Dual purpose S-trityl-linkers for glycoarray fabrication on both polystyrene and gold

Article abstract of DOI:10.1039/c2ob26118a

There is a wide range of immobilisation reactions to tether substrates to a variety of surfaces for array-based analysis. Most of these immobilisation strategies are specific for a particular surface and require an additional linker to be attached to the substrate or the surface. Furthermore, the analysis of functionalised surfaces is often restricted to certain analytical techniques and therefore, different immobilisation strategies for different surfaces are desirable. Here we have tested an S-tritylated linker for non-covalent or covalent immobilisation of mannosides to polystyrene or gold surfaces. S-Tritylated mannosides with varying linkers were readily synthesised and used to add to biorepulsive maleimide-terminated preformed SAMs after in situ deprotection of the S-trityl group. In addition, S-tritylated mannosides themselves formed stable glycoarrays on polystyrene microtiter plates. The glycoarrays were successfully analysed by MALDI-ToF mass spectrometry, SPR spectroscopy, and interrogated with GFP-transfected Escherichia coli cells. This work has shown that a dual purpose linker can be used on multiple surfaces to form arrays allowing for different testing as well as analytical approaches.

Full text of DOI:10.1039/c2ob26118a

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PAPER
Dual purpose S-trityl-linkers for glycoarray fabrication on both polystyrene
and gold†
Johannes W. Wehner,a Martin J. Weissenborn,b Mirja Hartmann,a Christopher J. Gray,b Robert Šardzík,b
Claire E. Eyers,b Sabine L. Flitsch*b and Thisbe K. Lindhorst*a
Received 11th June 2012, Accepted 21st September 2012
DOI: 10.1039/c2ob26118a
There is a wide range of immobilisation reactions to tether substrates to a variety of surfaces for
array-based analysis. Most of these immobilisation strategies are specific for a particular surface and
require an additional linker to be attached to the substrate or the surface. Furthermore, the analysis of
functionalised surfaces is often restricted to certain analytical techniques and therefore, different
immobilisation strategies for different surfaces are desirable. Here we have tested an S-tritylated linker for
non-covalent or covalent immobilisation of mannosides to polystyrene or gold surfaces. S-Tritylated
mannosides with varying linkers were readily synthesised and used to add to biorepulsive maleimide-
terminated preformed SAMs after in situ deprotection of the S-trityl group. In addition, S-tritylated
mannosides themselves formed stable glycoarrays on polystyrene microtiter plates. The glycoarrays were
successfully analysed by MALDI-ToF mass spectrometry, SPR spectroscopy, and interrogated with
GFP-transfected Escherichia coli cells. This work has shown that a dual purpose linker can be used on
multiple surfaces to form arrays allowing for different testing as well as analytical approaches.
via native chemical ligation (NCL),17 1,3-dipolar cyclo-
Introduction
addition,18 or Diels–Alder cycloaddition.19,20 Each surface type,
Microarrays are valuable tools in the analysis of biological inter-
actions in fundamental research and in high-throughput screen-
ing and have promising applications as diagnostic devices in the
clinic.1,2 Among many different microarrays, glycoarrays are
carbohydrate-functionalised surfaces, which have received much
attention in the glycosciences for the investigation of the mole-
cular details of carbohydrate–protein interactions,3–7 and to
study cellular adhesion such as in the context of carbohydrate-
specific bacterial colonisation of surfaces.8
A key step in the preparation of glycoarrays, which can
consist of a variety of materials such as gold, glass or poly-
styrene, is the immobilisation of the glycoconjugates on the
respective surface.9–14 A variety of methods have been utilised
for this step, involving covalent or non-covalent attachments.
Common covalent immobilisation techniques include the amide
formation via direct amine coupling into activated esters15,16 or
however, requires specific methods for functionalisation.
For example, for the formation of self-assembled monolayers
(SAMs) on gold, thiol-functionalised derivatives are needed, in
order to form Au–S bonds on the surface. Hence, immersion of
gold wafers in a solution of thiol-functionalised glycosides has
been developed into a common method for the preparation of
glyco-SAMs.18,19,21 Moreover, carbohydrate thiols have been
utilised in the formation of glycoarrays through Michael-type
addition to maleimide-terminated surfaces.22 For the formation
of glycoarrays on polystyrene, prefunctionalised microtiter plates
have been employed for covalent immobilisation.8,23 However,
direct non-covalent array formation on polystyrene is especially
appealing as it requires no additional immobilisation agents. As
polystyrene is inherently hydrophobic, hydrophobic interactions
or π–π interactions between the glycoconjugate and the polymer
surface can be used in this case to produce robust
glycoarrays.3,9,13,24–26
In the course of our work on the preparation and biological
testing of glycoamino acids, we have found that S-trityl-
protected low molecular weight glycoconjugates are readily
made and purified.27,28 This has prompted us to test the direct
application of S-tritylated carbohydrate derivatives for the
preparation of glycoarrays on different surfaces. De-tritylation
would lead to thiol-modified glycoconjugates to allow immobil-
isation on plain gold or on preformed maleimide-terminated
biorepulsive SAMs. On the other hand, the trityl protecting
aOtto Diels Institute of Organic Chemistry, Christiana Albertina
University of Kiel, Otto-Hahn-Platz 3/4, 24098 Kiel, Germany.
E-mail: tklind@oc.uni-kiel.de; Fax: +49-431-880-7410
bSchool of Chemistry & Manchester Institute of Biotechnology, The
University of Manchester, 131 Princess Street, Manchester, M17DN,
UK. E-mail: sabine.flitsch@manchester.ac.uk;
Fax: +44 (0)161-275-1311; Tel: +44 (0)161-306-5172
†Electronic supplementary information (ESI) available: Experimental
procedures and analytical details (NMR, mass spectrometry, characteris-
ation and formation of glycoarrays). See DOI: 10.1039/c2ob26118a
This journal is © The Royal Society of Chemistry 2012
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2-aminoethyl mannoside 129 with the commercially available
cysteine derivatives Fmoc–Cys(Trt)–OH (2) or 11-tritylsulphanyl-
undecanoic acid (3),9,30 leading to the peptide-coupled manno-
sides 427 and 6,9 respectively. Removal of the Fmoc protecting
group and N-acetylation can be effected in one pot yielding the
known S-tritylated glycoamino acid 5.27 Then, it was important
to add S-tritylated mannoside derivatives to the collection having
a longer spacer, because this usually facilitates immobilisation
of the respective compound on a surface. Therefore, 6-amino-
4-thiahexyl mannoside 78,31 was made and subjected to peptide
coupling with the S-tritylated thiols 2 and 3, using HATU and
DIPEA. In analogy to the preparation of 4 and 6, this reaction
led to the trityl-functionalised mannosides 8 and 11, having con-
siderably longer spacers than their analogues 4 and 6. Removal
of the Fmoc protecting group in 8 led to 9 and then acetylation
to the N-acetylated target molecule 10 (Scheme 1).
