Vancomycin dimer formation between analogues of bacterial peptidoglycan surfaces probed by force spectroscopy

Article abstract of DOI:10.1039/b919347b

Functionalised thiols presenting peptides found in the peptidoglycan of vancomycin-sensitive and -resistant bacteria were synthesised and used to form self-assembled monolayers (SAMs) on gold surfaces. This model bacterial cell-wall surface mimic was used to study binding interactions with vancomycin. Force spectroscopy, using the atomic force microscope (AFM), was used to investigate the specific rupture of interfacial vancomycin dimer complexes formed between pairs of vancomycin molecules bound to peptide-coated AFM probe and substrate surfaces. Clear adhesive contacts were observed between the vancomycin-sensitive peptide surfaces when vancomycin was present in solution, and the adhesion force demonstrated a clear dependence on antibiotic concentration. The Royal Society of Chemistry.

Full text of DOI:10.1039/b919347b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Vancomycin dimer formation between analogues of bacterial peptidoglycan
surfaces probed by force spectroscopy†
Matthew Batchelor,*‡a Dejian Zhou,b Matthew A. Cooper,c Chris Abella and Trevor Raymentd
Received 17th September 2009, Accepted 26th November 2009
First published as an Advance Article on the web 8th January 2010
DOI: 10.1039/b919347b
Functionalised thiols presenting peptides found in the peptidoglycan of vancomycin-sensitive and
-resistant bacteria were synthesised and used to form self-assembled monolayers (SAMs) on gold
surfaces. This model bacterial cell-wall surface mimic was used to study binding interactions with
vancomycin. Force spectroscopy, using the atomic force microscope (AFM), was used to investigate the
specific rupture of interfacial vancomycin dimer complexes formed between pairs of vancomycin
molecules bound to peptide-coated AFM probe and substrate surfaces. Clear adhesive contacts were
observed between the vancomycin-sensitive peptide surfaces when vancomycin was present in solution,
and the adhesion force demonstrated a clear dependence on antibiotic concentration.
vancomycin dimerisation has been shown to be cooperative with
that of vancomycin–DADA binding and it has been suggested
Introduction
Vancomycin is a powerful antibiotic used to treat infections
that dimerisation plays a role in substrate binding in vivo and is
caused by Gram-positive bacteria. This glycopeptide antibiotic
important for the overall efficacy of the antibiotic. Furthermore,
molecule acts by reversibly binding to D-alanyl-D-alanine (DADA)
a synthetic vancomycin dimer has been shown to be more
terminating mucopeptides that are present as intermediates in the
effective against certain vancomycin-resistant organisms than
vancomycin alone.19 To date, all experiments on dimer formation
have been performed with solution-phase mimics of mucopeptides,
using techniques such as nuclear magnetic resonance (NMR)
spectroscopy13–15,20 and isothermal titration calorimetry (ITC),21
which may not accurately represent the binding topology found
on a bacterial cell surface.
biosynthesis of bacterial cell-walls. The bound complex contains
five specific hydrogen-bonds as well as hydrophobic interactions
between the alanine methyl groups and hydrocarbon components
of vancomycin (see ESI† Fig. S1 for a binding model).1 This
binding process sterically inhibits the action of cell-wall cross-
linking enzymes (transpeptidases and transglycosylases), limiting
its growth and thereby preventing microbial growth.1,2 Research
into the properties of vancomycin is currently of particular
interest due to its use in the treatment of methicillin-resistant
Staphylococcus aureus (MRSA) infections, and because of the
worrying increase in examples of vancomycin-resistant bacteria,3,4
which can result from a single amino acid mutation of the terminal
D-alanine to D-lactate in the growing bacterial cell-wall.
