A 'dual click' strategy for the fabrication of bioselective, glycosylated self-assembled monolayers as glycocalyx models

Article abstract of DOI:10.1039/c3ob40386f

Solid surfaces decorated with specific saccharide patterns can serve as a model for the chemically and structurally highly complex glycocalyx of eukaryotic cells. Here we present an approach based on self-assembled monolayers on gold, which are built up in a three-step manner to provide a solid basis, a biorepulsive oligoethylene glycol part, and a specific carbohydrate terminus in a modular way. Of the different reaction sequences, the one with two consecutive 'click reactions' (the copper(i)-catalysed 1,3-dipolar cycloaddition of alkynes with azides and the thiourea-bridging of isothiocyanates with amines) directly 'on SAM' results in the densest layers, as demonstrated by infrared absorption reflection spectroscopy and ellipsometry. As a 'real life' test, the surfaces obtained this way were used for bacterial adhesion experiments. Here the biorepulsivity of the middle part of the SAMs as well as specific binding to the carbohydrate termini could be clearly demonstrated. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob40386f

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
A ‘dual click’ strategy for the fabrication of bioselective,
glycosylated self-assembled monolayers as glycocalyx
models†
Cite this: Org. Biomol. Chem., 2013, 11,
4006
Carsten Grabosch,a Martin Kind,b Yasmin Gies,b Felix Schweighöfer,b
Andreas Terfort*b and Thisbe K. Lindhorst*a
Solid surfaces decorated with specific saccharide patterns can serve as a model for the chemically and
structurally highly complex glycocalyx of eukaryotic cells. Here we present an approach based on self-
assembled monolayers on gold, which are built up in a three-step manner to provide a solid basis, a bi-
orepulsive oligoethylene glycol part, and a specific carbohydrate terminus in a modular way. Of the
different reaction sequences, the one with two consecutive ‘click reactions’ (the copper(I)-catalysed 1,3-
dipolar cycloaddition of alkynes with azides and the thiourea-bridging of isothiocyanates with amines)
directly ‘on SAM’ results in the densest layers, as demonstrated by infrared absorption reflection spectros-
copy and ellipsometry. As a ‘real life’ test, the surfaces obtained this way were used for bacterial adhesion
experiments. Here the biorepulsivity of the middle part of the SAMs as well as specific binding to the
carbohydrate termini could be clearly demonstrated.
Received 22nd February 2013,
Accepted 12th April 2013
DOI: 10.1039/c3ob40386f
www.rsc.org/obc
regard to the well-defined monolayer which is formed in the
self-assembly process, glyco-SAMs are unmatched by any other
Introduction
Eukaryotic cells are covered by a nanometre-thick molecular glycoarray concepts regarding the regularity and controllability
layer that consists of a complex multitude of structurally very of the formed monolayer. In addition, SAMs are amenable to a
diverse glycoconjugates. This so-called glycocalyx is of essen- number of biophysical methods that facilitate their character-
tial importance for fundamental biochemical processes such isation, and the precise investigation of molecular interactions
as cell–cell communication and cell adhesion.1 To study the with the SAM surface.7
details of the molecular recognition processes occurring at the
The most frequently used class of SAMs are thiolate mono-
glycosylated cell surface, suitable synthetic models are layers on gold, which are conveniently formed by immersion
required.2 These ideally allow for the construction of defined of gold substrates in solutions of thiols,8 thioacetates,9 or dis-
glycoarrays and their structural modification on demand. ulphides,10 respectively. For biological adhesion studies, it is
Thus, many different glycoarrays and methods for the prepa- essential to use so-called biorepulsive SAMs, which suppress
ration of carbohydrate-coated surfaces have been introduced.3 the unspecific adsorption of proteins to the surface. Such
However, among the different types of glycoarrays that are protein-repelling properties can be achieved by the introduc-
used in glycobiology, systems based on glycosylated self- tion of oligoethylene glycol (OEG) moieties into the employed
assembled monolayers (glyco-SAMs) have unique advantages.4 molecules.11 Biorepulsive properties of glyco-SAMs are a pre-
SAMs offer the possibility to fabricate reliable as well as struc- requisite for the investigation of carbohydrate recognition by
