Unraveling the protein targets of vancomycin in living S. aureus and E. faecalis cells

Article abstract of DOI:10.1021/ja2039979

Vancomycin is a potent glycopeptide antibiotic that has evolved to specifically bind to the d-Ala-d-Ala dipeptide termini of nascent peptidoglycans. Although this mode of action is well established, several studies indicate that vancomycin and analogues e

Full text of DOI:10.1021/ja2039979

ARTICLE
pubs.acs.org/JACS
Unraveling the Protein Targets of Vancomycin in Living S. aureus
and E. faecalis Cells
J€urgen Eirich, Ronald Orth, and Stephan A. Sieber*
Center for Integrated Protein Science Munich CIPSM, Department of Chemistry, Institute of Advanced Studies IAS,
Technische Universit€at M€unchen, Lichtenbergstrasse 4, 85747 Garching, Germany
S Supporting Information
b
ABSTRACT: Vancomycin is a potent glycopeptide antibiotic
that has evolved to specifically bind to the D-Ala-D-Ala
dipeptide termini of nascent peptidoglycans. Although this
mode of action is well established, several studies indicate
that vancomycin and analogues exploit noncanonical target
sites. In order to address all vancomycin targets in clinically
relevant Staphylococcus aureus and Enterococcus faecalis strains
we developed a series of small-molecule photoaffinity probes
based on vancomycin. Proteomic profiling revealed the
specific labeling of two previously unknown vancomycin
targets that are likely to contribute to its antibiotic activity. The specific inhibition of the major staphylococcal autolysin
Atl confirms previous observations that vancomycin alters S. aureus cell morphology by interaction with the autolytic
machinery. Moreover, in E. faecalis the vancomycin photoprobe specifically binds to an ABC transporter protein, which likely
impedes the uptake of essential nutrients such as sugars and peptides. The labeling of these two prominent membrane targets in
living cells reveals a thus far unexplored mode of vancomycin binding and inhibition that could allow a rational design of
variants with improved activity.
’ INTRODUCTION
vancomycin analogue action have been reported, indicating that
the complex structure of this compound class is suitable to
interact with other targets than just D-Ala-D-Ala dipeptides.
Studies by Kahne et al. revealed the transglycosylase domain of
PBP1b as a specific binding site for a chlorobiphenyl-subsituted
vancomycin derivative that leads to inhibition of transglycosyla-
tion and therefore exerts a potent antibiotic effect even against
vancomycin-resistant enterococcal strains (VRE).11 Affinity
chromatography further verified the direct interaction of immo-
bilized vancomycin derivatives with this enzyme and other
enzymes in cell lysates.11,12 Moreover, recent investigations with
biotinylated vancomycin photoprobes indicate that vancomycin
binds to VanSsc kinase and induces the expression of resistance
genes in Enterococcus faecalis.13 Although this study was carried
out in Escherichia coli lysates and not in VRE, the labeling of the
whole proteome indicated the specific interaction with several
other proteins that remained unexplored.
Vancomycin is a potent glycopeptide antibiotic that specifi-
cally binds to the D-Ala-D-Ala dipeptide termini of nascent
peptidoglycan. The molecule forms a series of hydrogen bonds
with the peptide backbone that block its further processing by
penicillin binding proteins (PBPs) and thus the final steps of
peptidoglycan and cell wall biosynthesis.1ꢀ3 Although vancomy-
cin was kept as an antibiotic of last resort, resistances to this
unique mechanism of action have been observed which currently
limit its application as a drug to treat serious methicillin-resistant
Gram-positive infections. One prominent form of resistance,
observed in enterococcal strains, redesigns cell wall biosynthesis
to incorporate D-Ala-D-Lac (lactate) depsipeptide termini which
significantly reduces vancomycin binding due to a lack of a crucial
hydrogen bond.1 Multiresistant Staphylococcus aureus (MRSA, e.
g. Mu50 strain) have also been reported to display reduced
susceptibilities to vancomycin.4,5 One important feature of these
cells is a thickened cell wall that is rich in muropeptide compo-
nents which retain intact D-Ala-D-Ala binding sites and therefore
sequester substantial amounts of vancomycin distant to the cell
wall biosynthesis site.4ꢀ9 The binding to muropeptides further
leads to an inhibition of the S. aureus autolytic system by blocking
the access of staphylococcal hydrolases to the cell wall substrate
that is relevant for cell wall turnover, cell division, and cell
separation.5,7,10 Vancomycin-treated S. aureus cells therefore
display a lack of daughter cell separation, leading to multicellular
clusters. In addition, several other modes of vancomycin or
Although useful for the evaluation of compound selectivity,
profiling of cell lysates can cause artificial labeling effects due to
induced protein degradation and corresponding changes in
enzyme activities as well as the unrestricted access of molecules
to cytosolic proteins.14ꢀ16 We here introduce a novel generation
of vancomycin photoprobes that are capable of labeling protein
Received: May 1, 2011
Published: July 07, 2011
r
2011 American Chemical Society
12144
dx.doi.org/10.1021/ja2039979 J. Am. Chem. Soc. 2011, 133, 12144–12153
|

Journal of the American Chemical Society
ARTICLE
Figure 1. Structures of vancomycin-derived probes 1ꢀ3 and a benzophenone minimal probe (BP) carrying the photo-cross-linker (green) and the
alkyne handle (blue) on different positions.
targets in living bacterial cells that are crucial for vancomycin
activity.
introduces modifications at easy entry points such as the
C-terminal carboxylic acid as well as the free amine of the
vancosamine sugar. Second, the corresponding probes should
be detached from any bulky marker that would affect its inter-
action with targets during live cell labeling.17ꢀ19 We therefore
designed and synthesized three different probe scaffolds that
contain a benzophenone photo-cross-linker and a small alkyne
handle (Figure 1). The alkyne serves as a benign tag for the
modification with marker azides after live cell labeling via the
Huisgen/Sharpless/Meldal click chemistry (CC) reaction
(Figure 2).14,20ꢀ22 The benzophenone is necessary to establish
a covalent bond between the probe scaffold and the target
enzyme which is required for visualization, enrichment, and
isolation of the labeled proteins. Activation of hexynoic acid via
an NHS ester allowed a specific amide bond formation at the free
amine of the vancosamine sugar moiety (Scheme 1).23ꢀ28 Sub-
sequently, the free C-terminal carboxylic acid was modified with
a benzophenone amide under standard peptide coupling condi-
tions (probe 1). We utilized closely related synthetic procedures
to introduce another probe (probe 2) with the opposite decora-
tion (benzophenone at vancosamine sugar and alkyne at C-term-
inal carboxylic acid) as well as one probe (probe 3) in which both
functionalities are attached to the vancosamine sugar (Figure 1).
The in situ profiling of S. aureus (SA), methicillin-resistant
S. aureus (MRSA), vancomycin susceptible E. faecalis (VSE), and
vancomycin resistant E. faecalis (VRE) reveals the specific
labeling of two previously unknown vancomycin targets that
are likely to contribute to its antibacterial activity. In S. aureus, the
inhibition of the major staphylococcal autolysin Atl confirms
previous observations that vancomycin alters S. aureus cell
morphology.5,6,10 We here demonstrate that this is not solely
done by blocking the access of autolysin to their sequestered
peptidoglycan substrate but rather by a direct inhibition of the Atl
activity. Moreover, in VSE and VRE vancomycin specifically
binds to an ABC transporter protein, which likely impedes the
uptake of essential nutrients such as sugars and peptides. The
labeling of these two prominent membrane targets in living cells
reveals a, thus far, unexplored mode of vancomycin binding and
inhibition that could allow a rational design of variants with
improved activity.
’ RESULTS AND DISCUSSION
The design of vancomycin-based affinity probes has to fulfill
several prerequisites: First, we required a synthetic strategy that
12145
dx.doi.org/10.1021/ja2039979 |J. Am. Chem. Soc. 2011, 133, 12144–12153

Journal of the American Chemical Society
ARTICLE
Figure 2. Vancomycin-derived probes are covalently attached to the amino acid backbone of their target proteins via a benzophenone moiety as a photo-
activatable cross-linker. The alkyne group of the probes allows the identification of labeled enzymes by the introduction of a fluorescent tag via click
chemistry after cell lysis and SDS-gel electrophoresis.
Scheme 1. Synthetic Strategy Leading to Probe 1a
a The vancomycin glycopeptide core is depicted as a square in 5 and probe 1. Its peptide backbone runs as a horizontal diagonal (free carboxy terminus
left, methylated amino terminus right). The disaccharide is located on the top edge.