Many studies have been performed to investigate the interaction
of vancomycin with DADA in solution, and on cell-wall mimic
surfaces.5–12 Further studies have shown that vancomycin can form
a dimer complex in solution.13–15 Dimerisation occurs between
vancomycin monomers through four hydrogen bonds,1,16–18 and
these interactions occur on the opposite side of the molecule to
the site where the DADA ligand binds. The dimerisation constant
has been reported to be ~700 M-1 in solution.13 The process of
In this study vancomycin dimerisation has been investigated
between two antibiotic monomers that are both bound to cell-
wall mimic lysyl-D-alanyl-D-alanine (KDADA) surfaces. This rep-
resents a novel approach to measuring dimerisation constants,
and may prove a useful method to explore the process and
effects of dimerisation using a model of a cell-wall system in
which interactions are templated on surfaces. The model cell-
walls were prepared using the self-assembly of a mixture of
functionalised alkanethiols onto gold surfaces. These surfaces
were analysed using ellipsometry and contact angle measurements,
and surface plasmon resonance (SPR) spectroscopy was used
to characterise the binding profiles of vancomycin with surface-
bound ligands. Subsequently, force spectroscopy6,22–24 with the
atomic force microscope (AFM) was used to measure directly the
force required to rupture the dimer formed between two surface-
bound species, as shown schematically in Fig. 1.
aDepartment of Chemistry, University of Cambridge, Lensfield Road,
Cambridge, UK CB2 1EW. E-mail: m.r.batchelor.98@cantab.net
bSchool of Chemistry and the Astbury Centre for Structural Molecular
Biology, University of Leeds, Leeds, UK LS2 9JT
Results and discussion
cInstitute for Molecular Bioscience, The University of Queensland, Brisbane,
4072, Australia
For these studies, gold-coated AFM probes and substrates were
functionalised with self-assembled monolayers (SAMs) of peptidic
alkanethiols25 (the molecular structures of the alkanethiols are
given in Scheme 1). Mixed SAMs were composed of alkanethiols
that contained a tri(ethylene glycol) spacer with a distal KDADA
group (1), which were diluted with a background of alkanethiols
terminating in tri(ethylene glycol)-OH (PEG-OH, 4).26 The SAMs
dSchool of Chemistry, University of Birmingham, Birmingham, UK B15
2TT
† Electronic supplementary information (ESI) available: Binding model
and supporting figures; synthesis of compounds and relevant spectra. See
DOI: 10.1039/b919347b
‡ Current address: Royal Society of Chemistry, Thomas Graham House,
Science Park, Cambridge, UK, CB4 0WF.
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Fig. 1 Schematic for a force spectroscopy investigation of the interacting vancomycin dimer system. (a) KDADA ligands project from the tip and surface.
Note: only two KDADA ligands are shown on both the tip and surface for simplicity; the exact number of ligands within the contact region when the tip
approaches is likely to be more than shown. The number of ligands depends on the sharpness of the tip, and the contact force used in force spectroscopy
collection, and therefore varies for different tip–surface combinations. (b) Vancomycin, added to the intervening solution, binds to ligands on tip and
surface. (c) During force spectroscopy measurement, the tip and substrate are pushed together and then pulled apart and the adhesive force between
bound vancomycin monomers from the tip and surface is measured.
Scheme 1 Molecular structures of functional alkanethiols used in this study.
were formed from a mixed solution of 1 and 4 in a molar ratio of
1 : 9 in ethanol.5 Alternative peptide-functionalised surfaces were
also prepared in an equivalent manner by using alkanethiols with
a distal lysyl-D-alanyl-L-alanine (KDALA, 2) or lysyl-D-alanyl-D-
lactate (KDADLac, 3) group. The surfaces were characterised on
flat gold surfaces using ellipsometry (SAM thickness measure-
ments) and water contact angle (CA) goniometry (surface wetting
properties)—results are given in Table 1.
The thickness and CA values for the pure PEG-OH monolayer
compare very well with those reported in the literature: CA(adv.)