turally diverse, well-ordered glycoarrays,5 in particular when specific proteins, called lectins.12
systems with long alkyl chains at the basis are used.6 With
Lectins occur ubiquitously in all organisms, but serve very
different functions. In vertebrates they play essential roles in
cell–cell recognition and signalling,13 while microorganisms
on the other hand utilise own lectins to attach to the glycocalyx
of their host cells.14 For example, pathogenic bacteria such as
UPEC (uropathogenic Escherichia coli) utilise fimbrial lectins to
adhere to glycosylated surfaces and eventually form biofilms.15
These bacterial adhesion and colonisation processes can cause
severe infections,16 which are often life-threatening especially
for young children. It is therefore important to study and
aOtto Diels Institute of Organic Chemistry, Christiana Albertina University of Kiel,
Otto-Hahn Platz 3/4, D-24098 Kiel, Germany. E-mail: tklind@oc.uni-kiel.de;
Fax: +49 431 880 7410; Tel: +49 431 880 2023
bInstitute of Inorganic and Analytic Chemistry, Goethe-University Frankfurt,
D-60438 Frankfurt, Germany. E-mail: aterfort@chemie.uni-frankfurt.de;
Fax: +49 69 798 29188; Tel: +49 69 798 29180
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c3ob40386f
4006 | Org. Biomol. Chem., 2013, 11, 4006–4015
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
understand the details of fimbriae-mediated, carbohydrate- mediated ‘click chemistry’, we decided to protect the thiol
specific bacterial adhesion, in order to develop antiadhesive groups. Several protective groups permitting the formation of
surfaces, such as for transplantation medicine, and means for thiolate-anchored SAMs on gold surfaces for thiols exist. Here
prevention of bacterial adhesion.17
the acetyl group was selected. The thioacetate-functionalised
Hence it has become our goal to utilise glyco-SAMs to study alkyne 1 has been used in a previous study both for the for-
carbohydrate-specific bacterial adhesion employing live E. coli mation of SAMs and for ‘click reactions’ in solution.21b Here it
cells. Owing to our expertise with type 1-fimbriated E. coli, a was also used to optimise the ‘click reactions’ in solution
respective GFP-tagged strain (pPKL1162) was employed.18 In before transferring them to the surface. For the Cu(I)-catalysed
this fluorescent strain, some hundred copies of type 1 fimbriae 1,3-dipolar cycloaddition reaction, the difunctional amino-hexa-
are projected from the bacterial surface. They carry an α-D- ethylene glycol azide was employed, since it provides the bi-
mannoside-specific lectin at their tips, called FimH. So far, orepulsive OEG unit later to be used in the SAMs. Surprisingly,
FimH is the best-investigated bacterial lectin involved in bac- the desired amino-terminated 1,4-triazole 2 could only be
terial adhesion.19 Its structure and carbohydrate specificity are obtained in 46% yield even under optimised conditions. The
well understood from several X-ray studies.20
reason for this relatively low yield is a migration of the S-acetyl
To achieve a selective recognition of a surface by these enti- group towards the amino group under the reaction conditions
ties, it is necessary not only to expose α-D-mannoside residues, (cf. ESI†). The second ‘click reaction’ was performed between
but also to tailor the surface in such a way that the non- the amine 2 and the p-isothiocyanato-functionalised manno-
specific adhesion of this and other proteins becomes sup- side 3.23 Under optimised reaction conditions (diisopropyl-
pressed. In principle, this is possible by the synthesis of a ethylamine (DIPEA) in DMF), the thiourea-bridged thioacetate
molecule consisting of three blocks, that is (1) a mercaptoalkyl 4 could be obtained in a pure form and excellent yield
chain (for the formation of a stable SAM), (2) an OEG chain (Scheme 2).
(for the suppression of non-specific interactions), and (3) an
To avoid the problems with S → N-acetyl group migration,
α-D-mannoside terminus (for the specific interaction with the we switched to the corresponding disulphides, which can be
bacterial lectin). During an optimisation project, this would regarded as self-masked thiols, capable of forming thiol mono-
require many multi-step syntheses of the different molecules, layers of comparable quality as the ones derived from thiols.5b
so we rather wished to develop a convergent strategy to assem- For this, commercially available 11-mercaptoundecanoic acid
ble all the required molecular parts directly at the surface was oxidatively dimerised by treatment with sulphuryl chloride
(‘on SAM’ synthesis).