To test if and how the structural modifications influence the
antibiotic potency of all vancomycin probes we determined their
individual minimal inhibitory concentrations (MIC) against
S. aureus, MRSA, VSE, and VRE (Table 1). Unmodified vanco-
mycin and all three probes displayed potent activities against
S. aureus, MRSA, and VSE. As expected, a slight drop in potency
was observed for VRE. Interestingly, the MIC of probe 3 in VSE
drops below 0.6 μM, making it at least 4-fold more potent than
vancomycin itself. These intriguing results indicate that the new
probes exhibit all suitable traits for successful proteomic tools to
unravel vancomycin protein targets.23
To evaluate labeling properties of the vancomycin probes we
first tested conditions with bacterial lysates and finally optimized
probe concentration and time of irradiation with the pathogenic
strains VRE and MRSA in situ (Figure S1 in Supporting
Information [SI], Figure 3 AꢀC). The cells were treated with
various concentrations of probes and irradiated at 366 nm for
different durations. Subsequently, bacteria were lysed, rhodamine
12146
dx.doi.org/10.1021/ja2039979 |J. Am. Chem. Soc. 2011, 133, 12144–12153

Journal of the American Chemical Society
ARTICLE
Table 1. MIC Values (μM) for Vancomycin Hydrochloride
and the Probes 1ꢀ3
Atl pro-peptide is cleaved into several processing intermediates
that contain the two active domains together with different
repeat units.33,34 Since vancomycin probe incubation with SA
and MRSA revealed a strong labeling of an Atl fragment of about
63 kDa it is most likely that vancomycin selectively interacts
solely with the amidase unit of Atl. This was confirmed by protein
overexpression where all three probes labeled not only the full
length (ATLfl) but also the recombinant amidase functional
domain (ATLam) (Figure S2, SI).
strain
vancomycin ꢁ HCl probe 1 probe 2 probe 3
S. aureus Mu50
1.3
1.3
2.5
5
2.5
1.3
1.3
2.5
2.5
2.5
2.5
5
1.3
2.5
S. aureus NCTC 8325
E. faecalis OG1RF (VSE)
E. faecalis V583 (VRE)
<0.6
2.5
Previous phenotypic characterization of a S. aureus Atl dele-
tion mutant by G€otz et al. demonstrated that mutant cells grow in
large cell clusters which suggests that Atl is responsible for the
splitting of the septum during cell division to allow the separation
of daughter cells.31 This mutation was not lethal since a related
Aaa amidase partially recovered the loss of Atl activity. A double
mutation of both enzymes, however, significantly impaired
bacterial growth.35,36 In a different study by Tomasz et al. a
similar ΔAtl phenotype of cell clustering was observed when sub-
MIC concentrations of vancomycin were added to the culture
medium of S. aureus which strongly suggests an inhibition of the
autolytic process.5,10 The authors suggest that this inhibtion is
mediated by binding of vancomycin to muropeptides that
sterically blocks access of all cell wall hydrolytic enzymes to the
peptidoglycan substrate. Our results strongly emphasize that
vancomycin also directly binds to the amidase unit of Atl and
inhibits its enzymatic activity. This is further supported by an Atl
activity assay in which recombinant ATLam was incubated with
the natural peptidoglycan substrate. Atl degrades peptidoglycan,
which can be photometrically followed at 600 nm. The depen-
dence of the reaction rate on substrate concentration followed
MichaelisꢀMenten kinetics with a KM of 0.75 mM and a kcat of
710 sꢀ1. Enzyme inhibition occurred in a concentration-dependent
manner (Figure 4). Concentrations ranging from 75 nM to
300 μM of vancomycin show a slight to strong inhibition with an
IC50 value of 95 μM and a KI of 30 μM.
Taken together, our results show that vancomycin and corre-
sponding probes directly target the Atl amidase domain in the
presence and absence of peptidoglycan and also inhibit its
activity. It is therefore most likely that this direct interaction
further enhances the observed cell wall clustering phenotype that
is caused by steric clashes with the autolytic machinery. This dual
strategy of Atl amidase inhibition and limiting the access of all
hydrolytic enzymes to their peptidoglycan substrates ensures a
fine-tuned regulation of autolysis that may allow a basal level of
cell wall remodeling which is probably essential for bacterial
survival. One additional function of the autolytic machinery is to
trigger antibiotic induced cell degradation. Downregulation of auto-
lysis provides S. aureus a unique advantage to survive low-level
vancomycin concentrations in its microenvironment.10 In addition,
inhibition of the autolytic machinery increases the thickness of cell
walls that further sequester vancomycin molecules and also limits the
access of antibiotics to reach their cell wall biosynthesis targets. In
fact, these changes in cell morphology and autolytic activity are
observed among many strains of vacomycin-resistant S. aureus.4,8,9
While Atl was the most prominent target in staphylococci,
E. faecalis does not encode an amidase containing Atl, which
accounts for the lack of its detection by our affinity probes. In
contrast, probe labeling in living VRE and VSE revealed a peptide
ABC transporter as the major proteome target. Peptide ABC
transporters have a diverse array of functions in bacteria.37,38
Some transporters are specialized for the import of essential
nutrients such as sugars and peptides, while others carry out the
azide was attached to the probe scaffold under CC conditions,
and labeled proteins were visualized via fluorescent SDS gel
analysis. A proteome incubation with 1 μM of probe and an
irradiation time of 1 h turned out to be optimal in order to
achieve saturated target labeling (Figure 3 C). In situ labeling of
staphylococcal proteomes revealed that all three probes labeled a
protein in the membrane with an apparent molecular mass of
about 65 kDa (Figure 3B). Labeling of enteroccocal cells with all
probes under in situ conditions revealed labeling of a single
protein band with a molecular weight of about 40 kDa
(Figure 3A). As there were no observed differences between
S. aureus and MRSA as well as VSE and VRE, the labeled proteins
likely do not represent resistance-associated targets but rather
general interaction partners of vancomycin. All three probes
exhibited the same protein target preferences, which indicates
that protein labeling is driven solely by the scaffold and not by the
attached modifications. We therefore conducted all subsequent
studies with probe 1 as a representative example.
To unravel the identity of these two proteins we performed a
quantitative proteome analysis by attaching a trifunctional rho-
damineꢀbiotinꢀazide tag under CC conditions for the visuali-
zation, enrichment, and identification of proteins by SDS-gel
electrophoresis and mass spectrometry.29 Analysis of the peptide
fragments by the SEQUEST algorithm revealed strong hits for
the bifunctional autolysin (ATL) in case of MRSA and a peptide
ABC transporter (pABC) in case of VSE (Table S1, SI). The MS
results were independently confirmed by the recombinant over-
expression of both target proteins in E. coli and subsequent
labeling with probe 1 (Figure 3D). Atl was cloned and expressed
in full length (ATLfl) as well as in a shorter version with its
amidase domain (ATLam), which was sufficient for successful
probe interaction. Labeling occurred only with the native, folded
proteins and not with heat-treated unfolded proteins, emphasiz-
ing a specific interaction with the probes (Figure 3D). Moreover,
these proteins were not labeled by a benzophenone-alkyne
control peptide (Figure 1) which shows that target binding is
not mediated by the photo-cross-linker but solely by the vanco-
mycin scaffold (Figure S2, SI).30 Unmodified vancomycin com-
peted for ATLfl, ATLam, and pABC labeling with probe 1, which
further emphasizes a direct and selective interaction of the
unmodified natural product with these proteins (Figure 3E,
Figure S3, SI). Interestingly, these interactions are likely to
contribute to vancomycin antibiotic activity since both targets
are important for bacteria to maintain their cell wall (ATL) and
import nutrition (pABC).
In order to maintain cell integrity, viability, division, and
growth, peptidogylcan must be continuously synthesized and
degraded.31 Peptidoglycan hydrolases are crucial for the degra-
dation process and Atl represents the predominant hydrolase in
staphylococci. Atl is a 147 kDa bifunctional enzyme with a
62 kDa N-acetylmuramyl-L-alanine amidase (am) and a 51 kDa
endo-β-N-acetylglucosaminidase domain.32 After translation, the
12147
dx.doi.org/10.1021/ja2039979 |J. Am. Chem. Soc. 2011, 133, 12144–12153

Journal of the American Chemical Society
ARTICLE
Figure 3. Labeling of bacterial pathogens by vancomycin-based probes 1, 2 and 3. Membrane proteins of VRE (A) and MRSA (B) after in vitro (left) and
in situ (right) labeling. (C) Time- and concentration-dependent labeling in VRE and MRSA. (D) Recombinantly expressed ATLfl, ATLam, and pABC.
NI = before induction, I = induced, Δ = heat control, P = protein in the proteome. (E) Competitive, concentration-dependent displacement of probe 1
by unmodified vancomycin in labeling experiments with overexpressed target proteins.