34◦, CA(rec.) 22◦, thickness 2.0 nm.27 The small increase in
thickness and reduced receding CA change observed when the
peptide thiols are present in the monolayer are reasonable given
the size and polarity of the peptides. The values obtained were
subsequently used as a quality-control for the SAMs used in the
spectroscopy measurements.
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Table 1 Measured advancing and receding water contact angles (CAs),
as well as the ellipsometric thickness, of the pure 4 monolayer, and the
peptide-modified monolayers (prepared from a 1 : 9 solution mixture of 1,
2 or 3 with 4). Errors represent standard deviations from n measurements
analysis of equilibrium binding levels. A reduction of the 1 : 4
molar ratio to 1 : 29 gave a Kd of ~1.1 mM—a value that is still
in line with the values observed previously, and gives a more
accurate lower limit estimate for the dissociation constant for the
system used here. Additional control experiments were carried out
which showed that the surface-binding behaviour of vancomycin
could be blocked by the addition of free acetyl-lysyl-D-alanyl-
D-alanine (AcKDADA) to solution (see ESI† Fig. S4). It was
also demonstrated that vancomycin did not bind significantly to
alternative peptide sequences or to the background surface (e.g.
ESI† Fig. S5).
SAM
CA(adv.)
CA(rec.)
Thickness/nm
PEG-OH (4)
KDADA (1)
KDALA (2)
KDADLac (3)
36 1◦ (n = 11) 28 1◦ (n = 5)
2.1 0.3 (n = 8)
35 2◦ (n = 7)
34 3◦ (n = 4)
34◦
Low/wet (n = 7) 2.4 0.5 (n = 3)
Low/wet (n = 4) 2.3 0.4 (n = 2)
20◦
2.4
Further characterisation was achieved by using surface plas-
mon resonance spectroscopy to monitor the binding behaviour
of different concentrations of vancomycin with the surfaces.
Examples of typical vancomycin binding profiles are shown in
Fig. 2. Scatchard analysis of the equilibrium binding levels (e.g.
see ESI† Fig. S2) gave a dissociation constant (Kd) of ~0.4 mM,
which is consistent with values reported in the literature for both
solution1,10 and surface-phase5,7 studies. The kinetic behaviour was
flow-rate dependent (see ESI† Fig. S3) and, due to the fast inherent
on/off-rate of the interaction, accurate rate constants could not
be obtained by this technique and the analysis was limited to
equilibrium binding levels.7 Reducing the density of KDADA
ligands on the surface resulted in an increase in the observed
off-rate and an increase in the Kd determined by Scatchard
Force–distance curves were recorded between KDADA-
functionalised AFM probes and surfaces in phosphate-buffered
saline (PBS) solutions (see Fig. 1 for details about the experi-
mental procedure). No significant adhesion was observed when
vancomycin was absent from the solution. The curves all had the
appearance of those in Fig. 3a, where approach and retract profiles
overlap throughout. When vancomycin (1 mM) was present in the
solution the force curves (>90%) showed an enhanced adhesive
contact with respect to the above control (see the force curve
example in Fig. 3b). Distributions of adhesion forces, recorded
using a single probe–surface combination, are shown in Fig. 4.
A control experiment with the PBS buffer only represents the
measurement of the maximum background noise level (Fig. 4a,
average adhesion force
standard deviation of 43
12 pN).
The average adhesion force in 1 mM vancomycin was 180 90
pN. The process was highly reproducible: when the liquid cell was
thoroughly rinsed with buffer and the surface-bound vancomycin
was washed away, the adhesion force disappeared (Fig. 4c). Re-
introduction of vancomycin (1 mM) to the solution again gave an
adhesion force of 185 90 pN (Fig. 4d). Several repetitions of this
Fig. 2 Overlaid sensorgrams showing typical SPR responses to: (a) 3 min
injections of vancomycin at 20 ml min-1 across a KDADA chip prepared
from a 1 : 9 molar ratio 1 : 4 solution and (b) 5 min injections of vancomycin
at 20 ml min-1 across a KDADA chip prepared from a 1 : 29 molar ratio 1 : 4
solution (vancomycin concentrations were two-fold dilutions between the
values shown; the bottom line in (b) resulted from an injection of buffer).
