lL of 0.5 M solution) and DIPEA (125 lmol,
l
L of 0.5 M solution). The peptide was washed with DMF
(3 Â 1 mL) between each deprotection and coupling step. A typical
cycle consisted of deprotection, DMF wash, coupling and a further
DMF wash. The N-a-Fmoc protection at the final residue was re-
moved at the end of the synthesis under the usual conditions. After
synthesis was complete a preactivated solution of (2S,3S)-3-(eth-
oxycarbonyl)oxirane-2-carboxylic acid (12.01 mg, 3 equiv), HCTU
(51.7 mg, 3 equiv) and DiPEA (19.3 mg, 6 equiv) in DMF (1 mL)
was added to each well and agitated for 1 h. The peptidyl resin
was removed from the synthesizer, washed several times
(3 Â 2 mL DMF, 3 Â 2 mL DCM, 3 Â 2 mL methanol, 3 Â 2 mL dieth-
ylether) and dried in vacuo.
4. Experimental section
All general laboratory chemicals obtained from chemical sup-
pliers (Novabiochem UK or Aldrich Chemical Co.) were used with-
out further purification. Flash chromatography was performed on
Screening Devices Silica Gel 60 (0.04–0.063 mm). TLC-analysis
was conducted on DC-alufolien (Merck, Kiesel gel 60, F254) with
detection by UV-absorption (254 nm). 1H and 13C NMR spectra
were recorded on a Brüker AV-400 (400/100 MHz) spectrometer.
Chemical shifts (d) are given in ppm relative to tetramethylsilane
and chloroform, H2O or DMSO residual solvent peak was used as
internal standard. Coupling constants are given in Hertz (Hz).
Infrared spectra were obtained on PerkinElmer Spectrum 100 FT-
IR spectrophotometer, from a thin film deposited onto a sodium
chloride plate from dichloromethane. The IR spectra were then
analyzed with Spectrum Express, Version 1.01.00 (PerkinElmer).
Mass spectra and accurate mass data were obtained by J. Barton
at the Chemistry Department Mass Spectrometry Service (Imperial
College London) by electrospray ionisation or chemical ionisation
techniques.
4.1.2. Deprotection and cleavage from resin
Cleavage and deprotection of peptides was achieved by add-
ing 1.5 mL of deprotection mixture to dry peptidyl resin (25
lmol). The mixture was agitated on an orbital shaker for up to
3 h and then filtered. The resin was washed twice with a small
volume of TFA and the combined washings and filtrate were pre-
cipitated with 10 mL ice cold tert-butylmethylether (tBME). The
mixture was centrifuged at 5000 rpm for 15 min at 0 °C, the
supernatant discarded and the remaining peptide washed with
a fresh aliquot of tBME. The process was repeated three times
to ensure complete removal of all organic impurities. The crude
peptide was dried in a desiccator over silica gel to yield an off-
white solid.
Solid phase resins: Rink-amide resin and Biotin-PEG-Novatag re-
sin were purchased from Novabiochem UK. The resin substitution
ratios were as follows: Rink-amide resin: 0.71 mmol/g; Biotin-
PEG-Novatag resin: 0.48 mmol/g; Universal-PEG-Novatag resin:
0.33 mmol/g.