Figure 4. Interaction of vancomycin with ATLam via a peptidoglycan turnover assay. (A) LineweaverꢀBurk plot for the degradation of peptidoglycan
by ATLam to determine KM and kcat (B) Concentration-dependent inhibition of ATLam by vancomycin.
export of toxins or, in the case of virulent or drug-resistant bacteria,
antibiotics.39 E. faecalis encodes a large number of sugar-uptake
systems such as ABC transporters that support bacteria to ferment
nonabsorbed sugars in the gastrointestinal tract.40 The ABC trans-
porter identified here represents a thus far uncharacterized protein
that is most likely not involved in antibiotic resistance as it is present
in both VRE and VSE strains.41 This is supported by gene expression
data that show that there is no increased transcription upon
incubation of bacteria with vancomycin (see Table S2 ([SI]).
utilizes affinity-based photo-cross-linking probes. Three func-
tional vancomycin probes were designed, and two unique
structural modifications even exceeded the antibiotic potency
of the unmodified natural product. It is well established that the
binding of vancomycin to the D-Ala-D-Ala motif of nascent
peptidoglycan and the corresponding inhibition of cell wall
biosynthesis comprise the primary mode of action, which leads
to autolysin-triggered cell rupture and death. However, the direct
inhibition of the Atl amidase domain represents a mode of action
that causes massive defects in cell morphology and enhances the
tolerance of S. aureus to low concentrations of vancomycin. This
provides S. aureus with an efficient strategy to evade low
vancomycin concentrations without acquiring resistance on the
’ CONCLUSION
In conclusion, we unraveled the protein targets of vancomycin
in living bacterial cells by a chemical proteomic strategy that
12148
dx.doi.org/10.1021/ja2039979 |J. Am. Chem. Soc. 2011, 133, 12144–12153

Journal of the American Chemical Society
ARTICLE
genetic level. The binding of vancomycin to Atl provides an
intriguing perspective to design improved vancomycin inhibitors
that exhibit lower affinity for this target and therefore increase
antibacterial susceptibility in S. aureus and MRSA.
1H NMR (600 MHz, d6-DMSO, δ/ppm): 9.45 (s, 1H), 9.18 (s, 1H),
9.08 (s, 1H), 8.70 (d, J = 18.9 Hz, 1H), 8.57 (s, 1H), 7.89ꢀ7.79 (m, 1H),
7.68 (s, 1H), 7.52 (dd, J = 14.6, 8.4 Hz, 1H), 7.47 (s, 1H), 7.44 (d, J = 8.4
Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.17ꢀ7.13
(m, 1H), 7.05 (s, 1H), 6.99 (s, 1H), 6.77 (dd, J = 8.4, 2.0 Hz, 1H), 6.71
(d, J = 8.5 Hz, 2H), 6.68 (s, 1H), 6.58 (s, 1H), 6.40 (d, J = 6.6 Hz, 1H),
6.25 (d, J = 2.2 Hz, 1H), 5.98 (d, J = 6.5 Hz, 1H), 5.93 (s, 1H), 5.75 (d, J =
7.9 Hz, 1H), 5.67 (s, 1H), 5.60 (s, 1H), 5.35 (d, J = 5.0 Hz, 1H), 5.31 (dd,
J = 6.7, 3.6 Hz, 1H), 5.28 (d, J = 7.7 Hz, 1H), 5.17 (s, 1H), 5.10 (d, J = 6.6
Hz, 1H), 5.08 (d, J = 5.1 Hz, 1H), 5.04 (d, J = 5.1 Hz, 1H), 4.92 (s, 1H),
4.69ꢀ4.63 (m, 1H), 4.43 (s, 1H), 4.42 (s, 1H), 4.32 (s, 1H), 4.18 (s,
1H), 4.05 (t, J = 5.4 Hz, 1H), 3.69 (d, J = 5.2 Hz, 1H), 3.53 (d, J = 8.1 Hz,
1H), 3.49 (d, J = 6.0 Hz, 1H), 3.44 (d, J = 6.3 Hz, 1H), 3.30ꢀ3.22 (m,
3H), 3.22 (s, 1H), 2.77 (t, J = 2.6 Hz, 2H), 2.63ꢀ2.60 (m, 1H),
2.28ꢀ2.21 (m, 5H), 2.16ꢀ2.06 (m, 4H), 1.97 (d, J = 13.0 Hz, 1H), 1.79
(d, J = 12.7 Hz, 1H), 1.70ꢀ1.51 (m, 2H), 1.40 (s, 3H), 1.10 (d, J = 6.3
Hz, 3H), 0.91 (d, J = 5.1 Hz, 3H), 0.86 (d, J = 5.6 Hz, 3H).
’ EXPERIMENTAL SECTION
General. All chemicals were of reagent grade or better and used
without further purification. Chemicals and solvents were purchased
from various commercial sources. For all reactions, only commercially
available solvents of purissimum grade, dried over molecular sieves and
stored under an argon atmosphere were used. Solvents for chromatog-
raphy and workup purposes were generally of reagent grade and distilled
before use. In all reactions, temperatures were measured externally. All
NMR spectra of probe 1 and its precursor 5 were recorded with devices
1
of the Bavarian NMR Center (for H and 13C spectra see Schemes
13C NMR (151 MHz, d6-DMSO, δ/ppm): 174.1, 172.6, 171.2, 170.8,
170.4, 169.5, 169.1, 167.8, 166.8, 157.2, 156.5, 155.1, 152.3, 151.3, 149.9,
148.2, 142.5, 139.8, 136.1, 135.7, 134.6, 131.9, 128.6, 127.3, 127.2, 126.2,
125.5, 124.3, 123.4, 121.6, 118.0, 116.2, 107.2, 105.7, 104.6, 102.3, 101.3,
96.7, 84.3, 78.1, 77.0, 76.7, 71.6, 71.3, 70.7, 70.1, 69.7, 63.1, 61.8, 61.2,
61.0, 58.4, 56.7, 54.9, 53.9, 53.7, 50.9, 41.0, 37.1, 33.2, 32.5, 27.8, 24.0,
23.8, 22.7, 22.4, 22.2, 17.7, 16.9.
S1ꢀS3 in SI). Other 1H, 13C, and two-dimensional (2D) NMR spectra
were measured with Bruker Avance 250, Avance 360, or Avance 500 (in
cases of 7 and probes 2 and 3 equipped with a CryoProbe) with B-ACS-
60/120 auto-samplers and referenced to the residual proton or carbon
signal of the deuterated solvent, respectively. ESI mass spectra were
recorded with Thermo LTQ Orbitrap XL or LTQ FT Ultra equipped
with Dionex UltiMate 3000 Nano or Rapid Separation LC Systems.
HPLC analysis was accomplished with a Waters 2695 separations
module, a X-Bridge BEH130 C18 column (4.6 mm ꢁ 100 mm), and
a Waters 2996 PDA detector.
HR-ESI-MS, positive mode (m/z): 1542.4782 [M + H]+ (calc:
1542.4793).
Synthesis of N-(3-Aminopropyl)-4-benzoylbenzamide
6.26,28 4-Benzoylbenzoic acid (500 mg, 2.2 mmol, 1 equiv) was
dissolved with DMAP (5 mg, cat.) in 5 mL of DMF and stirred at
22 °C for 10 min with DIC (379 μL, 2.4 mmol, 1.1 equiv) for
preactivation. 1,3-Diaminopropane (184 μL, 2.2 mmol, 1 equiv) was
added, and the reaction was stirred 2 h at 22 °C.
Synthetic Strategy to Vancomycin-Based ABPP-Probes.
The synthesis was based on commercially available vancomycin hydro-
chloride. Standard peptide-coupling methods were used to modify the
glycopeptide antibiotic as previously described.23 Full characterization
by NMR and HR-MS/MS was carried out for the probes.
Synthesis of 2,5-Dioxopyrrolidin-1-yl hex-5-ynoate 4.24,25
N-Hydroxysuccinimide NHS (1.08 g, 9.35 mmol, 1.05 equiv) and N,N0-
diisopropylcarbodiimide (DIC) (1450 μL, 9.35 mmol, 1.05 equiv) were
added to a solution of 5-hexynoic acid (1.00 g, 984 μL, 8.92 mmol,
1 equiv) in 40 mL of CH2Cl2. The reaction was stirred for 16 h at rt. This
mixture was washed with 30 mL of H2O three times, and the combined
inorganic phases were re-extracted with 30 mL of DCM. The combined
organic phases were washed with sat. NaCl and dried over Na2SO4, and
the solvent was removed in vacuo. The crude product was purified by
column chromatography (SiO2, ethyl acetate/isoHexan = 2:3, Rf = 0.47)
yielding 1.19 g of a yellow oil (64%).
Thirty milliliters of 0.1% (v/v) TFA in H2O was added, and the aq
phase was extracted twice with 30 mL of EtOAc. The combined organic
phases were extracted with 50 mL of 0.1% (v/v) TFA in H2O. The aq
phases were pooled, and the solvent was removed in vacuo; 447 mg of a
yellow foam was obtained (72%) and used without further purification.