Fig. 1 Dual linkers: thio-functionalised bioprobes (e.g. carbohydrates
as shown for α-D-mannosides) can be directly attached to polystyrene
surfaces in their S-tritylated form (left), or added as thiols to gold or
maleimide-terminated surfaces (right).
Fabrication and interrogation of glycoarrays on gold
group should also allow preparation of glycoarrays on simple
polystyrene microtiter plates, by hydrophobic interactions
between the molecule’s trityl fragment and the hydrophobic
surface (Fig. 1). In this account we report the synthesis of S-trity-
lated glycosides, their utilisation in glycoarray fabrication on
gold as well as on polystyrene and the interrogation of the pre-
pared surfaces with lectins as well as live bacterial cells.
Initially we tested, if the prepared S-tritylated glycosides can be
immobilised on gold with concomitant removal of the trityl pro-
tecting group. Thus, in situ de-tritylation of 5, 6, 10, and 11 was
effected overnight by treatment with trifluoroacetic acid (TFA)
and triethylsilane (TES) in dichloromethane.28 Then, the solvent
was removed and the crude free thiol dissolved in PBS buffer,
centrifuged and the solution applied to the gold surface accord-
ing to the standard protocol for preparation of SAMs.17 To test if
the immobilisation of in situ deprotected mannosides was
successful, the prepared glycoarrays were analysed by MALDI-
ToF mass spectrometry.32 This mass spectrometric protocol is a
reliable method for the analysis of SAMs on gold, in which typi-
cally the masses of the disulphides of the respective thiols are
detected.17 Also here, the detected peaks correspond to the disul-
phides of the thiols derived from 5, 6, 10, and 11 (cf. ESI,
Fig. S16–S19†). Thus, the MS analysis showed the success of
glycoarray formation after in situ de-tritylation. In addition,
Results and discussion
Synthesis of S-tritylated glycoconjugates
As we have long-standing interest in the investigation of
mannose-specific lectins, in particular mannose-specific bacterial
adhesion, we have made a selection of four S-tritylated manno-
side derivatives for this study, 5, 6, 10, and 11 (Scheme 1).
We have shown earlier that preparation of mannosides 5 and 6
is readily accomplished by coupling of the well-known
Scheme 1 Synthesis of S-tritylated α-D-mannosides 5, 6, 10, and 11. Reaction conditions: (a) HATU, DIPEA, DMF, 0 °C → room temp., overnight,
81% (4, from 1 and 2), 74% (6, from 1 and 3), 84% (8, from 7 and 2), 78% (11, from 7 and 3); (b) (i) morpholine, DMF, room temp., quant. (ii)
Ac2O, DIPEA, room temp., 4 h, quant.; (c) morpholine, DMF, room temp., 67%; (d) pyridine, Ac2O, room temp., overnight, 96%.
8920 | Org. Biomol. Chem., 2012, 10, 8919–8926
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Fabrication and interrogation of glycoarrays on polystyrene
tritylated 6 was deprotected and purified to deliver the pure thiol
6-SH. When pure 6-SH was employed for glycoarray fabri-
cation, mass spectrometric analysis gave very similar results as
when the in situ deprotection–immobilisation approach was
employed (cf. ESI, Fig. S15†). The efficiency of immobilisation
of in situ deprotected thiols greatly simplifies fabrication of
glycoarrays on gold. S-Tritylated derivatives are much easier to
purify than free thiols, owing to their greater hydrophobicity.
In addition, free thiols are prone to oxidation, forming the
respective disulphides, a problem which is circumvented in the
in situ deprotection approach.
The same in situ de-tritylation protocol was employed on pre-
formed maleimide-terminal self-assembled monolayers (SAMs)
on gold (cf. Fig. 1). These SAMs include biorepulsive oligoethyl-
eneglycol units,33 which are important in biological studies to
avoid nonspecific protein adsorption. The S-tritylated bioprobes
5, 6, 10, and 11 were treated as above and the non-purified mix-
tures directly applied to the maleimide-functionalised surface.
After 1.5 h reaction time, the surface was rinsed with ethanol
and again analysed by MALDI-ToF MS. The MALDI MS
spectra showed the corresponding masses of the coupled ligands
(ESI, Fig. S24–S27†). Comparison with the coupling results
obtained with previously deprotected and purified thiols revealed
that glycoarray formation after in situ deprotection is similarly
effective.
As an additional method to test glycoarray formation on gold,
SPR spectroscopy was used. Here, the mannose-specific lectin
concanavalin A (ConA) was employed for interrogation of
glycoarrays prepared after in situ de-tritylation of a pair of com-
parable mannosides, 10 having the shorter spacer, and 11 having
a longer spacer incorporated. The crude thiols were added to a
maleimide-terminated biorepulsive SAM on gold and ConA was
allowed to interact with the formed glycoarray. In both cases, the
expected carbohydrate–lectin interactions were detected,
suggesting glycoarray formation (Fig. 2). For the array formed
from thiol derived from 11, a much stronger interaction
with ConA was measured than for the analogous case using 10.
This suggests that (tritylated) thiols having the bioprobe
attached to a rather long spacer are better suited for surface
immobilisation.
The next step was to employ the S-trityl group in mannosides 5,
6, 10, and 11 (Scheme 1) to anchor these molecules to a hydro-
phobic polystyrene surface, as it has been shown earlier for
another type of tritylated molecules.13 For the non-covalent func-
tionalisation of polystyrene microplates hydrophobic molecules
have been used regularly. From these studies it is known that the
π–π interactions established between the polystyrene surface and
the aromatic trityl fragment are strong enough to guarantee a
robust direct immobilisation on polystyrene microtiter plates.
First, the reaction conditions for glycoarray fabrication on
polystyrene were optimised and methanol was identified as
the most suitable solvent for immobilisation of the prepared
tritylated glycosides. Then, in order to determine the stability
of produced glycoarrays against different washing conditions,
a colorimetric phenol–sulphuric acid assay was performed
(Fig. 3).34–36 This assay allows quantification of glycoconjugates
immobilised on surfaces. Washing with ethanol removed the
immobilised glycosides completely, as expected. In contrast,
washing with twice distilled water and/or PBST buffer led to
negligible reduction of the carbohydrate content on the surface
in the case of the glycoarrays formed from the tritylated glyco-
sides 6, 10, and 11. However, the mannoside with the shortest
spacer, compound 5, formed the least stable glycoarray on poly-
styrene which was washed out by water or buffer to over 50%
according to the phenol–sulphuric acid assay.