Fig. 3 Raw force–distance curves typical of those recorded between
KDADA-functionalised surfaces in buffered solution (top), and in van-
comycin-containing buffered solutions (bottom).
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and surface. Removal of one partner (i.e. the surface monomer)
led to complete suppression of the specific adhesion force. In
a second control experiment, the tip–surface adhesion between
KDADA surfaces in 1 mM vancomycin was completely eliminated
by the addition of excess (50 mM) free AcKDADA to the solution
(see ESI† Fig. S7). The free ligand effectively competes for and
blocks the site that vancomycin would use to bind to the surface,
thus greatly reducing the amount of vancomycin on the surface.
The process is again reversible, as removal of the free AcKDADA
from the system led to the complete recovery of specific adhesive
contacts.
Having established that the observed forces were dependent
on surface-bound vancomycin, we wished to determine if the
system could be used to evaluate surface-templated dimerisation
of vancomycin. The adhesion force was observed to be dependent
on the concentration of vancomycin in solution (Fig. 5). Both the
proportion of recorded curves showing adhesion and the mag-
nitude of adhesion forces measured were affected (representative
distributions are given in the ESI† Fig. S8). Few adhesive contacts
were observed at low vancomycin concentration (<1 mM). The
interactions that were observed were small in magnitude. The
largest adhesion forces occurred at ~10 mM vancomycin, and
nearly all curves collected showed an adhesive interaction. Above
10 mM, the number and the magnitude of adhesive forces observed
were reduced.
Fig. 4 Histograms showing the adhesion force distributions from ~500
force–distance curves recorded between KDADA-functionalised surfaces
(a) in buffer, (b) in 1 mM vancomycin, (c) in buffer after rinsing with buffer,
(d) with a regenerated probe surface after introducing 1 mM vancomycin
(repeat). The data presented were collected using the same probe–substrate
combination. The adhesion values in buffer (~50 pN) correspond to a
maximum baseline noise level in the absence of any resolvable adhesion.
No effect was observed by altering the histogram bin size.
experiment using different AFM probes and substrates showed
similar results, with the average adhesion force ranging from ~150–
250 pN, depending on the tip–surface combination used. The same
tip–surface combination consistently gave near identical average
adhesion forces. This variation in adhesion force between different
probes is probably due to differences in the tip radius of the AFM
probes, and therefore different numbers of KDADA ligands (and
hence bound vancomycin molecules) within the contact region.
To determine if the observed adhesion force was specific to the
interaction of vancomycin monomers when bound to KDADA
1, several control experiments were carried out. Firstly, whilst
using the same KDADA-modified tip, the KDADA ligands on the
surface were exchanged for alternative peptide sequences KDALA
2 or KDADLac 3, or the peptide was removed leaving a monolayer
of PEG-OH 4 only. Vancomycin is reported to not bind with
KDALA,28 only weakly bind with KDADLac5 and should not
adsorb non-specifically onto the PEG-OH surface.25 For each of
these control surfaces, no adhesion was observed in the presence
of 1 mM vancomycin, and the force histograms were similar to
those in Fig. 4a and 4c (see ESI† Fig. S6). The average adhesion
forces for KDALA, KDADLac and PEG-OH were 47 13 pN,
49 14 pN and 47 14 pN respectively, effectively identical to the
background noise level. This control experiment confirmed that
the observed adhesion force was highly specific to the KDADA
peptide, and strongly suggests that the observed adhesion was
indeed due to the formation of inter-surface vancomycin dimers
between monomers bound to mucopeptide analogues on the probe
Fig. 5 A plot of the average adhesion forces from ~500 force–distance
curves for increasing concentrations of vancomycin in solution. The data
presented were collected using the same probe–substrate combination;
repeat experiments showed the same trend. The initial sigmoidal response
is followed by a reduction in adhesion at high concentration. Rinsing the
surfaces with buffer resulted in the loss of adhesive contacts. Error bars
show the standard deviation for each distribution.