1H NMR (360 MHz, d6-DMSO, δ/ppm): 8.01 (d, J = 8.3 Hz, 2H,
Harom), 7.79 (d, J = 8.4 Hz, 2H, Harom), 7.66 (dd, J = 6.3, 2.3 Hz, 2H, 2 ꢁ
Harom), 7.44 (dd, J = 6.6, 3.6 Hz, 1H, 1 ꢁ Harom), 7.12 (dd, J = 6.3, 3.0
Hz, 2H, 2 ꢁ Harom), 3.44ꢀ3.01 (m, 2H, 2 ꢁ CdONHCH2), 2.88 (t, J =
11.8 Hz, 2H, 2 ꢁ CH2NH2), 1.88ꢀ1.73 (m, 2H, 2 ꢁ CH2CH2CH2).
13C NMR (91 MHz, d6-DMSO, δ/ppm): 196.7 (PhCdOPh), 162.3
(CdONH), 142.9 (CqCdO), 129.7 (2 ꢁ Carom), 129.6 (Carom), 128.6
(2 ꢁ Carom), 127.3 (Carom), 122.5 (2 ꢁ Carom), 118.1 (2 ꢁ Carom), 111.2
(Carom), 36.8 (NHCH2), 35.8 (CH2NH2), 23.3 (CH2CH2CH2).
HR-ESI-MS, positive mode (m/z): 283.1448 [M + Na]+ (calc:
283.1441).
N0-Hex-5-ynamide C-(3-Aminopropyl)-4-benzoylbenzamide
Vancomycin Probe 1.23 Vancomycin derivative 5 (20 mg, 13 μmol,
1 equiv) was dissolved together with 6 (7.3 mg, 26 μmol, 2 equiv) in
600 μL of DMF. DIPEA (11.4 μL, 65 μmol, 5 equiv), PyBOP (7.4 mg,
14 μmol, 1.1 equiv), and HOBT (13% H2O, 2.1 mg, 14 μmol, 1.1 equiv)
were added in sequence, and the reaction mixture was shaken for 16 h at
22 °C in the dark. The reaction was crashed into 6 mL of ice-cold Et2O.
The precipitate that formed was centrifuged at 10000g and 4 °C for
15 min, and the clear supernatant was removed. The pellet was air-dried
for 30 min, and the crude product was purified via HPLC, yielding 1.7 mg
of a colorless solid (7%).
1H NMR (360 MHz, CDCl3, δ/ppm): 2.86 (s, 4H, 2 ꢁ NCOCH2),
2.79 (t, J = 7.4 Hz, 2H, COCH2), 2.37 (td, J = 6.9, 2.6 Hz, 2H, CqCH2),
2.03 (t, J = 2.6 Hz, 1H, CqCH), 1.98 (m, 2H CH2CH2CH2).
13C NMR (91 MHz, CDCl3, δ/ppm): 169.0 (2 NCdO), 168.2
(1 OCdO), 82.4 (1 Cq), 69.8 (1 CH), 29.7 (1 CH2), 25.6 (2 CH2), 23.3
(1 CH2), 17.6 (1 CH2).
HR-ESI-MS, positive mode (m/z): 232.0591 [M + Na]+ (calc:
232.0580).
Synthesis of N0-Hex-5-ynamide Vancomycin 5.27 Vancomy-
cin hydrochloride (300 mg, 202 μmol, 1 equiv) was dissolved in 2 mL of
DMF. N,N0-Diisopropylethylamine (DIPEA) (176 μL, 1.0 mmol,
5 equiv) and a solution of 4 (134 mg, 58 μL, 343 μmol, 1.7 equiv) in
1 mL of DMF were added. The reaction was stirred 24 h at rt and was
subsequently poured into 30 mL of ice cold Et2O. The resulting
precipitate was centrifuged at 10000g at 4 °C for 15 min. The colorless
supernatant was decanted and the pellet air-dried for 30 min. The
residue was purified by HPLC, yielding 135 mg of a colorless solid
(43%).
HPLC analysis, mobile phase (HPLC grade): A = water with 0.1% (v/v)
TFA, B = acetonitrile with 0.1% (v/v) TFA. Gradient: T0: B = 0%; T70:
B = 70%. Retention time: 33.0 min.
HPLC analysis, mobile phase (HPLC grade): A = water with 0.1% (v/v)
TFA, B = acetonitrile with 0.1% (v/v) TFA. Gradient: T0: B = 0%; T70:
B = 70%. Retention time: 18.0 min.
1H NMR (500 MHz, d6-DMSO, δ/ppm): 9.33 (s, 1H), 9.05 (s, 1H),
8.79 (s, 1H), 8.67 (s, 1H), 8.52 (s, 1H), 8.01 (d, J= 8.4 Hz, 2H), 7.86 (s, 1H),
12149
dx.doi.org/10.1021/ja2039979 |J. Am. Chem. Soc. 2011, 133, 12144–12153

Journal of the American Chemical Society
ARTICLE
7.81 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 7.1 Hz, 2H), 7.71 (s, 1H), 7.60
(s, 1H), 7.58 (s, 2H), 7.57 (s, 1H), 7.45 (s, 1H), 7.43 (s, 1H), 7.31 (d, J =
8.3 Hz, 1H), 7.22 (s, 1H), 7.17 (s, 1H), 6.98 (s, 2H), 6.76 (d, J = 8.4 Hz,
1H), 6.71 (s, 1H), 6.69 (s, 1H), 6.56 (s, 1H), 6.36 (d, J = 1.8 Hz, 1H),
6.27 (d, J = 2.0 Hz, 1H), 5.93 (s, 1H), 5.79 (s, 1H), 5.75 (s, 1H), 5.69 (s,
1H), 5.62 (s, 1H), 5.32 (s, 1H), 5.31 (s, 1H), 5.31 (s, 1H), 5.30 (s, 1H),
5.18 (s, 1H), 5.17 (s, 1H), 5.16 (s, 1H), 4.93 (s, 1H), 4.66 (d, J = 6.7 Hz,
1H), 4.46 (s, 1H), 4.45 (s, 1H), 4.37 (d, J = 5.4 Hz, 1H), 4.24 (d, J = 10.4
Hz, 1H), 4.05 (s, 1H), 3.68 (d, J = 10.1 Hz, 1H), 3.55 (d, J = 8.8 Hz, 1H),
3.52 (d, J = 10.9 Hz, 1H), 3.40 (covered by H2O, 3H), 3.30ꢀ3.23 (m,
3H), 3.20 (s, 1H), 3.03ꢀ2.99 (m, 2H), 2.75 (t, J = 2.6 Hz, 1H), 2.65 (s,
1H), 2.52 (s, 1H), 2.27ꢀ2.13 (m, 5H), 2.14ꢀ2.04 (m, 8H), 1.98 (d, J =
13.0 Hz, 1H), 1.79 (d, J = 9.8 Hz, 1H), 1.65 (s, 1H), 1.60ꢀ1.55 (m, 1H),
1.44ꢀ1.39 (m, 1H), 1.23 (s, 1H), 1.02 (d, J = 6.3 Hz, 3H), 0.91 (d, J =
6.1 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H).
63.17, 61.85, 61.27, 59.60, 58.92, 54.90, 53.99, 53.79, 51.12, 40.12, 35.51,
32.77, 31.28, 24.76, 23.74, 22.81, 22.35, 17.47.
ESI-MS, positive mode (m/z): 1658.4966 [M + H]+ (calc: 1658.4972).
N0-Benzoylbenzamide C Propargylamide Vancomycin
Probe 2.23 To a solution of vancomycin derivative 7 (50 mg,
30 μmol, 1.05 equiv) in 300 μL of DMF 3-amino-1-propyne (1.9 μL,
29 μmol, 1.0 equiv) were added HOBT (13% H2O, 5 mg, 32 μmol, 1.1
equiv), PyBOP (17 mg, 32 μmol, 1.1 equiv), and DIPEA (15 μL,
90 μmol, 3.2 equiv), and the reaction was stirred 2 h at 22 °C. The
reaction mixture was subsequently poured into ice-cold Et2O and
treated as described before for probe 1.
The residue was purified by HPLC, yielding 38 mg of a colorless solid
(75%).
HPLC analysis, mobile phase (HPLC grade): A = water with 0.1% (v/v)
TFA, B = acetonitrile with 0.1% (v/v) TFA. Gradient: T0: B = 0%; T70: B =
70%. Retention time: 22.2 min.