In the next step, the prepared glycoarrays were tested with live
bacterial cells in a GFP-assisted adhesion assay, which was
established earlier.8 Here, the genetically engineered E. coli
strain PKL1162 was used.8,37 Protocols for cellular adhesion
assays on polystyrene microplates usually involve a blocking
step with BSA or skimmed milk for example, to prevent
unspecific binding of the cells to the microtiter plate surface.
However, we could show that when the tritylated mannosides
Fig. 3 Removal of compounds 5, 6, 10 and 11 from polystyrene sur-
faces using different washing steps. Microtiter plate wells were functio-
nalised with 25 mM methanolic solutions of tritylated mannosides 5, 6,
10, and 11, then it was washed with water and/or buffer and the remain-
ing glycoside content on the surface determined by the phenol–sulphuric
colorimetric acid assay. The glycoside content without washing was
defined as 100%. Six-fold washing with ethanol removed the glycoarray
completely (not shown).
Fig. 2 SPR spectroscopy on surfaces functionalised with 10 and 11
using the lectin ConA was used to prove the formation of glycoarrays
after in situ deprotection of tritylated mannosides 10 and 11.
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Fig. 5 Inhibition curves of competitive bacterial adhesion inhibition
assays using polystyrene glycoarrays prepared from 6 (top) and 11
(bottom). Methyl α-D-mannoside was used as an inhibitor and type 1
fimbriated E. coli cells (PKL1162) were used to adhere to the surface.
Fig. 4 Bacterial adhesion curves (GFP-tagged E. coli PKL1162)
obtained by application of glycoarrays consisting of compounds 6 (top)
and 11 (bottom) after 1 h incubation and fluorescence readout.
were used for modification of polystyrene plates, no blocking
step was necessary (ESI, Fig. S28†).
glycoarray formation. This finding is in line with earlier results,
which have indicated that mannosides having a thiahexyl
aglycon moiety show a higher affinity to the mannose-specific
lectin of E. coli than mannosides having an ethyl aglycon.38
Glycoarrays on polystyrene were prepared using tritylated
mannosides 6 and 11 at different concentrations. Concentration
dependency of bacterial adhesion to these two glycoarrays was
tested and was found as expected in both cases, with the inten-
sity of the GFP fluorescence increasing with higher con-
centrations of the applied mannoside solutions (Fig. 4). A
plateau was reached at concentrations between 20 mM and
25 mM. Mannosides 5 and 10 were less suited in this assay.
Only little adhesion could be detected and no consistent concen-
tration dependency of bacterial adhesion could be observed in
the case of these mannosides linked via short spacers (ESI,
Fig. S31†).
After having shown that tritylated mannosides such as 6 and
11 form stable glycoarrays on polystyrene microtiter plates upon
direct treatment, testing of inhibition of bacterial adhesion to
these surfaces could be done next. From the results obtained
in the adhesion experiments, 25 mM concentrations appeared
optimal to form microarrays for competitive bacterial adhesion
inhibition assays. As described earlier,38 serial dilutions
Conclusions
S-Tritylated mannosides were synthesised and shown to be suit-
able for the fabrication of glycoarrays on different surfaces such
as gold and polystyrene. An in situ deprotection protocol has
allowed us to apply tritylated carbohydrate derivatives on plain
gold as well as on maleimide-terminal preformed biorepulsive
SAMs. As SAMs on gold on the one hand and polystyrene
microtiter plates on the other can be used in quite different appli-
cations, S-tritylated glycoconjugates can be regarded as facile
derivatives for orthogonal immobilisation on surfaces of opposite
character.
The prepared glycoarrays were analysed by MALDI-ToF
MS and were shown to be robust and suited for interrogation
with lectins and live bacterial cells. Next, we will further employ
this methodology in a 384 well polystyrene microtiter plate
format to facilitate inhibitor screening in bacterial adhesion
assays.
of methyl α-D-mannoside (MeMan),
a standard inhibitor
of mannose-specific bacterial adhesion, were applied to inhibit
bacterial adhesion to the two different glycoarrays formed with 6
and 11, respectively. The obtained inhibition curves are depicted
in Fig. 5. After sigmoidal fitting of the testing results, IC50
values could be deduced, with IC50 (MeMan) ∼2.9 mM for the
inhibition of bacterial adhesion to the surface, modified with
mannoside 6 and IC50 (MeMan) ∼5.3 mM for 11. Thus, the
surface prepared from mannosides 11 appears to be slightly
more adhesive in this testing system than when 6 was used for
Experimental
Commercially available starting materials and reagents were
used without further purification. Reactions requiring dry con-
ditions were performed under an atmosphere of nitrogen using
8922 | Org. Biomol. Chem., 2012, 10, 8919–8926
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General procedure for in situ de-tritylation of S-protected
mannosides
oven-dried glassware. Anhydrous DMF was purchased from
Acros. All other used solvents were purified by distillation.
ConA was purchased from Vector labs. Bovine serum albumin
(BSA), methyl α-D-mannopyranoside (MeMan) and polyethy-
lene glycol sorbitan monolaurate (Tween® 20) were obtained
from Sigma-Aldrich. Microtiter plates with a hydrophobic
surface (Corning, no. 3540, low volume 384 wells, flat clear
bottom, black polystyrene, nontreated and Corning, no. 3631, 96
wells, flat clear bottom, black polystyrene, nontreated) were
obtained from Corning. 2-Aminoethyl α-D-mannopyranoside
(1),29 6-amino-4-thiahexyl α-D-mannopyranoside (7),8,31
N-(fluoren-9-yl-methoxycarbonyl)-S-(triphenylmethyl)-L-cysteine-
[2-(α-D-mannopyranosyloxy)ethyl]amide (4),27 11-tritylsulphanyl-
undecanoic acid, and 2-(11-tritylsulphanyl-undecanoyl)-
aminoethyl α-D-mannopyranoside (6)9 were prepared according
to the literature.
The S-tritylated mannosides (5, 6, 10, or 11, 3 μmol) were dis-
solved in dichloromethane (1 mL). Then, triethylsilane (5 equiv.)
and trifluoroacetic acid (5 equiv.) were added and the reaction
mixture was left for 1.5 h at room temperature and was sub-
sequently treated with further trifluoroacetic acid (5 equiv.) and
left overnight. Thereafter, the solvent was removed in vacuo and
the residue dissolved in 10 mM PBS to obtain a final concen-
tration of the corresponding free thiol of 10 mM. This solution
was centrifuged and the supernatant directly applied to the differ-
ently modified gold surfaces.