Below the Kd for vancomycin–KDADA binding, found to be
around 1 mM here and in previous solution and surface-based
studies,1,5,7,10 relatively few vancomycin molecules will be bound
to ligands on the probe and surface. Thus the likelihood of a
bound vancomycin molecule on the tip interacting with another
bound molecule on the surface to form an inter-surface dimer
is low, and hence there is no concomitant increase in adhesion
forces. The amount of vancomycin bound to both surfaces will
have increased until all the accessible sites were occupied as the
concentration rose above the Kd (e.g. at about ten times the Kd, 90%
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of the available binding sites would be occupied). As the surfaces
were pushed together, the likelihood of an interaction being
observed between vancomycin molecules bound to the tip and
surface greatly increased, leading to the observed maximum
adhesion force. The subsequent reduction in average adhesion
force observed at even higher vancomycin concentrations may be
attributed to the introduction of a population of dimers bound to
either of the surfaces (i.e. ‘on-surface’ dimers which form when
ligand-bound vancomycin binds a free vancomycin molecule from
solution, see Fig. 6a) prior to the force spectroscopy measurement.
The number of sites for inter-surface dimers (between the tip and
surface) to form would be reduced, leading to reduced adhesion
forces. A recent study has shown that, at higher vancomycin
concentrations than used here, ligand-mediated supramolecular
complexes can form through alternative dimerisation modes. This
could also be a factor influencing the adhesion levels observed at
100 mM.18
KDADA-terminating thiols on gold, and the vancomycin–surface
binding properties were characterised using SPR. Using force
spectroscopy with an AFM, clear adhesive contacts were observed
between two of these KDADA surfaces when vancomycin was
present in solution. The adhesion was dependent upon the
concentration of vancomycin and specific to KDADA. We attribute
the adhesion force to the breakage of vancomycin dimers which
form a bridge between the two KDADA surfaces. The adhesion
showed a dependence on concentration that initially followed the
thermodynamics of vancomycin–KDADA binding, but at high
vancomycin concentrations a significant reduction of adhesion
was observed. The dimerisation constants13,21 for vancomycin are
reported to be Kdim < 700 M-1 in solution, and therefore high
proportions of dimers are only expected to form at concentrations
above ~1.4 mM. However, it has previously been shown that when
DADA ligands are present in solution the dimerisation constant is
increased by about a factor of ten.15,20,21 The significant decrease
in adhesion shown at 100 mM agrees well with these earlier
observations, and our results are therefore a further indication
of the cooperative nature of ligand binding and dimerisation.
Whilst more detailed investigation would be necessary, it is possible
that cooperativity could be further enhanced in our case because,
when ligands are fixed on a surface or in a growing cell-wall, the
close proximity of neighbouring surface ligands could reduce the
entropic penalty for dimer formation between a pair of ligand-
bound vancomycin monomers at neighbouring surfaces (see the
possible intra-surface dimer model in Fig. 6b).
By using the intimate proximity of the modified AFM tip
and substrate, the system can approximate the neighbouring
mucopeptides in a growing cell-wall surface. It therefore repre-
sents an improved model system (compared to purely solution-
based methods) for studying the formation and/or disruption
of dimer complexes of this type. The method could allow direct
investigation into the relationship between dimer formation and
ligand binding. Improving the force resolution, through use
of an AFM with lower thermal noise, using sharper tips and
reducing the ligand surface density, would allow the investigation
of energy barriers for single-dimer interactions through dynamic
force spectroscopy, and more generally, this approach could be
used to give greater insight into the behaviour of species exhibiting
bivalent binding interactions, including investigations into the
feature of cooperativity.