13C NMR (126 MHz, d6-DMSO, δ/ppm): 195.43, 170.80, 170.35,
170.20, 169.30, 169.22, 169.14, 168.40, 167.70, 166.07, 165.84, 157.07,
156.32, 155.10, 152.53, 151.12, 150.23, 148.27, 142.29, 140.34, 139.18,
137.73, 136.67, 135.61, 133.04, 129.73, 129.68, 129.56, 128.69, 127.43,
127.37, 127.28, 127.22, 127.02, 126.44, 126.25, 125.39, 125.36, 124.35,
123.34, 121.84, 118.34, 118.17, 116.25, 115.96, 107.64, 106.28, 104.83,
102.09, 101.13, 97.55, 84.26, 77.76, 77.13, 76.74, 71.89, 71.39, 70.28,
70.04, 69.66, 63.25, 62.04, 61.30, 61.15, 58.85, 57.87, 54.92, 53.92, 53.71,
51.09, 40.10, 36.79, 36.33, 34.99, 34.52, 31.25, 29.29, 25.95, 24.34, 23.68,
23.07, 22.73, 22.36, 17.41, 17.34.
1H NMR (500 MHz, d6-DMSO, δ/ppm): 9.34 (s, 1H), 9.00 (s, 2H),
8.69 (s, 1H), 8.64 (s, 1H), 8.48 (s, 1H), 8.39 (s, 2H), 7.87 (d, J = 8.0 Hz,
2H), 7.82 (d, J = 7.8 Hz, 2H), 7.77 (d, J = 7.7 Hz, 2H), 7.74 (d, J = 7.4 Hz,
2H), 7.65 (s, 1H), 7.58 (s, 1H), 7.56 (s, 1H), 7.46 (s, 1H), 7.34 (s, 1H),
7.29 (s, 1H), 7.20 (s, 1H), 7.12 (d, J = 8.2 Hz, 1H), 7.08 (d, J = 8.0 Hz,
1H), 6.75 (s, 1H), 6.71 (s, 1H), 6.69 (s, 1H), 6.66 (s, 1H), 6.38 (d, J = 7.4
Hz, 1H), 6.22 (s, 1H), 5.96 (s, 1H), 5.84 (s, 1H), 5.76 (s, 1H), 5.63
(s, 1H), 5.52 (s, 1H), 5.34 (s, 1H), 5.32 (s, 1H), 5.30 (s, 1H), 5.25
(s, 1H), 5.24 (s, 1H), 5.19 (s, 1H), 4.94 (s, 1H), 4.77 (d, J = 6.2 Hz, 1H),
4.55 (s, 1H), 4.45 (s, 1H), 4.40 (s, 1H), 4.22 (s, 1H), 3.96 (s, 1H),
3.91ꢀ3.84 (m, 1H), 3.69 (s, 1H), 3.28 (s, 3H), 3.13 (s, 1H), 2.81
(s, 1H), 2.64 (s, 1H), 2.36 (s, 1H), 2.26ꢀ2.11 (m, 6H), 2.01ꢀ1.93 (m,
1H), 1.80ꢀ1.64 (m, 2H), 1.58 (s, 1H), 1.28 (s, 1H), 1.07 (s, 1H), 0.96
(d, J = 6.3 Hz, 3H), 0.91 (d, J = 5.7 Hz, 3H), 0.86 (d, J = 5.9 Hz, 3H).
13C NMR (126 MHz, d6-DMSO, δ/ppm): 195.46, 170.92, 170.10,
169.13, 169.10, 169.05, 168.19, 167.72, 166.13, 165.00, 158.15, 157.15,
155.11, 152.55, 151.18, 150.19, 148.23, 142.38, 139.24, 138.83, 137.47,
136.72, 136.34, 133.06, 129.87, 129.72, 129.45, 128.71, 127.47, 127.44,
127.27, 127.16, 126.93, 126.42, 126.18, 125.46, 125.41, 124.42, 123.43,
121.82, 118.09, 117.92, 116.33, 115.72, 109.58, 106.46, 104.79, 102.15,
101.26, 96.81, 81.25, 77.21, 77.13, 76.81, 73.00, 71.36, 70.85, 70.28,
69.97, 63.15, 62.02, 61.30, 61.24, 58.96, 57.48, 55.02, 53.87, 53.77, 51.12,
40.19, 35.47, 32.74, 31.23, 28.16, 24.79, 22.82, 22.35, 17.48, 16.81.
ESI-MS, positive mode (m/z): 1696.5234 [M + H]+ (calc:
1696.5225).
HR-ESI-MS, positive mode (m/z): 1806.6060 [M + H]+ (calc:
1806.6056).
N0-Benzoylbenzamide Vancomycin 7.23 To a solution of
vancomycin hydrochloride (100 mg, 68 μmol, 1.0 equiv) in 600 μL of
DMF was added DIPEA (33 μL, 201 μmol, 3.0 equiv). 4-Benzoylben-
zoic acid (16 mg, 71 μmol, 1.05 equiv) was preactivated with PyBOP
(39 mg, 74 μmol, 1.1 equiv), HOBT (13% H2O, 11 mg, 74 μmol, 1.1
equiv), and DIPEA (11 μL, 68 μmol, 1 equiv) in 200 μL of DMF. After
10 min the two solutions were combined and shaken for 2 h at 22 °C.
The reaction mixture was subsequently poured into 20 mL of ice-cold
Et2O. The resulting precipitate was treated as described before in the
case of probe 1. The residue was purified by HPLC, yielding 62 mg of a
colorless solid (56%).
HPLC analysis, mobile phase (HPLC grade): A = water with 0.1%
(v/v) TFA, B = acetonitrile with 0.1% (v/v) TFA. Gradient: T0: B = 0%;
T70: B = 70%. Retention time: 21.6 min.
1H NMR (500 MHz, d6-DMSO, δ/ppm): 9.47 (s, 1H), 9.19 (s, 1H),
9.09 (s, 1H), 8.70 (s, 1H), 8.57 (s, 1H), 7.95 (d, J = 5.9 Hz, 2H), 7.86 (s,
1H), 7.82 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 8.1 Hz, 2H), 7.67 (s, 1H), 7.59
(s, 1H), 7.58 (s, 2H), 7.56 (s, 1H), 7.46 (s, 1H), 7.45 (s, 1H), 7.35
(s, 1H), 7.29 (d, J = 4.1 Hz, 1H), 7.20 (s, 1H), 7.11 (s, 2H), 6.77 (s, 1H),
6.73 (s, 1H), 6.72 (s, 1H), 6.70 (s, 1H), 6.40 (d, J = 7.8 Hz, 1H), 6.25
(d, J = 4.5 Hz, 1H), 5.97 (br s, 1H), 5.77 (s, 1H), 5.76 (s, 1H), 5.62
(s, 1H), 5.52 (s, 1H), 5.34 (s, 1H), 5.32 (s, 1H), 5.31 (s, 1H), 5.24
(s, 1H), 5.23 (s, 1H), 5.20 (s, 1H), 5.11 (s, 1H), 4.94 (s, 1H), 4.79ꢀ4.74
(m, 1H), 4.67 (d, J = 5.3 Hz, 1H), 4.46 (s, 1H), 4.43 (d, J = 5.4 Hz, 1H),
4.20 (s, 1H), 3.95 (s, 1H), 3.70 (d, J = 10.3 Hz, 1H), 3.58 (s, 1H), 3.53
(s, 1H), 3.36 (s, 1H), 3.30ꢀ3.17 (m, 3H), 3.14 (s, 1H), 2.81 (s, 1H),
2.67ꢀ2.62 (m, 2H), 2.28ꢀ2.12 (m, 5H), 2.00ꢀ1.86 (m, 6H),
1.78ꢀ1.52 (m, 5H), 1.09ꢀ1.06 (m, 1H), 0.96 (d, J = 6.4 Hz, 3H),
0.91 (d, J = 5.9 Hz, 3H), 0.86 (d, J = 6.0 Hz, 3H).
3-(4-Benzoylphenyl)-2-(hex-5-ynamido)propanoic Acid 8:
BP Minimal Probe.30 2-Amino-3-(4-benzoylphenyl)propanoic acid
(300 mg, 1.1 mmol, 1.0 equiv) was dissolved in 4 mL of CH2Cl2 with
DIPEA (474 μL, 2.8 mmol, 2.5 equiv). 1 (242 μL, 1.5 mmol, 1.3 equiv)
was added and the reaction was stirred 1.5 h at 22 °C. The solvent was
removed in vacuo and the crude product purified via HPLC yielding 342
mg of a yellow foam (85%).
HPLC analysis, mobile phase (HPLC grade): A = water with 0.1% (v/v)
TFA, B = acetonitrile with 0.1% (v/v) TFA. Gradient: T0:B=0%; T1: B= 60%;
T7: B = 80%. Retention time: 3.5 min.