Fabrication of glycoarrays on gold and maleimide-terminated
SAMs and their MALDI-ToF MS analysis
Reactions were monitored by thin-layer chromatography using
either silica gel 60 GF254 on aluminium foil (Merck) or RP-18
F254s on aluminium foil (Merck) with detection by UV light
and charring with sulphuric acid in EtOH (10%). Merck silica
gel 60 (0.040–0.063 mm) was used for flash chromatography.
Analytical HPLC was performed on a Merck Hitachi LaChrom
L-7000 series apparatus with a LiChrospher 100 RP-8 (5 μm,
Merck) column (for HPLC chromatograms see the ESI†). Pre-
parative MPLC was performed on a Büchi apparatus using a
LiChroprep RP-18 column (40–60 μm, Merck) for reversed-
phase and a LiChroprep Si 60 column (40–60 μm, Merck) for
normal-phase silica gel chromatography. 1H and 13C NMR
spectra were recorded on a Bruker DRX-500 or a Bruker AV-600
instrument. NMR spectra were calibrated with respect to the
solvent peak (in the case of CDCl3 the reference was tetra-
methylsilane (TMS)). 2D NMR techniques (COSY, HSQC,
HMBC) were used for full assignment of the spectra. ESI MS
measurements were performed on a Mariner ESI-ToF 5280
instrument (Applied Biosystems). MALDI-ToF mass spectra
were recorded on a Bruker Biflex-III 19 kV instrument with Cl-
CCA (4-chloro-α-cyanocinnamic acid) or DHB (2,5-dihydroxy-
benzoic acid) as matrix. Optical rotation was measured on a
Perkin-Elmer polarimeter 341 (Na-D-line: 589 nm, length of cell
1 dm). IR spectra were recorded on a Perkin-Elmer Paragon
1000 FT-IR instrument. For sample preparation a Golden Gate
diamond ATR unit with a sapphire stamp was used. The SPR
experiments were performed on a Biacore 3000 system (GE
Healthcare, Sweden) using a gold sensor chip (GE Healthcare).
For bacterial adhesion studies and phenol–sulphuric acid assays,
a TECAN infinite 200 multifunction microplate reader was
employed. The wavelengths of the band pass filters for excitation
and emission were 485 and 535 nm, respectively. For the
phenol–sulphuric acid assay absorbance at 492 nm was
measured.
A disposable 64-well gold plate (Applied Biosystems) was
cleaned with a Piranha solution (12 mL, 3 : 1 conc. H2SO4/30%
H2O2) for 30 min, rinsed with distilled water and ethanol
and dried under a stream of nitrogen. A solution of carboxylic
acid-terminated linkers [HS–(CH2)17–(OC2H4)6–OCH2–COOH]
and tri(ethylene glycol)-terminated alkanethiol spacers
[HS–(CH2)17–(OC2H4)3–OH] in dry DMSO (final concentration
0.4 mg mL−1, molar ratio 1 : 4) was applied on the plate (∼1 μL
per well) and left overnight at room temperature to form a mixed
SAM. The plate was washed with ethanol and dried under nitro-
gen. The carboxylic acid groups were activated by spotwise
treatment with a solution of EDC, NHS and N-(2-aminoethyl)-
maleimide (all Sigma-Aldrich, 0.180 M, 0.174 M and 0.174 M,
respectively) in dry DMF for 1–2 h, followed by washing with
water and ethanol and drying as above. The product formation
was analysed by MALDI-ToF MS. Unless otherwise stated all
MALDI-ToF MS experiments on gold surfaces were carried out
on an Ultraflex II instrument (Bruker Daltonics) in positive
reflection mode. A solution of matrix (2,4,6-trihydroxyacetophe-
none, 10 mg mL−1 in acetone) was applied on the gold and
allowed to dry before analysis.
Surface plasmon resonance (SPR) analysis using 10 and 11
The gold-coated sensor was cleaned with a Piranha solution,
rinsed with water and ethanol and dried in a stream of nitrogen.
The formation of self-assembling monolayers was performed in
the same way as described above. The chip was washed with
ethanol, dried under nitrogen and mounted onto a chip holder
following the instructions in the supplier’s manual. After
docking in the instrument the sensor was equilibrated with PBS
buffer (10 mM, degassed and filtered) at a flow rate of 10 μL
min−1. For surface activation, 70 μL of a 1 : 1 mixture of
freshly prepared solutions of NHS (0.4 M) and EDC (0.1 M) in
water were injected. The reference spot (channel 1) was blocked
by injecting 70 μL of aminoethanol-hydrochloride (1 M).
Additional channels were modified with N-(2-aminoethyl)-
maleimide (10 mM, flow rate 10 μL min−1) for 10 min and then
treated with in situ deprotected mannosides 10 and 11 for about
E. coli bacteria (PKL1162)8,37 were used and grown in
LB-media + AMP + CAM (100 mg ampicillin, 50 mg
chloramphenicol L−1) at 37 °C under slight agitation. Buffers
were used as follows: PBS buffer solution (pH 7.2): sodium
chloride (8.00 g), potassium chloride (200 mg), sodium hydro-
gen phosphate-dihydrate (1.44 g), and potassium dihydrogen
phosphate (200 mg) were dissolved in bidist. water (1.00 L).
PBST buffer solution (pH 7.2): PBS buffer + 0.05% v/v
Tween® 20.
an hour at a flow rate of 3 μL min−1
.
Binding studies were carried out using the lectin ConA (10 μg
mL−1, 250 μL) in buffer (0.15 M NaCl, 1 mM CaCl2, 1 mM
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MnCl2, pH 7.0) at a flow rate of 25 μL min−1. After the injection
and the wells were then washed with PBS buffer (3 × 20 μL per
a 600 s dissociation sequence was followed.
well). Fluorescence was read out at 485/535 nm.
2-(11-Sulphhydryl-undecanoyl)aminoethyl
Fabrication of glycoarrays on polystyrene
α-D-mannopyranoside (6-SH)
A series of 5 to 50 mM stock solutions of tritylated mannosides
5, 6, 10, and 11 (in MeOH) were prepared. A 12 μL sample of
each solution was pipetted into a 384-well polystyrene microtiter
plate, which was dried by standing overnight at ambient temp-
erature. Each well was then washed three times with deionised
water and three times with PBST buffer (20 μL per well each).