4.1.3. Peptide purification
Peptide analysis and purification was performed either on
Gilson RP-HPLC system or Waters LC–MS system.Purification of
crude peptides was performed on a Gilson semi-preparative
RP-HPLC system (Anachem Ltd, Luton, UK) equipped with 306

T. H. Tam Dang et al. / Bioorg. Med. Chem. 20 (2012) 614–621
619
pumps and a Gilson 155 UV/Vis detector. Analytical RP-HPLC
was performed on a Gilson analytical HPLC system (Anachem
Ltd, Luton, UK) equipped with a Gilson 151 UV/Vis detector
and Gilson 234 auto injector. For both HPLC systems, the peptide
bond absorption was detected at 223 nm. The following elution
methodwas used: 2–98% MeCN in H2O over 40 min; all solvents
used were supplemented with 0.1% TFAand were degassed with
helium. LC–MS analysis was performed on Waters HPLC system
(Waters 2767 autosampler for samples injection and collection;
Waters 515 HPLC pump to deliver the mobile phase to the
source; XBridge C18 column (Waters, 4.6 mm D Â 100 mm L for
analytical and 19 mm D Â 100 mm L for preparative); Waters
3100 mass spectrometer with ESI and Waters 2998 Photodiode
Array (detection at 200–600 nm)). The following elution method-
was used: 5–95% MeOH in H2O over 15 min. All solvents used
were supplemented with 0.1% formic acid and were degassed
with helium.
(OtBu)Ser-H (6.210 g) in THF (60 mL) at rt. The resulting mixture
was stirred for 24 h and then concentrated under reduced pres-
sure. The residue was purified by silica gel flash column chroma-
tography (Hexane–EtOAc, 9:1) to afford a white solid (0.662 g,
37% yield from Fmoc-L
-(OtBu)Ser-SBn). Rf = 0.51 (Hexane–EtOAc,
2:1); mp 50–53 °C; dH/ppm (400 MHz; CDCl3): 7.80 (2H, d,
J = 7.49, Fmoc-4 and 5), 7.63(2H, d, J = 7.12, Fmoc-1 and 8), 7.43
(2H, t, J = 7.40, Fmoc-3 and 6), 7.35 (2H, t, J = 7.11, Fmoc-2 and
7), 6.96 (1H, dd, J = 4.64, 15.63, S
a-CH), 6.00 (1H, d, J = 15.66,
CHCOOEt), 5.31 (1H, d, J = 7.85, NH), 4.49 (1H, s, S
a
), 4.45 (2H, d,
J = 6.88, Fmoc-CH2), 4.28 (1H, t, J = 6.92, Fmoc-9), 4.23 (2H, q,
J = 7.08, COOCH2CH3), 3.54–3.47 (2H, m, Sb), 1.33 (3H, t, J = 7.15,
COOCH2CH3), 1.21 (9H, s, tBu). mmax (neat)/cmÀ1: 3342, 1725
(CO), 1697, 1541, 1182, 1032, 881, 738. ESI-MS: m/z = found
438.2276 ([M+H]+), 460.2100 ([M+Na]+), (required 460.2100,
C26H31NO5Na).
4.2.4. Ethyl-3-Fmoc-
L-Ser-(E)-propenoate
-Ser(t-
4.2. Solution-phase synthesis and characterization
TFA (2 mL) was added to a solution of ethyl-3-[Fmoc-
L
Bu)]-(E)-propenoate (0.318 g, 0.833 mmol) in DCM (8 mL) at rt.
The reaction mixture was stirred for 2 h, and then the volatiles
were removed under reduced pressure. The residue was triturated
with a 1:1 mixture of Et2O and hexanes (3 mL), and the resulting
white solid was collected by filtration and washed thoroughly with
cold Et2O–Hexane (1:1) to afford the product (0.197 g, 0.518 mmol,
62% yield). Rf = 0.51 (Hexane–EtOAc, 2:1); mp 110–113 °C; dH/ppm
(400 MHz; CDCl3): 7.78 (2H, d, J = 7.50, Fmoc-4 and 5), 7.60 (2H, d,
J = 7.05, Fmoc-1 and 8), 7.41 (2H, t, J = 7.40, Fmoc-3 and 6), 7.32
4.2.1. Fmoc-L
-Ser(tBu)-SBn
DMAP (0.159 g, 1.30 mmol, 0.1 equiv) and DCC (2.820 g,
13.69 mmol, 1.05 equiv) were added sequentially to a solution
of Fmoc-L-Ser(tBu)-OH (5.000 g, 13.04 mmol, 1 equiv) and benzyl
mercaptan (3.06 mL, 26.08 mmol, 2 equiv) in THF (65 mL) at rt.