1H NMR (500 MHz, CDCl3, δ/ppm): 10.00 (s, 1 H, CO2H), 7.75 (d,
J = 7.1 Hz, 2 H, Harom.), 7.71 (d, J = 8.2 Hz, 2 H, Harom), 7.58 (t, J = 7.4
Hz, 1 H, Harom), 7.46 (t, J = 7.7 Hz, 2 H, Harom), 7.29 (d, J = 8.1 Hz, 2 H,
Harom), 6.61 (d, J = 7.6 Hz, 1 H, NH), 4.94 (ps. q, J = 6.1 Hz, 1 H,
CHꢀCO2H), 3.35 (dd, J = 14.1, 5.7, 1 H, CaromꢀCHaHb), 3.19 (dd,
1 H, J = 14.0, 6.4 Hz, CaromꢀCHaHb), 2.36 (t, J = 7.3, 2 H,
NHCdOCH2), 2.25ꢀ2.15 (m, 2 H, HCtCCH2), 1.95 (t, J = 2.6
Hz, 1 H, HCtC), 1.81 (ps. quint, J = 6.8 Hz, 2 H, HCtCꢀCH2CH2).
13C NMR (90 MHz, CDCl3, δ/ppm): 196.6, 173.8, 172.9, 141.0,
137.4, 136.4, 132.6, 130.4, 130.1, 130.0, 129.8, 129.4, 128.5, 128.3, 128.3,
83.2, 69.5, 53.1, 37.4, 34.7, 24.0, 17.7.
13C NMR (126 MHz, d6-DMSO, δ/ppm): 195.50, 172.62, 170.46,
169.19, 169.16, 167.89, 167.80, 167.58, 166.18, 165.05, 158.26, 157.24,
156.59, 155.13, 152.62, 151.22, 148.31, 142.49, 139.28, 138.85, 137.98,
136.77, 135.70, 133.09, 129.90, 129.75, 129.48, 128.74, 127.49, 127.43,
127.41, 127.35, 127.30, 126.97, 126.50, 126.28, 125.63, 124.41, 123.45,
121.63, 118.11, 118.02, 116.24, 115.80, 113.44, 105.71, 104.85, 102.36,
101.29, 97.62, 78.30, 77.13, 76.77, 71.68, 71.44, 70.32, 70.12, 63.27,
ESI-MS, positive mode (m/z): 364.1544 [M + H]+ (calc: 364.1549).
12150
dx.doi.org/10.1021/ja2039979 |J. Am. Chem. Soc. 2011, 133, 12144–12153

Journal of the American Chemical Society
ARTICLE
N0-3-(4-benzoylphenyl)-2-(hex-5-ynamido)propanoic amide
vancomycin Probe 3.23 To a solution of Vancomycin hydrochloride
(100 mg, 68 μmol, 1.0 equivz) in 700 μL of DMF, DIPEA (33 μL,
201 μmol, 3.0 equiv) was added. PyBOP (39 mg, 74 μmol, 1.1 equiv)
and HOBT (13% H2O, 11 mg, 74 μmol, 1.1 equiv) were dissolved in
DMF. DIPEA (11 μL, 68 μmol, 1 equiv) and a solution of 3-(4-
benzoylphenyl)-2-(hex-5-ynamido)propanoic acid 8 (210 mg/mL in
DCM, 120 μL, 71 μmol, 1.05 equiv) were added. After 10 min of
preactivating the carboxylic acid, the two solutions were combined and
shaken for 2 h at 22 °C. Before HPLC purification, the reaction mixture
was treated as described before for probe 1. 76 mg of a colorless solid
(63%) were obtained.
22 °C. Reactions were initiated by addition of 1 μM of probe and allowed
to incubate (additional) 15 min at 22 °C. Then they were irradiated in
aliquots of 20 μL in open polystyrene microwell plates for 120 min on ice
with 366 nm UV light (Benda UV hand lamp NU-15 W). For heat
controls the proteome was denatured with 2% SDS (4 μL of 21.5% SDS)
at 95 °C for 6 min and cooled to 22 °C before the probe was applied. Follow-
ing incubation, reporter-tagged azide reagents (13 μM rhodamineꢀ
azide (1 μL) for analytical or 20 μM rhodamineꢀbiotinꢀazide for
preparative scale) were added followed by 1 mM TCEP (1 μL) and
100 μM ligand (3 μL). Samples were gently vortexed, and the
cycloaddition was initiated by the addition of 1 mM CuSO4 (1 μL).
The reactions were incubated at 22 °C for 1 h. For analytical gel
electrophoresis, 50 μL of 2 ꢁ SDS loading buffer was added, and 50 μL
applied on the gel. Fluorescence was recorded in a Fujifilm Las-4000
luminescent image analyzer with a Fujinon VRF43LMD3 lens and a
575DF20 filter.
Preparative experiments were carried out in a final volume of 2 mL of
proteome sample. Reactions for enrichment were carried out together
with a control lacking the probe to compare the results of the
biotinꢀavidin-enriched samples with the background of unspecific
protein binding on avidin-agarose beads. After CC proteins were
precipitated using a 5-fold volume of prechilled acetone, samples were
stored on ice for 60 min and centrifuged at 16000g for 10 min. The
supernatant was discarded and the pellet washed two times with 200 μL
of prechilled methanol and resuspended by sonication. Subsequently,
the pellet was dissolved in 1 mL of PBS with 0.4% SDS by sonication and
incubated under gentle mixing with 50 μL of avidin-agarose beads
(Sigma-Aldrich) for 1 h at room temperature. The beads were washed
three times with 1 mL of PBS/0.4% SDS, twice with 1 mL of 6 M urea,
and three times with 1 mL of PBS. Fifty microliters of 2ꢁ SDS loading
buffer was added, and the proteins were released for preparative SDS-
PAGE by 6 min incubation at 95 °C. Gel bands were isolated, washed,
and tryptically digested as described previously.45
In situ Experiments. For analytical in situ studies, bacteria were
grown in BHB medium and harvested 1 h after reaching stationary phase
by centrifugation. After washing with PBS, the cells were resuspended in
200 μL of PBS. Bacteria were incubated 15 min at 22 °C with 10 μM of
probe followed by irradiation at 366 nm on ice for 2 h. Competitive
in situ experiments with vancomycin were performed as described for the
in vitro case. Subsequently, the cells were washed twice with PBS
and lysed by sonication in 100 μL of PBS. The proteomes were separated
into cytosolic and membrane fractions, followed by CC as described above.
Mass Spectrometry and Bioinformatics. Tryptic peptides
were loaded onto a Dionex C18 Nano Trap Column (100 μm) and
subsequently eluted and separated by a Dionex C18 PepMap 100 mm
(3 μm) column for analysis by tandem MS followed by high-resolution
MS using a coupled Dionex Ultimate 3000 LC-Thermo LTQ Orbitrap
XL system. The mass spectrometry data were searched using the
SEQUEST algorithm against the corresponding databases via the soft-
ware “bioworks”. The search was limited to only tryptic peptides, two
missed cleavage sites, monoisotopic precursor ions, and a peptide
tolerance of <10 ppm. Filters were set to further refine the search
results. The Xcorr vs charge state filter was set to Xcorr values of 1.5, 2.0,
and 2.5 for charge states +1, +2, and +3, respectively. The number of
different peptides has to be e2, and the peptide probability filter was set
to <0.001. These filter values are similar to others previously reported for
SEQUEST analysis. Minimum P-values and Xcorr values of each run as
well as the total number of obtained peptides are reported in Table S1,
SI.
1H NMR (500 MHz, d6-DMSO, δ/ppm): 9.48 (s, 1H), 9.20 (s, 1H),
9.00 (s, 1H), 8.68 (s, 1H), 8.53 (s, 1H), 8.15 (s, 2H), 7.85 (d, J = 10.0 Hz,
1H), 7.71 (d, J = 7.7 Hz, 2H), 7.66 (d, J = 7.0 Hz, 2H), 7.62 (d, J = 7.9 Hz,
1H), 7.57 (s, 1H), 7.56 (s, 2H), 7.54 (s, 1H), 7.45 (s, 1H), 7.38 (s, 1H),
7.33 (s, 1H), 7.25 (s, 1H), 7.17 (s, 1H), 7.08ꢀ6.99 (m, 2H), 6.77 (d, J =
8.3 Hz, 1H), 6.71 (d, J = 8.7 Hz, 1H), 6.61 (s, 1H), 6.49 (s, 1H), 6.40 (s,
1H), 6.25 (s, 1H), 5.98 (s, 1H), 5.76 (s, 1H), 5.74 (s, 1H), 5.60 (s, 1H),
5.52 (s, 1H), 5.32 (s, 1H), 5.31 (s, 1H), 5.26 (s, 1H), 5.24 (s, 1H), 5.22
(s, 1H), 5.17 (s, 1H), 5.11 (s, 1H), 4.94 (s, 1H), 4.94 (s, 1H), 4.68 (d, J =
7.2 Hz, 1H), 4.47 (s, 1H), 4.43 (s, 1H), 4.26 (s, 1H), 4.19 (s, 1H), 3.95
(s, 1H), 3.67 (s, 1H), 3.37 (s, 1H), 3.28ꢀ3.25 (m, 5H), 2.86 (s, 1H),
2.77ꢀ2.71 (m, 1H), 2.65 (s, 1H), 2.25 (s, 5H), 2.13 (s, 6H), 2.00 (s,
1H), 1.78ꢀ1.50 (m, 3H), 1.40 (s, 1H), 1.28 (s, 1H), 1.07 (s, 1H), 0.91
(d, J = 5.5 Hz, 3H), 0.86 (d, J = 5.7 Hz, 3H), 0.80 (d, J = 6.2 Hz, 3H).