The tritylated mannoside 6 (50 mg, 75.1 μmol) was dissolved in
dichloromethane (1 mL), triethylsilane (60 μL, 376 μmol) and
trifluoroacetic acid (58 μL, 751 μmol) were added and the reac-
tion mixture was stirred for 2 h at ambient temperature. The
solvent was removed under reduced pressure and the crude
product was purified by RP-MPLC (120 g RP-18, A: methanol,
B: water, A: 60% → 95%, 90 min) yielding the deprotected title
compound 6-SH (31.3 mg, 73.6 μmol, 98%) after lyophilisation.
Rf 0.33 (methanol–water, 3 : 1); [α]2D6 = +40.4 (c = 0.5, metha-
Phenol–sulphuric acid assay
1
3
To test the stability of glycoarrays formed by immobilisation of
5, 6, 10 or 11 against different washing conditions, these com-
pounds were immobilised as described above followed by
6 washing cycles with ethanol (20 μL per well each) followed
by phenol–sulphuric acid assay. 12 μL of solutions of trityl-
protected carbohydrate (50 mM, 25 mM, 12.5 mM solutions in
MeOH) were pipetted into a 384-well microtiter plate and the
plate was allowed to dry by standing overnight at ambient temp-
erature. The wells were then washed three times with deionised
water and three times with PBST (20 μL per well each). The
phenol–sulphuric acid assay was performed according to a litera-
ture-known method.35 A 5% phenol solution (4.2 μL per well)
was pipetted to the wells, followed by the addition of concen-
trated H2SO4 (21 μL per well). The mixture was incubated for
30 min at room temperature, and the absorbance measured at
492 nm (A492) to determine the amount of carbohydrate
immobilised on the microtiter plate. The amount of immobilised
5, 6, 10 and 11 was estimated from the ratio of the absorption at
492 nm of immobilised compounds (subjected to 3 washing
cycles with deionised water and/or PBST) to the A492 of
the corresponding control (unwashed). Washings with ethanol
(6 washing cycles using 20 μL ethanol per well) removed the
glycoarrays completely according to the phenol–sulphuric acid
assay.
nol); H NMR (500 MHz, CD3OD, 300 K): δ = 4.76 (d, J =
2
3
1.7 Hz, 1H, H-1Man), 3.83 (dd, J = 11.7 Hz, J = 2.3 Hz, 1H,
3
3
H-6aMan), 3.81 (dd, J = 1.7 Hz, J = 3.4 Hz, 1H, H-2Man), 3.75
(mc, 1H, OCHHCH2NH), 3.72–3.67 (m, 2H, H-3Man, H-6bMan),
3.60 (dd ∼ t, 3J = 9.5 Hz, 1H, H-4Man), 3.56–3.51 (m, 2H,
H-5Man, OCHHCH2NH), 3.45–3.32 (m, 2H, OCH2CH2NH),
3
3
2.49 (t, J = 7.1 Hz, 2H, CH2CH2SH), 2.19 (t, J = 7.5 Hz,
2H, HN(O)CCH2CH2), 1.59 (mc, 4H, HN(O)CCH2CH2,
OCH2CH2CH2S), 1.40 (mc, 2H, CH2CH2CH2), 1.35–1.25 (m,
12H, CH2CH2CH2) ppm; 13C NMR (125 MHz, CD3OD,
300 K): δ = 176.5 (C(O)NH), 101.7 (C-1Man), 74.8 (C-5Man),
72.6 (C-3Man), 72.1 (C-2Man), 68.6 (C-4Man), 67.3
(OCH2CH2NH), 62.9 (C-6Man), 40.2 (OCH2CH2NH), 37.1 (HN
(O)CCH2CH2), 35.2 (OCH2CH2CH2S), 30.6, 30.5, 30.4, 30.3,
30.2, 29.4 (6 CH2CH2CH2), 27.0 (HN(O)CCH2CH2), 25.0
(CH2CH2SH) ppm; HR-ESI MS: calcd for C38H72N2NaO14S2
(disulphide): m/z 867.4317 [M + Na]+; found: m/z 867.4309
[M + Na]+; IR (ATR): ν˜ = 3308, 2918, 2850, 1637, 1554, 1463,
1132, 1057, 1031, 975 cm−1
.
N-(Fluoren-9-yl-methoxycarbonyl)-S-(triphenylmethyl)-L-
cysteine-[6-(α-D-mannopyranosyloxy)-3-thiahexyl]amide (8)
A mixture of Fmoc-L-Cys(Trt)-OH (2, 2.46 g, 4.18 mmol),
mannoside 1 (1.37 g, 4.60 mmol), and HATU (1.91 g,
5.02 mmol) was dried for 1 h under vacuum and then dissolved
in dry DMF (40 mL). It was cooled to 0 °C, DIPEA (853 μL,
5.02 mmol) was added and the reaction mixture stirred overnight
at ambient temperature under a nitrogen atmosphere. The solvent
was removed under reduced pressure and the crude product was
purified by column chromatography (methanol–ethyl acetate,
1 : 12 → 1 : 9) yielding the title compound 8 (3.01 g, 3.48 mmol,
84%) as a colourless foam. Rf 0.38 (methanol–ethyl acetate,
GFP-based bacterial adhesion assay
Determination of bacterial adhesion. Trityl-protected carbo-
hydrates (5, 6, 10, and 11) were immobilised on 384-well micro-
titer plates as described above. The wells were incubated with
E. coli PKL1162 (2 mg mL−1 PBS buffer) for 1 h (37 °C,
120 rpm), and subsequently washed with the same buffer
(3 × 20 μL per well). E. coli binding to the mannoside-
functionalised surface was monitored by fluorescence measure-
ments at 485/535 nm using a microplate reader.
1
1 : 9); [α]2D2 = +21.8 (c = 0.5, methanol); H NMR (500 MHz,
CD3OD, 300 K): δ = 7.78 (mc, 2H, H-arylFmoc), 7.66 (d, 3J
= 6.8 Hz, 2H, H-arylFmoc), 7.40–7.34 (m, 8H, H-arylTrt
,
Inhibition of bacterial adhesion with methyl α-D-mannoside
(MeMan). Compounds 6 and 11 (12 μL per well, 25 mM) were
immobilised on 384-well microtiter plates as described above.