The cloudy reaction mixture was stirred at rt for 18 h and then
filtered. After concentrating the filtrate it was partitioned be-
tween EtOAc (70 mL) and 1.0 M HCl (40 mL). The organic layer
was dried over Na2SO4 and concentrated. Subsequent addition
of Et2O (15 mL) resulted in a white precipitate that was collected
by vacuum filtration and washed with petroleum ether. The
product was obtained as a yellow solid and used without further
purification (5.826 g, 8.18 mmol, 91% yield). Rf = 0.66 (Hexane–
EtOAc, 2:1); mp 163–165 °C; dH/ppm (400 MHz; CDCl3): 7.79
(2H, d, J = 7.50, Fmoc-4 and 5), 7.66–7.63 (2H, m, Fmoc-1 and
8), 7.41 (2H, app td, J = 2.72, 7.38, 7.35, Fmoc-3 and 6), 7.36–
7.30 (7H, m, Bn, Fmoc-2 and 7), 5.78 (1H, d, J = 8.94, NH), 4.55
(2H, t, J = 7.26, Fmoc-2 and 7), 6.92 (1H, dd, J = 4.19, 15.67, S
CH), 6.00 (1H, d, J = 15.70, CHCOOEt), 5.50 (1H, d, J = 7.80, NH),
4.50–4.36 (3H, m, Fmoc-CH2, S ), 4.29–4.09 (3H, m,CH2CH3,
a-
a
Fmoc-9), 3.75 (2H, s, Sb), 1.31 (3H, t, J = 7.14, COOCH2CH3). dC/
ppm (100 MHz; CDCl3): 166.26, 156.23 (CO), 145.10 (CH-Fmoc),
143.72, 141.33 (C-Fmoc), 127.78, 127.12, 125.00, 122.67, 120.03
(CH-Fmoc, CHCH), 66.91, 64.03, 60.76 (CH2), 53.80 (CH-Fmoc),
47.19 (Ca
), 14.21 (CH3). mmax (neat)/cmÀ1: 3320, 2974, 2878,
2839, 1716 (CO), 1692, 1535, 1461, 1377, 1287, 1212, 1016, 736.
ESI-MS: m/z = 382.1650 ([M+H]+), 405.1526 ([M+Na]+), (required
382.1576, C22H24NO5).
(2H, dd, J = 6.94, 10.40, Fmoc-CH2), 4.42–4.35 (1H, m, Sa), 4.29
(1H, t, J = 6.99, Fmoc-9), 4.16 (2H, dd, J = 13.76, 35.67, Sb), 3.96
(1H, dd, J = 2.50, 9.02, Bn-CH2), 3.57 (1H, dd, J = 3.44, 9.03, Bn-
CH2), 1.15 (9H, s, tBu). mmax (neat)/cmÀ1: 3322, 2954, 2936,
2908, 2853, 1724 (CO), 1684, 1625, 1554, 1509, 1392, 1331,
1234, 1174, 1071, 965, 698. HRMS (ESI): m/z = found 490.2039
([M+H]+), 512.1859 ([M+Na]+), (required 490.2052; C29H31NO4S
[M+H]+).
4.2.5. Ethyl-3-[Fmoc-
2-Chlorotrityl chloride resin (0.199 g, 1 equiv) was added to a
solution of ethyl-3-Fmoc- -Ser-(E)-propenoate (0.197 g, 0.518
mmol, 2 equiv) and pyridine (83.73 L, 1.035 mmol, 4 equiv) in
L-Ser(ClTrt resin)]-(E)-propenoate
L
l
dry THF (2 mL). The reaction mixture was heated at 60 °C with stir-
ring for 6 h. The resin was filtered and washed with DCM–MeOH–
DiPEA (17:2:1) and 3 Â DCM, 2 Â DMF, 2 Â DCM. Finally, the resin
was dried in vacuo over KOH. The yield of the loading was 72% as
determined by the Fmoc-test.