13C NMR (126 MHz, d6-DMSO, δ/ppm): 195.74, 172.66, 171.64,
170.96, 170.24, 170.13, 169.94, 169.24, 167.94, 167.48, 166.20, 157.28,
156.62, 155.17, 152.65, 151.26, 150.27, 148.37, 142.49, 139.56, 139.27,
137.34, 136.16, 135.76, 132.64, 129.84, 129.59, 129.47, 128.62, 127.55,
127.45, 127.31, 127.06, 126.61, 126.49, 126.32, 125.64, 125.52, 124.47,
123.52, 121.65, 118.19, 118.06, 116.33, 115.82, 107.53, 105.75, 104.90,
102.41, 101.40, 97.65, 84.14, 77.67, 77.13, 76.75, 71.73, 71.53, 70.82,
70.41, 70.12, 63.18, 61.91, 61.31, 61.27, 59.67, 58.99, 56.74, 54.01, 53.78,
53.31, 51.11, 40.22, 35.89, 35.04, 34.00, 31.30, 30.02, 24.65, 24.40, 23.78,
22.90, 22.37, 17.37, 17.25.
ESI-MS, positivemode(m/z):1795.5807[M+ H]+ (calc: 1795.5812).
Bacterial Strains. Staphylococcus aureus strains NCTC 8325 and
Mu50/ATCC 700699 (both from Institute Pasteur, France), and
Enterococcus faecalis strains V583/ATCC 700802 and OF1RF/ATCC
47077 (both form LGC Standards) were maintained in BHB (brain-
heart broth) medium at 37 °C.
MIC Measurments. Overnight cultures of the bacteria were diluted
in fresh BHB medium to OD600 = 0.01 and 100 μL was incubated in
Nunclon 96-well plates with round-shaped bottoms with 1 μL of the
corresponding DMSO stocks of vancomycin and the probes at varying
concentrations. The samples were incubated 12 h at 37 °C and the
optical densities obtained for MIC calculation. All experiments were
conducted at least in triplicates, and DMSO served as control.
Preparation of Proteomes for in Vitro Experiments. The
proteomes of the bacterial strains were prepared from 1-L liquid cultures
in BHB medium harvested 1 h after transition in the stationary phase by
centrifugation at 4000g. The bacterial cell pellet was washed with PBS,
resuspended in PBS buffer, and lysed by sonication with a Bandelin
Sonopuls under ice cooling. Membrane and cytosol were separated by
centrifugation at 18000g for 45 min.
In Vitro Analytical and Preparative Labeling Experiments.
Proteome samples were adjusted to a final concentration of 1 mg
protein/mL by dilution in PBS prior to probe labeling. Analytical
experiments were carried out in 43 μL total volume, such that once
CC reagents were added, a total reaction volume of 50 μL was reached.
In the case of competitive displacement experiments vancomycin
hydrochloride was added in various excess (1 μL of DMSO stocks)
and the proteoms were preincubated with the antibiotic for 15 min at
Recombinant Expression. The major hits of MS analysis were
recombinantly expressed in E. coli as an internal control of the MS results
by using the Invitrogen Gateway Technology. Target genes were
amplified from the corresponding genomes by PCR with an AccuPrime
Pfx DNA Polymerase kit with 65 ng of genomic DNA as template,
12151
dx.doi.org/10.1021/ja2039979 |J. Am. Chem. Soc. 2011, 133, 12144–12153

Journal of the American Chemical Society
ARTICLE
prepared by standard protocols. attB1 forward primer and attB2 reverse
primer were designed to yield attB-PCR Products needed for Gateway
Technology. The sequences of these primers can be found in Table S3, SI.
PCR products were separated and identified on 1% agarose gels
containing 0.2 μg/mL ethidium bromide, and gel bands were isolated
and extracted with an E.ZNA MicroElute Gel Extraction Kit. Concen-
trations of DNA were measured with a TECAN Infinite 200 PRO and
the corresponding NanoQuant Plate. One hundred femtomoles of
purified attB-PCR product and 50 fmol of attP-containing donor vector
pDONR201 in TE buffer were used for in vitro BP recombination
reaction with BP Clonase II enzyme mix to yield the appropriate attL-
containing entry clone. After transformation in chemically competent
One Shot TOP10 E. coli (Invitrogen), cells were plated on LB agar plates
containing 25 μg mLꢀ1 kanamycin. Clones of transformed cells were
selected and grown in LB medium supplemented with kanamycin. Cells
were harvested, and plasmids were isolated using an E.ZNA Plasmid
Mini Kit. The corresponding attB-containing expression clone was
generated by in vitro LR recombination reaction of approximately
50 fmol of the attL-containing entry clone and 50 fmol of an attR-
containing destination vector pDEST007 or Invitrogen Champion
pET300 NT-DEST using LR Clonase II enzyme mix in TE buffer.46
The expression clone was transformed in chemically competent BL21
E. coli cells (Novagen) and selected on LB agar plates containing 100
μg mLꢀ1 ampicillin. Validity of the clones was confirmed by plasmid
sequence analysis. Recombinant clones were grown in ampicillin LB
medium, and target gene expression was induced with anhydrotetracy-
cline or IPTG, depending on the expression vector used.
unskirted PCR plates in a Bio-Rad CFX96 real-time PCR detection
system. The polymerase was activated in an initial denaturation step for
30 s at 95.0 °C. Forty cycles were then run, each consisting of 5 s at
95.0 °C, followed by 10 s annealing and elongation at 61 °C. A melting
curve was measured to ensure specific replication of the target sequences
only.GAP and LAS genes were used as internal standards for ΔΔCq
analysis. Primer sequences can be found in Table S3, SI. Cq and Cq
mean values are shown in Table S2, SI.
’ ASSOCIATED CONTENT
S
Supporting Information. Complete ref 40. Additional
b
figures containing fluorescent gel scans, primer sequences, mass
spectrometry, real time PCR data, and 1H and 13C NMR-Spectra
of Probes 1, 2 and 3. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
stephan.sieber@tum.de
’ ACKNOWLEDGMENT
We thank Matt Nodwell for scientific advice, Mona Wolff for
excellent support, and G. Gemmecker and M. Sattler for help
with NMR analysis. J.E. was supported by the FOR1406 and the
TUM Graduate School (Faculty Center Chemistry). S.S. was
supported by the Deutsche Forschungsgemeinschaft (Emmy
Noether), SFB749, FOR1406, an ERC starting grant and the
Center for Integrated Protein Science Munich CIPSM.
ATLam, that was overexpressed using the pET300 NT-DEST, was
purified with Profinity IMAC Resin from BioRad corresponding to the
manufacture’s manual. The protein was desalted into the assay buffer
€
using a GE Healthcare AKTApurifier system equipped with a HiTrap
desalting column (5 mL). Purity was checked via SDS-PAGE and
Coomasie stain.
’ REFERENCES
(1) Kahne, D.; Leimkuhler, C.; Lu, W.; Walsh, C. Chem. Rev. 2005,
105, 425–448.
(2) Anderson, J. S.; Matsuhashi, M.; Haskin, M. A.; Strominger, J. L.
Proc. Natl. Acad. Sci. U.S.A. 1965, 53, 881–889.
(3) Perkins, H. R. Biochem. J. 1969, 111, 195–205.
(4) Hanaki, H.; Kuwahara-Arai, K.; Boyle-Vavra, S.; Daum, R. S.;
Labischinski, H.; Hiramatsu, K. J. Antimicrob. Chemother. 1998,
42, 199–209.
Peptidoglycan Assay. To evaluate the effect of vancomycin on
the degradation of peptidoglycan via ATL the following assay was
performed according to published procedures.47,48 Peptidoglycan from
Staphylococcus aureus (Sigma-Aldrich) was suspended in ddH2O to yield
3.3 mg/mL. The molarities given were calculated with respect to the
molecular weight of the murein monomers. The measurement of OD600
was carried out with a Tecan Infinite 200 PRO plate reader in 96-well
plates with lid and flat well shape at 30 °C.