Then, 5 μL of a serial dilution of the standard inhibitor MeMan
(1 μM–1000 mM) were pipetted to the plate followed by
addition of 5 μL of E. coli (PKL1162) solution (4 mg mL−1
PBS buffer). The plate was incubated for 1 h (37 °C, 120 rpm)
H-arylFmoc), 7.30–7.19 (m, 14H, H-arylTrt, H-arylFmoc), 4.72 (d,
2
3
3J = 1.6 Hz, 1H, H-1Man), 4.41 (dd, J = 10.6 Hz, J = 7.1 Hz,
1H, CHHFmoc), 4.30 (dd, 2J = 10.6 Hz, 3J = 6.8 Hz, 1H,
3
3
CHHFmoc), 4.23 (dd ∼ t, J = 6.8 Hz, 1H, CHFmoc), 3.93 (dd, J
= 8.4 Hz, 3J = 5.5 Hz, 1H, H-αCys), 3.82 (dd, 2J = 11.7 Hz, 3J =
3
3
2.4 Hz, 1H, H-6aMan), 3.78 (dd, J = 3.3 Hz, J = 1.6 Hz, 1H,
H-2Man), 3.77–3.74 (m, 1 H, OCHHCH2CH2S), 3.71 (dd,
8924 | Org. Biomol. Chem., 2012, 10, 8919–8926
This journal is © The Royal Society of Chemistry 2012

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2J = 11.7 Hz, J = 5.8 Hz, 1H, H-6bMan), 3.67 (dd, J = 9.4 Hz,
3J = 3.3 Hz, 1H, H-3Man), 3.61 (dd ∼ t, 3J = 9.6 Hz, 1H,
H-4Man), 3.54–3.50 (m, 1H, H-5Man), 3.49–3.43 (m, 1H,
OCHHCH2CH2S), 3.34–3.23 (m, 2H, SCH2CH2NH), 2.62–2.49
(m, 6H, OCH2CH2CH2S, SCH2CH2NH, H-βCys), 1.79 (mc, 2H,
OCH2CH2CH2S) ppm; 13C NMR (125 MHz, CD3OD, 300 K):
δ = 172.7 (C(O)NH), 158.0 (OC(O)NH), 146.0 (C-arylTrt),
145.1, 142.6 (C-arylFmoc), 130.8, 129.0 (CH-arylTrt),
128.8, 128.2 (CH-arylFmoc), 127.9 (CH-arylTrt), 126.3, 120.9
(CH-arylFmoc), 101.6 (C-1Man), 74.7 (C-5Man), 72.7 (C-3Man),
72.2 (C-2Man), 72.0 (Cq,Trt), 68.6 (C-4Man), 68.1 (CH2,Fmoc),
66.9 (OCH2CH2CH2S), 62.9 (C-6Man), 55.7 (C-αys), 48.4
(CHFmoc), 40.1 (SCH2CH2NH), 35.2 (C-βCys), 31.9
(SCH2CH2NH), 30.6 (OCH2CH2CH2S), 29.3 (OCH2CH2CH2S)
ppm; MALDI-ToF MS (DHB): calcd for C48H52N2NaO9S2: m/z
887.30 [M + Na]+; found: m/z 887.50 [M + Na]+; calcd
for C48H52KN2O9S2: m/z 903.27 [M + K]+; found: m/z 903.48
[M + K]+; IR (ATR): ν˜ = 3316, 3055, 2924, 1705, 1660, 1521,
3
3
N-(Acetyl)-S-(triphenylmethyl)-L-cysteine-[6-(α-D-
mannopyranosyloxy)-3-thiahexyl]amide (10)
The glycoamino acid 9 was dissolved in pyridine (2 mL) and
acetic anhydride (110 μL, 1.17 mmol) was added. The reaction
mixture was stirred overnight at room temperature. Then solvents
were removed under reduced pressure, it was codistilled with
toluene three times (10 mL each) and the crude product was sub-
jected to RP-MPLC (60 g RP-18, A: methanol, B: water, A:
40% → 95%, 120 min) yielding the title compound 10 (154 mg,
225 μmol, 96%) after lyophilisation. Rf 0.31 (ethyl acetate);
[α]2D2 = +29.4 (c = 0.5, methanol); HPLCtR = 2.64 min (A =
water, B = methanol, A: 20%, 10 min, 1.2 mL min−1); 1H NMR
(500 MHz, CD3OD, 300 K): δ = 7.38 (mc, 6H, H-arylTrt), 7.30
(mc, 6H, H-arylTrt), 7.23 (mc, 3H, H-arylTrt), 4.73 (d, 3J =
1.6 Hz, 1H, H-1Man), 4.19 (mc, 1H, H-αCys), 3.83 (dd, 2J =
11.7 Hz, 3J = 2.4 Hz, 1H, H-6aMan), 3.81–3.77 (m, 2H,
2
3
OCHHCH2CH2S, H-2Man), 3.71 (dd, J = 11.7 Hz, J = 5.6 Hz,
3
1H, H-6bMan), 3.70–3.67 (m, 1H, H-3Man), 3.61 (dd ∼ t, J =
1490, 1445, 1318, 1230, 1130, 1084, 1029, 974, 739 cm−1
.
9.6 Hz, 1H, H-4Man), 3.55–3.50 (m, 1H, H-5Man), 3.50–3.45 (m,
1H, OCHHCH2CH2S), 3.36–3.24 (m, 2H, SCH2CH2NH),
3.62–2.56 (m, 4H, OCH2CH2CH2S, SCH2CH2NH), 2.56 (dd,
2J = 12.4 Hz, 3J = 6.3 Hz, 1H, H-βaCys), 2.49 (dd, 2J = 12.4 Hz,
3J = 7.7 Hz, 1H, H-βbCys), 1.91 (s, 3H, NH(O)CCH3), 1.82 (mc,
2H, OCH2CH2CH2S) ppm; 13C NMR (125 MHz, CD3OD,
300 K): δ = 173.0 (CH3C(O)NH), 172.4 (C(O)NH), 145.9
(C-arylTrt), 130.7, 129.0, 128.0 (CH-arylTrt), 101.6 (C-1Man),
74.7 (C-5Man), 72.7 (C-3Man), 72.2 (C-2Man), 68.6 (C-4Man), 68.0
(Cq,Trt), 66.9 (OCH2CH2CH2S), 62.9 (C-6Man), 54.0 (C-αCys),
40.1 (SCH2CH2NH), 34.9 (C-βCys), 31.9 (SCH2CH2NH), 30.7
(OCH2CH2CH2S), 29.3 (OCH2CH2CH2S), 22.5 (NHCOCH3)
ppm; HR-ESI MS: calcd for C35H44N2NaO8S2: m/z 707.2431
[M + Na]+; found: m/z 707.2426 [M + Na]+; MALDI-ToF MS
(Cl-CCA): calcd for C35H44N2NaO8S2: m/z 707.24 [M + Na]+;
found: m/z 707.33 [M + Na]+, calcd for C35H44KN2O8S2: m/z
723.22 [M + K]+; found: m/z 723.31 [M + K]+; IR (ATR): ν˜ =
3284, 2926, 1645, 1535, 1489, 1443, 1372, 1202, 1131, 1085,
S-(Triphenylmethyl)-L-cysteine-[6-(α-D-mannopyranosyloxy)-
3-thiahexyl]amide (9)
The Fmoc-protected cysteinyl mannoside 8 (406 mg, 467 μmol)
was dissolved in dry DMF (5 mL) under a nitrogen atmosphere
and morpholine (250 μL, 2.87 mmol) was added. The reaction
mixture was stirred for 1 h at ambient temperature and then
another portion of morpholine (250 μL, 2.87 mmol) was added.