Fig. 6 Schematics showing the possible reasons behind the decreased
adhesion force at high vancomycin concentrations. (a) The formation
of ‘on-surface’ dimers between ligand-bound vancomycins and free
vancomycin molecules from solution; (b) ‘intra-surface’ dimers from two
neighbouring ligand-bound vancomycin molecules on the same surface
(can equally happen on the tip). Both could prevent the formation of
‘inter-surface’ dimers between monomers bound on tip and surface, and
therefore decrease the adhesion force. We believe the major contribution
for the adhesion decrease at high vancomycin concentration is due to (a)
rather than (b), as (b) is less dependent on vancomycin concentration.
These data suggest that most of the interactions observed in
these studies were from the breakage of more than one dimer
interaction. At micromolar concentrations nearly all the curves
collected displayed a specific interaction. Poisson distribution
statistics indicate that, given this feature, the observation of a
single-molecule interaction is less probable than that of multiple
interactions. This is supported by the correlation between smaller
adhesion forces and reduced number of adhesive interactions in
proportion to the total number of curves collected. It is possible
that single-molecule interactions occur at forces on the cusp of the
resolution of the system.
Experimental
Materials
Buffer constituents, vancomycin and AcKDADA were from
Sigma (and used as received). Deionised water (18.2 MX cm
at source; Millipore, USA) was used throughout. The synthesis
of the 11-mercaptoundecyl tri(ethylene glycol) alcohol (4) has
been described previously.26 The KDADA-modified thiol (1) was
synthesized by solid phase methodology under argon using a
commercially available Wang resin and standard Fmoc-protecting
group chemistry (see ESI† Scheme S2). The cleaved product
was further purified by reverse phase high performance liquid
chromatography (HPLC) on a C18 column by varying the mobile
phase from 5% to 95% of acetonitrile in water (with 0.5%
trifluoroacetic acid). The peptide-modified thiols 2 (KDALA) and
Conclusion
In summary, cell-wall mimic KDADA-presenting surfaces were
prepared by using self-assembled monolayers of functional
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3 (KDADLac) were prepared by similar methods. A description of
the synthesis of the thiol-protected carboxylic acid7 (compound
5), used in the preparation of the peptide thiols, is given in the
ESI† Scheme S1. The peptide-thiols (1–3) and PEG-OH (4) were
characterized by NMR and high resolution mass spectrometry
(HRMS, +ESI, Q-TOF) as follows:
were performed in PBS (10 mM sodium phosphate, 138 mM NaCl,
2.7 mM KCl, pH 7.4). Solutions were passed through a 0.2 mm
syringe filter before use (Whatman, USA).
Monolayer characterisation
SAMs were characterised on thick thermally-evaporated gold
on silicon samples using water contact angle and ellipsometry
measurements. Thickness measurements were performed with an
EL X-02C High Precision Ellipsometer (DRE, Germany). D
and W measurements were recorded for five sites on bare gold
substrates, and a two-layer model, based on the average values,
was used to calculate the refractive index of the substrate. Further
D and W measurements were then taken on the gold substrates
after they had been incubated for at least 18 h in solutions
containing the appropriate mixture of alkanethiols (the samples
being thoroughly rinsed in ethanol and dried under nitrogen before
measurements recorded). The refractive index of the gold, and an
assumed organic refractive index of 1.45 were used in a three-layer
model to calculate the thickness of the SAM. Thickness values
were calculated for measurements at 5 sites on each sample. No
account was made for different functional tailgroups.
For contact angle measurements, a motorised syringe with
needle was fixed above the sample on an adjustable stage. Milli-
Q de-ionised water was added to, or removed from the SAM-
covered sample surface without removing the needle from the
droplet (method B, Bain et al.29). A CCD camera positioned at
sample level and perpendicular to the syringe was used to record
images. Advancing contact angles were recorded when the edge
of the droplet was seen to extend across the surface, and receding
contact angles when the periphery began to shrink. Measurements
of the contact angles were individually performed, by hand, for
both the left and right sides of the droplet using Scion Image
(Frederick, MD, USA). Reported values are the average of left
and right sides and n different samples. Contact angles were also
recorded, with similar results, on the gold-on-mica samples used
in force spectroscopy measurements.