For inhibitory studies the enzyme was preincubated with vancomycin
for 5 min and finally diluted to a concentration of 0.5 μg/mL (7.7 nM)
with the substrate suspension of OD600 ≈ 0.3. OD600 was measured over
30 min, and the 96-well was shaken constantly. The decrease of OD600
within this time was set to 100% for the uninhibited reaction. The other
reactions are given in relation to this value (Figure 4). The IC50 was
determined from the fitted curve shown in Figure 4 as the inhibitor
concentration where the protein shows half-maximal activity.
For MichaelisꢀMenten kinetics, substrate concentrations were var-
ied from 1 mM to 0.125 mM, and the corresponding enzyme turnover
was monitored as described above. The enzyme concentration was kept
constant at 7.7 nM. KM was determined via a LineweaverꢀBurk plot
with the x-intercept of the graph representing ꢀ1/KM (Figure 4). KI was
directly calculated from these values.49
Expression Analysis. E. faecalis V583 was grown overnight with
half maximal MIC of vancomycin and without antibiotic selection. Total
bacterial RNA was isolated with the Bio-Rad Aurum Total RNA mini kit
and transcribed to cDNA immediately by reverse transcriptase and a
random hexamer primer set using an iScript Select cDNA synthesis kit
(Bio-Rad). For RT PCR, specific primers were designed for pABC to
amplify a sequence specific for this gene of interest with Primer-BLAST.
RT PCR was conducted in a two-step thermal protocol using a SsoFast
EvaGreen Supermix kit (Bio-Rad) with white Low-Profile 96-well
(5) Sieradzki, K.; Tomasz, A. J. Bacteriol. 1997, 179, 2557–66.
(6) Utaida, S.; Pfeltz, R. F.; Jayaswal, R. K.; Wilkinson, B. J.
Antimicrob. Agents Chemother. 2006, 50, 1541–5.
(7) Sieradzki, K.; Tomasz, A. J. Bacteriol. 2003, 185, 7103–7110.
(8) Nannini, E.; Murray, B. E.; Arias, C. A. Curr. Opin. Pharmacol.
2010, 10, 516–521.
(9) Boyle-Vavra, S.; Challapalli, M.; Daum, R. S. Antimicrob. Agents
Chemother. 2003, 47, 2036–2039.
(10) Sieradzki, K.; Tomasz, A. Antimicrob. Agents Chemother. 2006,
50, 527–533.
(11) Chen, L.; Walker, D.; Sun, B.; Hu, Y.; Walker, S.; Kahne, D.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5658–5663.
(12) Sinha Roy, R.; Yang, P.; Kodali, S.; Xiong, Y.; Kim, R. M.;
Griffin, P. R.; Onishi, H. R.; Kohler, J.; Silver, L. L.; Chapman, K. Chem.
Biol. 2001, 8, 1095–1106.
(13) Koteva, K.; Hong, H. J.; Wang, X. D.; Nazi, I.; Hughes, D.;
Naldrett, M. J.; Buttner, M. J.; Wright, G. D. Nat. Chem. Biol. 2010,
6, 327–9.
(14) Speers, A. E.; Adam, G. C.; Cravatt, B. F. J. Am. Chem. Soc. 2003,
125, 4686–4687.
(15) Speers, A. E.; Cravatt, B. F. Chem. Biol. 2004, 11, 535–546.
(16) Blum, G.; von Degenfeld, G.; Merchant, M. J.; Blau, H. M.;
Bogyo, M. Nat. Chem. Biol. 2007, 3, 668–677.
(17) Evans, M. J.; Cravatt, B. F. Chem. Rev. 2006, 106, 3279–3301.
12152
dx.doi.org/10.1021/ja2039979 |J. Am. Chem. Soc. 2011, 133, 12144–12153

Journal of the American Chemical Society
ARTICLE
(18) B€ottcher, T.; Pitscheider, M.; Sieber, S. A. Angew. Chem., Int. Ed.
2010, 49, 2680–2698.
(19) Greenbaum, D.; Baruch, A.; Hayrapetian, L.; Darula, Z.;
Burlingame, A.; Medzihradszky, K. F.; Bogyo, M. Mol. Cell. Proteomics
2002, 1, 60–68.
(20) Huisgen, R. 1,3 Dipolar Cylcoaddition Chemistry; Wiley: New York,
1984.
(21) Rostovtsev, V. V.; Green, J. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596–2599.
(22) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057–3064.
(23) Griffin, J. H.; Linsell, M. S.; Nodwell, M. B.; Chen, Q.; Pace,
J. L.; Quast, K. L.; Krause, K. M.; Farrington, L.; Wu, T. X.; Higgins,
D. L.; Jenkins, T. E.; Christensen, B. G.; Judice, J. K. J. Am. Chem. Soc.
2003, 125, 6517–6531.
(24) Anderson, G. W.; Zimmermann, J. E.; Callahan, F. M. J. Am.
Chem. Soc. 1964, 86, 1655–1906.
(25) Orth, R.; Sieber, S. A. J. Org. Chem. 2009, 74, 8476–8479.
(26) Smith, M. D.; Gong, D.; Sudhahar, C. G.; Reno, J. C.; Stahelin,
R. V.; Best, M. D. Bioconjugate Chem. 2008, 19, 1855–1863.
(27) Kim, S. J.; Matsuoka, S.; Patti, G. J.; Schaefer, J. Biochemistry
2008, 47, 3822–3831.
(28) Ito, Y.; Kano, G.; Nakamura, N. J. Org. Chem. 1998, 63, 5643–5647.
(29) B€ottcher, T.; Sieber, S. A. Angew. Chem., Int. Ed. 2008, 47,
4600–4603.
(30) Kunzmann, M. H.; Staub, I.; Bottcher, T.; Sieber, S. A. Bio-
chemistry 2011, 50, 910–916.
(31) Biswas, R.; Voggu, L.; Simon, U. K.; Hentschel, P.; Thumm, G.;
Gotz, F. FEMS Microbiol. Lett. 2006, 259, 260–268.
(32) Oshida, T.; Sugai, M.; Komatsuzawa, H.; Hong, Y. M.; Suginaka,
H.; Tomasz, A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 285–289.
(33) Sugai, M. J. Infect. Chemother. 1997, 3, 113–127.
(34) Zoll, S.; Patzold, B.; Schlag, M.; Gotz, F.; Kalbacher, H.; Stehle,
T. PLoS Pathog. 2011, 6, e1000807.
(35) Antignac, A.; Sieradzki, K.; Tomasz, A. J. Bacteriol. 2007,
189, 7573–7580.
(36) Kajimura, J.; Fujiwara, T.; Yamada, S.; Suzawa, Y.; Nishida, T.;
Oyamada, Y.; Hayashi, I.; Yamagishi, J.; Komatsuzawa, H.; Sugai, M.
Mol. Microbiol. 2005, 58, 1087–1101.
(37) Garmory, H. S.; Titball, R. W. Infect. Immun. 2004, 72,
6757–6763.
(38) Davidson, A. L.; Dassa, E.; Orelle, C.; Chen, J. Microbiol. Mol.
Biol. Rev. 2008, 72, 317–364 and table of contents.
(39) van Veen, H. W.; Higgins, C. F.; Konings, W. N. Res. Microbiol.
2001, 152, 365–374.
(40) Paulsen, I. T.; et al. Science 2003, 299, 2071–2074.
(41) Burnie, J.; Carter, T.; Rigg, G.; Hodgetts, S.; Donohoe, M.;
Matthews, R. FEMS Immunol. Med. Microbiol. 2002, 33, 179–189.
(42) Shi, Z.; Griffin, J. H. J. Am. Chem. Soc. 1993, 115, 6482–6486.
(43) Vintonyak, V. V.; Maier, M. E. Angew. Chem., Int. Ed. 2007,
46, 5209–5211.
(44) Sundram, U. N.; Griffin, J. H. J. Org. Chem. 1995, 60, 1102–1103.
(45) Sieber, S. A.; Niessen, S.; Hoover, H. S.; Cravatt, B. F. Nat.
Chem. Biol. 2006, 2, 274–281.
(46) Ober, M.; Muller, H.; Pieck, C.; Gierlich, J.; Carell, T. J. Am.
Chem. Soc. 2005, 127, 18143–18149.
(47) Kuroda, A.; Sekiguchi, J. J. Gen. Microbio. 1990, 136, 2209–2216.
(48) Ayusawa, D.; Yoneda, Y.; Yamane, K.; Maruo, B. J. Bacteriol.
1975, 124, 459–469.
(49) Cer, R. Z.; Mudunuri, U.; Stephens, R.; Lebeda, F. J. Nucleic
Acids Res. 2009, 37, W441–W445.
12153
dx.doi.org/10.1021/ja2039979 |J. Am. Chem. Soc. 2011, 133, 12144–12153