to form 5.10b
We have recently started to elaborate a ‘dual click’ concept
Disulphide 5 was coupled with propargylamine using a
for the formation of glyco-SAMs.21 This approach combines 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexa-
two orthogonal, chemoselective reactions with 100% atom fluoro-phosphate (HATU)-mediated amide coupling reaction to
economy: first, the classical ‘click reaction’ of azides and form alkyne amide 6 (Scheme 3). ‘Click reaction’ with amino-
alkynes in the presence of Cu(I) yielding triazole ligation pro- hexaethylene glycol azide in degassed methanol using copper(I)
ducts,22 and second, thiourea-bridging, conjugating an amine iodide yielded 78% of the bistriazole 7. DMF turned out to
and an isothiocyanate by formation of a thiourea bridge, cata- be unsuitable for the thiourea conjugation – the second ‘click
lysed by a tertiary amine. Here we show for the first time that reaction’ – of the disulphide system. Instead disulphide 7 was
by such a strategy, three key achievements for a powerful glyco- dissolved in a small amount of DMSO. Addition of THF and a
biological tool can be accomplished: (i) fabrication of biorepul- catalytic amount of DIPEA gave the thiourea-bridged disul-
sive glyco-SAMs with inherent structural variability through a phide 8 in 62% yield after heating the mixture to 40 °C for
‘dual click’ chemoselective ligation chemistry, which can be 48 h.
performed ‘on SAM’ (Scheme 1); (ii) monitoring of all con-
After the reaction conditions for both ‘click reactions’ were
struction steps by characterisation of the resulting SAM optimised in solution, we tried to transfer these protocols to
systems via ellipsometry and infrared absorption reflection the surface reactions.
spectroscopy (IRRAS); and (iii) demonstration of stability and
Construction of the bioselective glyco-SAMs by ‘dual click’
reactions
selectivity of the achieved glyco-SAMs for the study of carbo-
hydrate-specific adhesion with live E. coli cells.
For the deposition of the SAMs, both disulphides and thio-
acetates have been employed. When deposited from ethanol,
both kinds of molecules form similar thiolate SAMs on gold.5a
In this study, typically the thioacetates were preferred due to
their better solubilities. A notable exception was the formation
of the amino-terminated SAM (step C in Scheme 1), for which
Results and discussion
Synthesis of the building blocks and test of the ‘click
reactions’ in solution
All reactions were performed in homogeneous solution to ela- the disulphide 7 was preferred over the thioacetate 2, since we
borate the coupling chemistry. Optimised reaction conditions found spectroscopic hints for migration of the S-acetyl group
were then transferred to the construction of the bioselective on the surface, too (cf. ESI†). These layers can also be prepared
glycol-SAMs. Since free thiols interfere e.g. with the Cu(I)- by the ‘on SAM click reaction’ of the monolayers formed from
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 4006–4015 | 4007

View Article Online
Paper
Organic & Biomolecular Chemistry
Scheme 1 Outline of the formation of glyco-SAMs by the ‘dual click’ approach either in solution, ‘on SAM’, or a combination thereof. The ‘dual click’ approach
facilitates the fabrication of many different biorepulsive glyco-SAMs by using a kit-like combination of building blocks. Note that the mannosyl residue, which has
been used in this work, represents a whole group of possible carbohydrate residues.
on the Au surface (for the detailed assignments of the bands
see Table S1 in the ESI†). Ellipsometry measurements with the
SAM formed with 7 yielded a layer thickness of 30 2 Å. Apply-
ing standard bond lengths, the maximum length of molecule
7 can be approximated to be 44 Å. Thus, the determined layer
thickness is in line with the assumption that a thiolate mono-
layer has formed from the amino-terminated disulphide. An
experimental layer thickness smaller than the theoretical
maximum can be caused either by a tilting of the chemisorbed
molecules or by conformational defects (deviation from all-
trans conformation). Information on such defects can be
gained by analysis of some vibrational bands, e.g. the one of
the asymmetric CH2 stretch mode. In spectra of stretched-out
Scheme 2 Synthesis of amino-functionalised thioacetate 2 and thiourea-brid- alkyl chains, this mode has wavenumber maxima at 2920 cm−1
ging in solution. (a) CuI, MeCN, 45 °C, 24 h, 46%; (b) DIPEA, DMF, rt, 12 h,
and below, while for disordered chains, the band undergoes a
quant.