1 (KDADA). 1H NMR (d ppm, 400 MHz, CD3OD): 1.25–
1.80 (m, 36H, [15CH2 + 2CH3]), 1.91 (s, 3H, CH3), 2.24 (t, 2H,
CH2), 2.47 (t, 2H, J = 7.1 Hz, CH2SH), 3.14 (t, 2H, J = 7.0 Hz,
NHCH2), 3.22 (t, J = 7.0 Hz, 2H, NHCH2), 3.44 (t, 2H, J =
6.7 Hz, -CH2(OEG)-), 3.54–3.69 (m, 12H, 3(EG)), 3.96 (s, 2H,
=
OCH2C O), 4.21 (t, 1H, J = 7.1 Hz, L-Lys-a-CH), 4.31–4.42
(m, 2H, 2 [D-Ala-a-CH]). 13C NMR (d ppm, 125 MHz, CDCl3):
176.24, 175.58, 174.46, 174.29, 173.21, 172.61 (6 C=O), 72.39,
71.95, 71.55, 71.52, 71.37, 71.22, 71.16 (PEG), 55.15, 50.00, 40.17,
39.83, 36.50, 35.22, 32.27, 30.72, 30.70, 30.64, 30.56, 30.27, 30.21,
29.40, 27.57, 27.21, 26.46, 24.97, 24.23, 22.57, 18.01, 17.50. HRMS
(+ESI, Q-TOF), found 842.4941; required for C39H73N5O11SNa [M
+ Na]+, 842.4925, dev. 1.86 ppm.
2 (KDALA). 1H NMR (d ppm, 400 MHz, CD3OD): 1.25–1.80
(m, 36H, [15CH2 + 2CH3]), 1.91 (s, 3H, CH3), 2.24 (t, 2H, J =
7.2 Hz, CH2), 2.47 (t, 2H, J = 7.1 Hz, CH2SH), 3.15 (t, 2H, J =
7.0 Hz, NHCH2), 3.22 (t, J = 7.0 Hz, 2H, NHCH2), 3.45 (t, 2H, J
= 7.0 Hz, -CH2(OEG)-), 3.54–3.67 (m, 12H, 3(EG)), 3.96 (s, 2H,
=
OCH2C O), 4.14 (t, 1H, J = 7.1 Hz, L-Lys-a-CH), 4.31-4.40 (m,
2H, 2 [Ala-a-CH]). 13C NMR (d ppm, 125 MHz, CDCl3): 176.22,
175.83, 174.67, 174.44, 173.21, 172.62 (6 C=O), 72.39, 71.96,
71.57, 71.53, 71.39, 71.22, 71.18 (PEG), 55.52, 50.24, 40.16, 39.82,
36.39, 35.24, 32.00, 30.74, 30.72, 30.65, 30.58, 30.28, 30.22, 30.06,
29.41, 27.58, 27.22, 26.45, 24.97, 24.28, 22.57, 17.93, 17.60. HRMS
(+ESI, Q-TOF), found 842.4916; required for C39H73N5O11SNa [M
+ Na]+, 842.4925, dev. -1.11 ppm.