This was repeated after 3 h and 4 h and stirred overnight. After
18 h the volatile compounds were removed under reduced
pressure and the crude product was purified by column chrom-
atography (methanol–ethyl acetate–TEA, 100 : 20 : 1) yielding
the title compound 9 (200 mg, 311 μmol, 67%) as a colourless
syrup. Rf 0.28 (RP-18, methanol–water, 4 : 1); [α]2D2 = +22.8 (c =
0.26, methanol); 1H NMR (500 MHz, CD3OD, 300 K): δ = 7.41
(mc, 6H, H-arylTrt), 7.29 (mc, 6H, H-arylTrt), 7.22 (mc, 3H,
H-arylTrt), 4.74 (d, 3J = 1.6 Hz, 1H, H-1Man), 3.85–3.77 (m, 3 H,
OCHHCH2CH2S, H-6aMan, H-2Man), 3.71 (dd, 2J = 11.7 Hz,
1055, 1031, 975, 742, 698, 675 cm−1
.
3
3
3J = 5.7 Hz, 1H, H-6bMan), 3.69 (dd, J = 9.6 Hz, J = 3.4 Hz,
1H, H-3Man), 3.61 (dd ∼ t, 3J = 9.6 Hz, 1H, H-4Man),
3.55–3.46 (m, 2H, H-5Man, OCHHCH2CH2S), 3.41–3.30 (m,
2H, SCH2CH2NH), 3.11 (dd ∼ t, 3J = 6.5 Hz, 1H, H-αCys),
6-(10-Tritylsulphanyl-undecanoyl)amino-4-thiahexyl-α-D-
mannopyranoside (11)
2
2.65–2.59 (m, 4H, OCH2CH2CH2S, SCH2CH2NH), 2.55 (dd, J
11-Tritylsulphanyl-undecanoic acid (3, 194 mg, 420 μmol) and
HATU (319 mg, 840 μmol) were dried for 1 h under vacuum,
dry DMF (2.50 mL) and DIPEA (288 μL, 1.68 mmol) were
added and the mixture was stirred for 20 min under a nitrogen
atmosphere at ambient temperature. Simultaneously in a different
reaction vessel 7 (150 mg, 504 μmol) was dried for 1 h under
vacuum, dissolved in dry DMF (2.50 mL) and stirred for 20 min
under a nitrogen atmosphere at ambient temperature. The reac-
tion mixture with the preactivated 11-tritylsulphanyl-undecanoic
acid (3) and HATU was cooled to 0 °C, the solution of manno-
side 7 was added and it was stirred under a nitrogen atmosphere
at ambient temperature overnight. Then, all volatile compounds
were removed under reduced pressure and the crude product was
subjected to MPLC (150 g silica column, A: ethyl acetate, B:
methanol, A: 99% → 85%, 120 min) and RP-MPLC (60 g
RP-18, A: methanol, B: water, A: 50% → 95%, 120 min) yield-
ing the title compound 11 (242 mg, 327 μmol, 78%) as a
= 12.1 Hz, 3J = 6.1 Hz, 1H, H-βaCys), 2.40 (dd, 2J = 12.1 Hz, 3J
= 7.0 Hz, 1H, H-βbCys), 1.90 (s, 3H, NHCOCH3), 1.83 (mc, 2H,
OCH2CH2CH2S) ppm; 13C NMR (125 MHz, CD3OD, 300 K): δ
= 175.5 (C(O)NH), 146.1 (C-arylTrt), 130.8, 129.0, 127.9 (CH-
arylTrt), 101.6 (C-1Man), 74.7 (C-5Man), 72.7 (C-3Man), 72.2
(C-2Man), 68.6 (C-4Man), 67.8 (Cq,Trt), 66.9 (OCH2CH2CH2S),
62.9 (C-6Man), 55.3 (C-αCys), 40.0 (SCH2CH2NH), 38.3
(C-βCys), 32.0 (SCH2CH2NH), 30.7 (OCH2CH2CH2S), 29.3
(OCH2CH2CH2S) ppm; HR-ESI MS: m/z = [Trt]+ 243.1242;
calcd for C33H43N2O7S2: m/z 643.2506 [M + H]+; found: m/z
643.2534 [M + H]+; calcd for C33H42NaN2O7S2: m/z 665.2326
[M + Na]+; found: m/z 665.2323 [M + Na]+; MALDI-ToF MS
(DHB): calcd for C33H42NaN2O7S2: m/z 665.23 [M + Na]+;
found: m/z 665.47 [M + Na]+; IR (ATR): ν˜ = 3303, 3062, 2921,
1658, 1519, 1488, 1443, 1318, 1261, 1129, 1084, 1054, 1024,
975, 803, 743 cm−1
.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 8919–8926 | 8925

View Article Online
colourless foam. Rf 0.29 (ethyl acetate), [α]2D2 = +25.3 (c = 0.5,
5 (a) K. R. Love and P. H. Seeberger, Angew. Chem., 2002, 114, 3733–
3736; K. R. Love and P. H. Seeberger, Angew. Chem., Int. Ed., 2002, 41,
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6 T. Feizi, F. Fazio, W. Chai and C.-H. Wong, Curr. Opin. Struct. Biol.,
2003, 13, 637–645.
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8 M. Hartmann, A. K. Horst, P. Klemm and T. K. Lindhorst, Chem.