marked blue shift.24 The maximum of the asymmetric CH2
stretch band in spectrum b (SAM of substance 7) lies at
2920 cm−1, indicating that the alkyl chain in the SAM is rather
alkyne 1 with the OEG azide following a protocol established
by us earlier (step B in Scheme 1).21b This led to layers of 7
which were characterised by ellipsometry and IRRAS. Fig. 1
compares the spectra of the bulk substance 7 (trace a) and the
one obtained from the surface (trace b).
well-ordered. Analysis of the ethylene glycol CH2 wagging band
provides information on the conformation of the OEG chain in
the SAM of substance 7. The band maximum is at 1351 cm−1
which leads to the conclusion that the conformation of this
chain is mainly gauche.25 This finding is a possible explanation
for an experimentally derived layer thickness that is clearly
smaller than the maximum possible length of molecule 7 as
Spectra a and b (Fig. 1) resemble each other in all major
bands, thus indicating that a monolayer of 7 has been formed
4008 | Org. Biomol. Chem., 2013, 11, 4006–4015
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
After the coupling reaction of the SAM of 7 with mannoside 3,
three new bands appear in the IR spectrum of the resulting
SAM (trace c), which can also be found in the bulk spectrum
(trace e) of the acetyl-protected thiol 4. Firstly, a broad signal
between 3400 and 3600 cm−1 indicates the presence of OH
groups which belong to the mannose moiety. This signal
occurs in the monolayer spectrum of 8 (c) as well as in the
spectra of neat substances 3 (f) and 4 (e). The second new
signal is located at 1508 cm−1 in the spectrum of the SAM of
8 (c) and can be assigned to a CH bending mode in the aromatic
ring (1498 and 1508 cm−1 in the spectra of neat substances
3 (f) and 4 (e)). The third new signal in the monolayer spectrum
of 8 lies at 1188 cm−1 and is not present in the spectra of neat
substances 3 and 7, yet at 1222 cm−1 in the spectrum (e) of 4.
This band can be assigned to the stretch mode of the CS
double bond formed in the thiourea-bridging reaction, thus
demonstrating that the latter has been successful. This is corro-
borated by an increase of the layer thickness as measured by
ellipsometry. After the thiourea-bridging step the SAM has a
thickness of 34.8 0.7 Å. Since molecule 3 has a length of
approximately 11 Å, a greater increase would have been
expected. We speculate that only a part of the NH2 moieties
exhibited by the 7 SAM reacted with substance 3, leading only
to a 5 Å increase of the average layer thickness.
Scheme 3 Synthesis of amino-functionalised disulphide 7 and thiourea-brid-
ging in solution. (a) HATU, DIPEA, DMF, 0 °C → rt, 6.5 h, 80%; (b) CuI, MeOH,
40 °C, 18 h, 78%; (c) DIPEA, THF, DMSO, 40 °C, 16 h, 62%.
For reasons of comparison, a ‘conventional’ SAM has been
prepared by immersion of an Au substrate into a solution of
substance 4 (corresponding to route E in Scheme 1) and
characterised by IR spectroscopy and ellipsometry. The IRRA
spectrum of the ‘conventional’ SAM of 4 (d) exhibits several
bands that can also be found in the respective neat substance
spectrum (e), e.g. the OH stretch band (3300–3600 cm−1), the
amide I and II bands (1653 and 1541 cm−1) and the OEG CO
band (1128 cm−1). The missing acetyl CO stretch band (located
at 1693 cm−1 in the neat substance spectrum (f) indicates
removal of the acetyl unit upon S–Au bond formation, as
expected. The layer thickness of this sample is 32 1 Å which
is somewhat smaller than the thickness of the ‘clicked’ SAM.
The asymmetric CH stretch band of the ‘conventional’ SAM
spectrum lies at a rather high wavenumber (2926 cm−1),
leading to the assumption of a mainly disordered alkylic chain
in this SAM. From the lower overall IR signal intensity com-
Fig. 1 FT-IR spectra of glyco-SAMs after different fabrication stages and of the
involved neat substances. (a) IR spectrum of neat substance 7, recorded with an
ATR unit, (b) IRRA spectrum of 7, after deposition onto an Au surface, (c) IRRA
spectrum of the same sample as in b, but after surface thiourea-coupling with
3, (d) IRRA spectrum of an Au surface after direct deposition of 4, (e) FT-IR spec-
trum of neat substance 4, recorded with an ATR unit, (f) FT-IR spectrum of neat pared to the spectrum of the ‘clicked’ SAM (trace c) and from
substance 3, recorded with an ATR unit. The absorbance scale for all ATR spectra
the smaller layer thickness, a lower concentration of molecules
at the substrate surface can be inferred, which also points to a
rather disordered monolayer.