3 (KDADLac). 1H NMR (d ppm, 500 MHz, CD3OD): 1.25–
1.80 (m, 36H, [15CH2 + 2CH3]), 1.92 (s, 3H, CH3), 2.24 (t, 2H,
J = 7.0 Hz, CH2), 2.47 (t, 2H, J = 7.0 Hz, CH2SH), 3.13–3.17
(m, 2H, NHCH2), 3.18–3.24 (m, 2H, NHCH2), 3.46 (t, 2H, J =
6.7 Hz, -CH2(OEG)), 3.55–3.68 (m, 12H, 3(EG)), 3.96 (s, 2H,
Surface plasmon resonance spectroscopy
=
OCH2C O), 4.30–4.40 (m, 1H, L-Lys-a-CH), 4.40–4.50 (m, 1H,
D-Ala-a-CH]). 5.00-5.10 (m, 1H, D-Lac-a-CH). 13C NMR (d ppm,
125 MHz, CD3OD): 175.99, 174.12, 173.34, 173.20, 172.84, 172.60
(6 C=O), 79.47, 72.40, 71.99, 71.58, 71.55, 71.39, 71.24, 71.19
(PEG), 54.37, 53.68, 40.19, 39.82, 36.73, 36.63, 35.23, 32.84, 32.22,
30.74, 30.71, 30.64, 30.57, 30.26, 30.21, 30.00, 29.85, 29.41, 27.58,
27.52, 27.22, 26.62, 26.56, 24.98, 24.17, 22.60 (CH3), 17.51 (CH3),
17.44 (CH3). HRMS (+ESI, Q-TOF), found 843.4761; required
for C39H73N5O11SNa [M + Na]+, 843.4765, dev. -0.53 ppm.
SPR spectroscopy studies were performed using a Biacore-X
instrument (Biacore, Sweden). Gold-coated chips were purchased
from Biacore (SIA Kit Au) and prior to use were cleaned for 1–
2 min in freshly prepared piranha solution. After extensive rinsing
with deionised water, the chips were dried with nitrogen gas and
placed into the appropriate thiol solution for at least 18 h. After
this incubation period, the chips were thoroughly rinsed with
ethanol and then with water, before mounting and docking in
the instrument. PBS was used as running buffer, using a fixed flow
rate, generally at 10 or 20 ml min-1. The chips were exposed to 3–
5 min injections of vancomycin in PBS. Before each vancomycin
injection the surface was regenerated using 10–20 ml of 10 mM
NaOH and the chip was exposed to flowing buffer until a stable
baseline was achieved. All SPR experiments were performed at
25 ◦C.
4 (PEG-OH). 1H NMR (250 MHz, CDCl3, d ppm): 1.20–
1.37 (m, 14H, 7CH2), 1.49–1.60 (m, 4H, 2CH2), 2.46 (q, 2H,
J = 7.0 Hz, HSCH2-), 3.05 (s, br, 1H, -OH), 3.40 (t, 2H, J =
7.0 Hz, -CH2PEG), 3.50–3.75 (m, 12H, 3(OCH2CH2). 13C NMR
(62.5 MHz, CDCl3, d ppm): 72.5, 71.4, 70.5, 70.3, 69.9, 61.5, 34.0,
33.7, 30.5, 29.5, 29.4, 29.0, 28.8, 28.7, 28.3, 26.0, 24.5. HRMS
(Q-TOF, ES+), found 359.2222; required for C17H36O4SNa [M +
Na]+, 359.2232.
Preparation of tips and surfaces
Solutions were prepared in absolute ethanol at a total thiol
concentration of 1 mM, and a 1 : 9 mixture of 1 and 4 (or 2/3 and
4) was used for the preparation of mixed SAMs. All experiments
Mica sheets (Agar Scientific, UK) were cut into 1 cm2 pieces and
then cleaved. AFM probes (DNP-S, Veeco, nominal k = 60 pN
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nm-1) were used as received. The probes and cleaved mica were
placed into an Edwards Auto 500 vacuum coater, and 5 nm of
chromium (Testbourne Ltd, UK, 99.99%) followed by 30–35 nm of
gold (Birmingham Metal, UK, 99.99%) were deposited by thermal
evaporation. Upon removal, the gold substrates were immediately
placed into the appropriate thiol solution and left for at least 18 h.
Before use the substrates were thoroughly rinsed with ethanol and
dried under nitrogen.
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1148 | Org. Biomol. Chem., 2010, 8, 1142–1148
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