4.2.2. Fmoc-
Triethylsilane (6.50 mL, 40.90 mmol, 5 equiv) was slowly added
to a suspension of Fmoc- -(OtBu)Ser-SBn (4 g, 8.18 mmol, 1 equiv)
L
-Ser(tBu)-H
L
and Pd/C (10%, 2.20 g) in acetone (200 mL) at rt previously de-
gassed with N2. The reaction mixture was stirred for 15 min and
then filtered through Celite. The filtrate was concentrated to afford
6.21 g of brown oily crude, which was used without further purifi-
cation. Rf = 0.57 (Hexane–EtOAc, 3:2); dH/ppm (400 MHz; CDCl3):
9.66 (1H, s, CHO), 7.81 (2H, d, J = 7.34, Fmoc-4 and 5), 7.65 (2H,
dt, J = 5.74, 11.49, Fmoc-1 and 8), 7.44 (2H, t, J = 7.29, Fmoc-3
and 6), 7.36 (7H, dd, J = 4.98, 9.52, Fmoc-2 and 7), 5.70 (1H, d,
J = 7.30, NH), 4.46 (2H, dd, J = 1.41, 6.38, Fmoc-CH2), 4.41 (2H, dd,
4.2.6. Ethyl-3-[-L-Ser-Lys-Thr-Glu-Leu-Ac]-(E)-propenoate (Ac-
LETKS-MA)
Ac-LETKS-MA was synthesised by standard SPPS, starting from
the resin above. dH/ppm (400 MHz; D2O): 6.84 (1H, ddd, J = 3.41,
4.81, 15.79, CHCHCOOEt), 5.90 (1H, ddd, J = 1.72, 11.57, 15.86,
CHCHCOOEt), 4.88 (2H, d, J = 3.76, OH), 4.55 (1H, dt, J = 3.12,
6.34, NHCHCO), 4.49 (1H, dd, J = 5.03, 9.04, NHCHCO), 4.36 (2H,
dd, J = 5.39, 8.99, NHCHCO), 4.30 (1H, dd, J = 5.55, 8.79, NHCHCO),
4.25–3.98 (6H, m, CH2(OEt), 4 Â NHCHCO), 3.70–3.56 (2H, m,
CH(Thr), NHCHCH), 2.91 (2H, t, J = 7.33, CH2), 2.78 (1H, t, J = 6.26,
CH2(Ser)), 2.60–2.55 (1H, m, CH2(Ser)), 2.44–2.25 (2H, m, CH2),
2.13–2.00 (1H, m, CH(Leu)), 1.94 (3H, s, CH3(Ac)), 1.81–1.29 (10H,
m, 5 Â CH2), 1.19 (3H, t, J = 7.18, CH3(Thr)), 1.16–1.09 (3H, m,
CH3(OEt)), 0.84 (3H, d, J = 6.24, CH3(Leu)), 0.80 (3H, d, J = 6.24,
CH3(Leu)). Rt: 5.26.
J = 5.84, 10.17, Sa), 4.29 (1H, t, J = 6.86, 13.98, Fmoc-9), 4.00 (1H,
dd, J = 3.10, 9.30, Sb), 3.67 (1H, dd, J = 4.20, 9.38, Sb), 1.20 (9H, s,
tBu). ESI-MS: m/z = found 390.1681 [M+Na]+, (required 390.1681;
C22H25NO4Na).