(a, e, f) is indicated by the bar next to spectrum a. The absorbance scale for all
monolayer spectra (b, c, d) is indicated by the bar next to spectrum b. Note that
some negative and positive bands between 2000 and 2400 cm−1 in the 7-SAM
In conclusion it can be stated that the thiourea ‘click reac-
spectrum (trace b) as well as in both other SAM spectra are due to gas phase
CO2 in the spectrometer beam path and due to the perdeuterated alkanethiol tion’ of the 7 SAM with substance 3 leads to a denser, better
SAM used as a reference.
ordered mannose-terminated OEG SAM than the direct depo-
sition of substance 4 onto the Au substrate.
Bacterial adhesion experiments
reaching the full length requires all-trans conformation. Since
OEG units with a gauche conformation have been associated
with biorepulsivity, it could be expected that the SAM of 7 has
this desired property.26
The next step in our ‘dual click’ strategy was the thiourea-
coupling reaction to build up a layer of 8 as described above.
As mentioned in the introduction, the aim of this project was
to develop a strategy permitting the selective adhesion of bio-
entities, such as bacteria. For this, the surfaces need not only
to expose recognisable binding groups but also possess a gen-
erally biorepulsive background. Due to the ‘dual click’ strategy
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 4006–4015 | 4009

View Article Online
Paper
Organic & Biomolecular Chemistry
Scheme 4 Adhesion of fluorescent bacteria to the different stages of the SAM during the ‘dual click’ approach. The GFP-transformed E. coli bacteria (pPKL1162)
enable a fast, direct fluorescence readout to investigate bacterial adhesion on surfaces. The native gold surface (I) was used as reference in each of the other experi-
ments. As can be seen in the epifluorescence micrographs, the (non-specific) adhesivity of the alkyne-terminated SAM II is comparable to the one of the native Au
surface. Introduction of the OEG chain reduces the adhesion significantly, while the α-mannosyl-terminated SAM is effectively recognised by the E. coli leading to
heavy adhesion.
presented here, all three parts of such a monolayer can be bacterial adhesion provided a high density of adhered bacteria
tested sequentially. As a probe we decided to use GFP-tagged on the glyco-SAM (8, Scheme 4, IV).
E. coli cells (strain pPKL1162), which expose multiple copies of
Adhesion was determined by counting the number of
type 1 fimbriae at their surface (Scheme 4). The lectin FimH, sessile bacteria per cm2 after gentle purging of the surfaces. As
located at the termini of these fimbriae, is sensitive to α-D- can be seen in Fig. 2, the adhesion at the alkyne-terminated
mannosides and therefore ideally suited to demonstrate the SAM of 1 (II) is comparable to the one at blank gold substrates
biorelevance of the glyco-SAM. For this, the three stages of the (I, used as a reference), which suggests non-specific inter-
SAM as well as pristine gold surfaces were exposed to suspen- actions. This interaction becomes effectively diminished for
sions of the bacteria for two hours each. The undecorated the amino-OEG-terminated SAM (II) indicating its biorepulsiv-
gold wafer (Scheme 4, I) and the alkyne-terminated surface ity. In contrast to this, the mannose-terminated surface III,
(1, Scheme 4, II) were used to control unspecific binding of the after the thiourea-bridging step, is significantly more adhesive
fluorescent E. coli bacteria. Furthermore, adhesion studies for the E. coli strain used in this study. The number of adher-
were performed on the biorepulsive amino-terminated SAM ent bacteria on this surface is three times the one on bare
(7, Scheme 4, III) and on the thiourea-bridged mannose-termi- gold, indicating a specific binding mechanism.
nated glyco-SAM (8, Scheme 4, IV). After fluorescence detection
With this, not only the successful attachment of the man-
of the GFP-tagged E. coli it emerged that the bacteria adhere nosyl moieties at the surface of the SAM could be demon-
with similar affinities to both control surfaces (Scheme 4, strated, but the viability of the ‘dual click’ approach as such.