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methanol); HPLCtR = 6.11 min (A = water, B = methanol, A:
1
20%, 10 min, 1.2 mL min−1); H NMR (500 MHz, CD3OD,
300 K): δ = 7.38 (mc, 6H, H-arylTrt), 7.26 (mc, 6H, H-arylTrt),
7.19 (mc, 3H, H-arylTrt), 4.75 (d, 3J = 1.7 Hz, 1H, H-1Man),
3
3.85–3.80 (m, 2H, H-6aMan, OCHHCH2CH2S), 3.79 (dd, J =
3
2
3
3.3 Hz, J = 1.7 Hz, 1H, H-2Man), 3.72 (dd, J = 11.7 Hz, J =
3
3
5.6 Hz, 1H, H-6bMan), 3.70 (dd, J = 9.3 Hz, J = 3.3 Hz, 1H,
H-3Man), 3.62 (dd ∼ t, J = 9.5 Hz, 1H, H-4Man), 3.56–3.48 (m,
2H, H-5Man
3
9 M. J. Weissenborn, J. W. Wehner, C. J. Gray, R. Šardzík, C. E. Eyers,
T. K. Lindhorst and S. L. Flitsch, Beilstein J. Org. Chem., 2012, 8, 753–
762.
,
OCHHCH2CH2S), 3.37–3.32 (m, 2H,
SCH2CH2NH), 2.63 (mc, 4H, OCH2CH2CH2S, SCH2CH2NH),
2.17 (t, 3J = 7.5 Hz, 2H, HN(O)CCH2CH2), 2.11 (t, 3J = 7.4 Hz,
2H, CH2STrt), 1.89–1.81 (m, 2H, OCH2CH2CH2S), 1.59
10 R. Šardzík, R. Sharma, S. Kaloo, J. Voglmeir, P. R. Crocker and
S. L. Flitsch, Chem. Commun., 2011, 47, 5425–5427.
11 S. Serna, S. Yan, M. Martin-Lomas, I. B. H. Wilson and N.-C. Reichardt,
J. Am. Chem. Soc., 2011, 133, 16495–16502.
3
(quint., J = 7.6 Hz, 2H, HN(O)CCH2CH2), 1.38–1.09 (m, 14H,
12 J. Voglmeir, R. Šardzík, M. J. Weissenborn and S. L. Flitsch, OMICS,
CH2CH2CH2) ppm; 13C NMR (125 MHz, CD3OD, 300 K): δ =
176.4 (C(O)NH), 146.5 (C-arylTrt), 130.8, 128.8, 127.7 (CH-
arylTrt), 101.6 (C-1Man), 74.7 (C-5Man), 72.7 (C-3Man), 72.2
(C-2Man), 68.6 (C-4Man), 67.6 (Cq,Trt), 66.9 (OCH2CH2CH2S),
62.9 (C-6Man), 40.1 (SCH2CH2NH), 37.1 (HN(O)CCH2CH2),
32.9 (CH2STrt), 32.1 (SCH2CH2NH), 30.7 (OCH2CH2CH2S),
30.5, 30.4, 30.4, 30.3, 30.1, 30.0, 29.7 (7 CH2CH2CH2), 29.3
(OCH2CH2CH2S), 27.0 (HN(O)CCH2CH2) ppm; HR-ESI MS:
calcd for C41H57NNaO7S2: m/z 762.3469 [M + Na]+; found: m/z
762.3453 [M + Na]+; MALDI-ToF MS (DHB): calcd for
C41H57NNaO7S2: m/z 762.35 [M + Na]+; found: m/z 762.52
[M + Na]+; calcd for C41H57KNO7S2: m/z 778.32 [M + K]+;
found: m/z 778.52 [M + K]+; IR (ATR): ν˜ = 3296, 2922, 2852,
1643, 1544, 1488, 1443, 1130, 1084, 1054, 1030, 975, 811, 741,
2010, 14, 437–444.
13 L. Zou, H. L. Pang, P. H. Chan, Z. S. Huang, L. Q. Gu and K. Y. Wong,
Carbohydr. Res., 2008, 343, 2932–2938.
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4400–4412.
17 M. J. Weissenborn, R. Castagnia, J. W. Wehner, R. Šardzík,
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18 M. Kleinert, T. Winkler, A. Terfort and T. K. Lindhorst, Org. Biomol.
Chem., 2008, 6, 2118–2132.
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1522–1531.
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and C.-H. Wong, Chem. Biol., 2002, 9, 713–720.
697, 676, 616 cm−1
.
Abbreviations
DIPEA N,N-diisopropylethylamine,
EDC
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,
25 F. Fazio, M. C. Bryan, O. Blixt, J. C. Paulson and C.-H. Wong, J. Am.
Chem. Soc., 2002, 124, 14397–14402.
26 C. Grabosch, K. Kolbe and T. K. Lindhorst, ChemBioChem, 2012, 13,
1874–1879.
27 A. Schierholt, M. Hartmann, K. Schwekendiek and T. K. Lindhorst,
Eur. J. Org. Chem., 2010, 3120–3128.
28 J. W. Wehner and T. K. Lindhorst, Synthesis, 2010, 3070–3082.
29 (a) M. Kleinert, N. Röckendorf and T. K. Lindhorst, Eur. J. Org. Chem.,
2004, 3931–3940; (b) T. K. Lindhorst, S. Kötter, U. Krallmann-Wenzel
and S. Ehlers, J. Chem. Soc., Perkin Trans. 1, 2001, 823–831;
(c) J. Dahmen, T. Frejd, G. Gronberg, T. Lave, G. Magnusson and
G. Noori, Carbohydr. Res., 1983, 116, 303–307.
30 D. Ryan, B. A. Parviz, V. Linder, V. Semetey, S. K. Sia, J. Su,
M. Mrksich and G. M. Whitesides, Langmuir, 2004, 20, 9080–9088.
31 D. Pagé and R. Roy, Bioorg. Med. Chem. Lett., 1996, 6, 1765–1770.
32 N. Laurent, J. Voglmeir, A. Wright, J. Blackburn, N. T. Pham,
S. C. Wong, S. J. Gaskell and S. L. Flitsch, ChemBioChem, 2008, 9,
883–887.
HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyl-
uronium hexafluorophosphate,
NHS
PBS
PBST
TEA
N-hydroxysuccinimide,
phosphate buffered saline,
phosphate buffered saline + 0.05% v/v Tween® 20,
triethylamine.
Acknowledgements
This work was supported by the Evonik Foundation (JWW), the
Royal Society (Wolfson Award to SLF), the European Commis-
sion (MJW), the EPSRC and SFB 677 (DFG collaborative
network). A further thanks goes to Corning for the kind support
with free samples.
33 E. Ostuni, R. G. Chapman, R. E. Holmlin, S. Takayama and
G. M. Whitesides, Langmuir, 2001, 17, 5605–5620.
34 M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers and F. Smith, Anal.
Chem., 1956, 28, 350–356.
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