4.2.3. Ethyl-3-[Fmoc-
(Carbethoxymethylene)-triphenylphosphane
6.135 mmol, 1.5 equiv) was added to a solution of crude Fmoc-
L
-Ser(tBu)]-(E)-propenoate
(2.137 g,
L
-

620
T. H. Tam Dang et al. / Bioorg. Med. Chem. 20 (2012) 614–621
4.3. Peptide characterization data
the mixture was centrifuged to remove bacteria (5 min at
10,000 g) and the supernatant neutralised by adding 2 M Tris por-
tion-wise (5 L at a time) until the desired pH was obtained.
l
Name
ESI-MS [M+H+]
Calculated
Rt
Yield
(%)
4.4.2. SDS–PAGE analysis
found
Standard glycine–Tris gels at 10% or 12% acrylamide were pre-
pared using mini-protean kits (Bio-Rad Laboratories). The separat-
ing gel (10 or 12% 39:1 acrylamide: bisacrylamide, 375 mM Tris pH
8.8, 0.1% SDS, 0.5% APS, 0.07% TEMED) was poured and ethanol
carefully layered on top. Once the gel was set, the ethanol was dis-
carded and the stacking gel (5% 39:1 acrylamide:bisacrylamide,
62.5 mM Tris pH 6.8, 0.1% SDS, 0.1% APS, 0.24% TEMED) was poured
and the comb put in place to form the wells. Once set, the comb
was removed and the gel was immersed in the running buffer. Pro-
tein samples were diluted in sample loading buffer. These, along
CAA-LR-PEG3-
Biotin
CAA-A-PEG3-
Biotin
CAA-R-PEG3-
Biotin
CAA-S-PEG3-
Biotin
FAA-LR-PEG3-
Biotin
FAA-A-PEG3-
Biotin
FAA-R-PEG3-
Biotin
FAA-S-PEG3-
Biotin
MA-LR-PEG3-
Biotin
MA-A-PEG3-
Biotin
MA-R-PEG3-
Biotin
MA-S-PEG3-
Biotin
792.4209
(C34H62ClN9O8S)
594.2728
(C25H45ClN5O7S)
679.3337
(C28H52ClN8O7S)
610.2677
(C25H45ClN5O8S)
776.4504
(C34H63FN9O8S)
578.3024
(C25H45FN5O7S)
663.3664
(C28H52FN8O7S)
594.2973
(C25H45FN5O8S)
842.4810
(C38H68N9O10S)
644.3329
(C29H50N5O9S)
729.3969
(C32H57N8O9S)
660.3278
792.4240 9.87 28
594.2730 9.45
679.3362 8.71 37
5
610.2681 9.16
776.4523 9.59
7
7
with a 10
lL aliquot of Broad Range Protein Marker (NEB), and a
578.3052 9.18 15
3.5 L aliquot of biotinylated protein ladder (Cell Signalling Tech-
l
nology) were heated to 100 °C for 5 min. Samples were loaded into
the wells of the gel and a constant voltage of 200 V applied for
40 min. For general visualisation of proteins, gels were stained in
Coomassie blue staining buffer for 4–16 h, followed by destaining.
Gels were image scanned and then rehydrated in 10% acetic acid
and dried between two cellulose membranes.
663.3661 8.46
594.2974 8.91
9
1
842.4819 10.39 10
644.3331 10.18 17
729.3961 9.33 39
660.3292 9.92 25
673.3775 5.26 1.5
832.4247 10.35 11
782.4479 10.55 4
4.4.3. Western blotting
Gels were transferred to PVDF membrane for immunoblotting.
SDS–polyacrylamide gels were transferred to Trans-blot™ transfer
medium (Bio-Rad Laboratories) PVDF membrane using a semi-dry
protocol. The membrane was prepared for transfer by incubation
sequentially in methanol (1–2 s), H2O (1–2 min) and finally in trans-
fer buffer (2 min). Gel and filter paper were pre-soaked in transfer
buffer. Filter paper, membrane and gel were stacked in the Trans-
blot™ SD semi-dry transfer cell (Bio-Rad) as a sandwich in the fol-
lowing order: filter paper-membrane-gel-filter paper. The transfer
was performed at 15 V for 15 min. After transfer, the membrane
was soaked in methanol (5 mL) and dried for 20 min, which avoids
the additional blocking step typically required for minimising non-
specific binding or back ground signal. The membrane was incu-
bated with primary antibody diluted to the specified ratio v/v in
10 mL PBS-T +3% (w/v) non-fat powdered milk for at least 1 h with
gentle rocking. After three washes in 10 mL PBS-T, secondary anti-
body diluted to the specified ratio v/v in 10 mL PBS-T+3% (w/v)
non-fat powdered milk was added and the membrane rocked gently
for at least 30 min. The membrane was washed three times in PBS-T.