I and II) by unspecific binding events. On the other hand, As a result of the fluorescence microscopic investigation of the
the biorepulsive amino-terminated surface of deposited 7 bacterial adherence we can conclude that the type 1 fimbriae-
(Scheme 4, III) indicated no significant bacterial adhesion mediated bacterial adhesion only occurs on the mannose-
due to the biorepulsive behaviour of the OEG moiety. terminated glyco-SAM. As expected, the bacterial adhesion is
After thiourea-bridging the mannose-specific FimH-mediated negative for the biorepulsive control experiments.
4010 | Org. Biomol. Chem., 2013, 11, 4006–4015
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
Experimental section
General remarks
Commercially available starting materials were used without
further purification: p-nitrophenyl α-D-mannopyranoside
(pNPMan) was purchased from Senn Chemicals, 11-mercapto-
undecanoic acid and propargylamine were from Aldrich,
N-Boc-ethylenediamine was from ABCR and amino-hexaethy-
lene glycol azide was from Polypure. Solvents were purified by
distillation prior to use. Reaction monitoring was performed
by TLC on silica gel F254 (Merck) or RP-18 (Merck) plates,
detection was achieved by UV light and/or by treatment of the
plates with a vanillin solution [vanillin (1.00 g) in methanol
(100 mL) and addition of glacial acetic acid (12.0 mL) and sul-
phuric acid (4.00 mL)] or ninhydrin solution [ninhydrin
(300 mg) in butanol (100 mL) and glacial acetic acid (3.00 mL)]
and subsequent heating. Flash chromatography was per-
formed on Merck silica gel 60 (0.040–0.063 mm). For NMR
spectroscopy Bruker DRX 500 or AV 600 instruments were
used. Chemical shifts (δ) are calibrated relative to the internal
solvent. For complete assignment the following two-dimen-
sional NMR techniques were used: 1H–1H COSY, 1H–13C HSQC
Fig. 2 Absolute bacteria density as determined by fluorescence microscopy on
differently functionalised SAMs (samples) in comparison to bare gold surface
substrates (references). Bacteria density data of alkyne-terminated, amino-OEG-
terminated, and mannose-OEG-terminated SAMs refer to gold surfaces functio-
nalised with 1, 7, and 8, respectively.
1
and H–13C HMBC. ESI-MS measurements were performed on
a Mariner instrument, MALDI-TOF mass spectra were recorded
with a Bruker Biflex III instrument with 19 kV acceleration
voltage and an ionization laser at 337 nm; 4-chloro-α-cyanocin-
namic acid (Cl-CCA) was used as a matrix. Optical rotation
values were measured on a Perkin-Elmer 241 polarimeter
(10 cm cells, Na-D-line: 589 nm) and are averaged from five
measurements. Elemental analyses were carried out with a
EuroEA3000 Elemental Analyzer from EuroVector.
Conclusions
In conclusion, it could be demonstrated that the ‘dual
click’ strategy provides an excellent tool to produce glyco-
SAMs suitable for bacteria-based assays. The use of two ‘click
reactions’ instead of one permits also the alteration of the
biorepulsive element between the basic SAM and the recog-
nition site, so that the system can easily become adopted
to the respective needs depending on the assay. This provides
a new dimension in the toolbox for the fabrication of
glycoarrays.
The possibility to thoroughly characterise the SAM-based
systems in each of the construction steps (e.g. by IRRAS and
ellipsometry) permits the optimisation of the individual ‘click
reactions’ and also detecting unexpected pit-falls, such as the
migration of protective groups previously considered as ‘inno-
cent’. We could also demonstrate that the SAMs formed by the
sequential build-up on the surface are denser, at least at their
basis, than those formed from long, completely preassembled
molecules, making them also more stable. As a first test for
the suitability of these systems for bio-assays with living bac-
teria, we could demonstrate that the introduction of the bio-
repulsive OEG-moiety not only suppressed the unspecific
adsorption of E. coli, but also that the final attachment of the
α-D-mannopyranoside enables the recognition of the surface by
the bacteria.
Our investigations currently focus on extending this
approach, e.g. for the fabrication of ‘mixed type’ SAMs expo-
sing more than one receptor molecule, the synthesis of photo-
switchable systems as well as the extension to true arrays with
many different surface chemistries on one substrate. These
systems will then be tested for their interactions with lectins
and, in particular, living cells.