Signal was detected using ECL reagents (Amersham Biosciences) or
SuperSignalÒ West Pico Chemiluminescent Substrate (Pierce) fol-
lowed by image capture using a LAS-3000 Image Reader (Fujifilm),
with appropriate sizing carried out using Microsoft Office Power-
(C29H50N5O10S)
673.3772
Ac-LTEKS-MA
(C30H53N6O11
Ac-K(Biotin)Y- 832.4279
MA (C40H62N7O10S)
Ac-K(Biotin)L- 782.4486
MA (C37H64N7O9S)
)
Note: all peptides were synthesized by standard SPPS (see above); %
overall yield is given relative to total resin load prior to loading of
the first amino acid.
4.4. Bacterial culture and biochemical methods
4.4.1. C. difficile culture conditions
C. difficile 630 was provided by Dr. Peter Mullany, Eastman Den-
tal Institute, London, and has been fully sequenced by the Well-
come Trust Sanger Institute.47 C. difficile was routinely cultured
either on blood agar base II (Oxoid Ltd, Basingstoke, UK) supple-
mented with 7% horse blood (TCS Biosciences, BotolphClaydon,
UK); or in brain-heart infusion (BHI) broth (Oxoid). Culture was
undertaken in an anaerobic cabinet (Don Whitley Scientific, Ship-
ley, UK) at 37 °C in a reducing anaerobic atmosphere (10% CO2,
10% H2, 80% N2). To obtain cells during exponential phase, an over-
night (ca. 16–18 h) liquid culture was inoculated 1:20 v/v into BHI
that had been pre-equilibrated (at least 2 h) to the temperature
and atmosphere of the anaerobic cabinet, and the culture was sha-
ken vigorously for 15 s to mix thoroughly. During growth, the cul-
ture liquid was agitated gently using the shaker at 200 rpm to
allow cells to disperse. In order to monitor the growth of C. difficile,
the OD600 of a sample was determined using WPA biowave CD8000
cell density meter (Isogen Life Science, UK). To extract surface layer
proteins C. difficile bacteria from a 16–18 h culture in BHI were har-
vested by centrifugation (15 min at 3500g) and resuspended in
0.04 volumes (relative to culture volume) of 0.2 M glycine–HCl
pH 2.2. After rotating incubation for 30 min at room temperature,
Point 2007.A rabbit
SLP, was used at 1/200,000 dilution to visualize SlpA as reported in
our previous work.34 Goat
-rabbit-HRP was used as secondary
a-LMW-SLP antibody, raised against 630 LMW
a
(DakoCytomation), at 1/2000 dilution.
4.4.4. NeutrAvidin™–HRP immunoblotting
SDS–polyacrylamide gels were transferred using the method
describe above, and the membranes were blocked for 1 h in
10 mL PBS-T containing 5% BSA (w/v) before washing for
3 Â 10 min in 10 mL PBS-T. The blots were probed using NeutrAvi-
din™-HRP at 1:5000 dilution in 10 mL PBS-T for 1 h at room tem-
perature. The membranes were then washed with 10 mL PBS-T for
3 Â 5 min before detection as described above.
Acknowledgments
We acknowledge financial support from the Medical Research
Council, UK (Grants G0701834 and G0900170) and the Department

T. H. Tam Dang et al. / Bioorg. Med. Chem. 20 (2012) 614–621
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