N-[(20-Amino-hexaethylene glycolyl)-1H-[1,2,3]-triazole-4-yl-
methyl]-11-thioacetyl-undecanoic acid amide (2)
Thioacetate 1 (74 mg, 211 μmol) and amino-hexaethylene
glycol azide (63 mg, 211 μmol) were dissolved in degassed
MeCN (3 mL), CuI (7.9 mg, 42 μmol, 0.2 eq.) was added and
the reaction mixture was stirred for 24 h at 45 °C. After remov-
ing the copper salt by filtration, the crude product was purified
by flash chromatography (CHCl3–MeOH 7 : 1) and lyophilised
to obtain 2 (61 mg, 94 μmol, 46%) as a colourless substance.
1H NMR (600 MHz, MeOH-d4): δ 7.87 (s, 1H, H-15), 4.58 (t, 2H,
J = 5.1 Hz, H-14), 4.42 (s, 2H, H-17), 3.89 (t, 2H, J = 5.1 Hz,
H-13), 3.72–3.59 (m, 22H, H-2–H-12), 3.09 (t, 2H, J = 5.1 Hz,
H-1), 2.86 (t, 2H, J = 7.4 Hz, H-28), 2.30 (s, 3H, H-30), 1.21
(t, 2H, J = 7.4 Hz, H-19), 1.61 (mc, 2H, H-27), 1.55 (quint, 2H,
J = 7.4 Hz, H-20), 1.38–1.27 (m, 12H, H-21–H-26) ppm; 13C NMR
(150 MHz, MeOH-d4): δ 197.6 (C-29), 176.2 (C-28), 146.3
(C-16), 124.9 (C-15), 71.6–70.8 (CH2OCH2), 67.9 (C-13),
51.3 (C-14), 40.5 (C-1), 36.9 (C-19), 35.6 (C-17), 30.8 (C-20),
30.5 (C-30), 30.3–29.8 (C-20–C-26, C-28), 26.9 (C-27) ppm; ESI
MS: [M + H]+ m/z calcd for C30H58N5O8S: 648.400; found:
648.397.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 4006–4015 | 4011

View Article Online
Paper
Organic & Biomolecular Chemistry
N-{20-[p-(α-D-Mannopyranosyloxy)-phenylthioureido-
hexaethylene glycolyl]-1H-[1,2,3]-triazole-4-yl-methyl}-
11-thioacetyl-undecanoic acid amide (4)
Bis-{N-[(20-amino-hexaethylene glycolyl)-1H-[1,2,3]-triazole-
4-yl-methyl]}-11,11′-disulphanediyl diundecanoic acid
diamide (7)
The amine 2 (3.9 mg, 6.0 μmol) was dissolved in dry DMF The dialkyne 6 (35 mg, 69 μmol) and amino-hexaethylene
(2 mL) and treated with isothiocyanatophenyl mannoside 3 glycol azide (48 mg, 138 μmol, 2.0 eq.) were dissolved in
(3.8 mg, 12 μmol, 2.0 eq.) and DIPEA (2.0 μL, 12 μmol, 1.9 eq.). degassed MeOH (5 mL). After CuI (11 mg, 55 μmol, 0.8 eq.)
The reaction solution was stirred at room temperature for was added, the reaction mixture was stirred at 40 °C for 18 h.
12 h, subsequently the solvent was removed in vacuo. The The solvent was removed in vacuo and the crude product puri-
crude product was purified by column chromatography (ethyl fied by flash chromatography (CHCl3–MeOH 2 : 1 + 1% Et3N)
acetate–MeOH 2 : 1) to obtain molecule 4 (5.7 mg, 6.0 μmol, to obtain 7 (65 mg, 54 μmol, 78%) as a colourless solid. m.
quant.) as a colourless syrup. 1H NMR (600 MHz, DMSO-d6): p. 103 °C; 1H NMR (500 MHz, DMSO-d6): δ 8.17 (br s, 2H, NH),
δ 9.60 (br s, 1H, NH), 8.42 (br s, 1H, NH), 7.84 (s, 1H, H-26), 7.83 (br s, 2H, 2 H-15), 4.48 (t, 4H, J = 5.3 Hz, 4 H-14), 4.28
7.28 (d, 2H J = 8.9 Hz, aryl-H), 6.99 (d, 2H, J = 8.9 Hz, aryl-H), (d, 4H, J = 5.6 Hz, 4 H-17), 3.80 (t, 4H, J = 5.3 Hz, 4 H-13),
5.29 (d, 1H, J1,2 = 1.7 Hz, H-1man), 4.99 (d, 1H, J = 4.1 Hz, OH), 3.56–3.48 (m, 44H, H-2, H-3, H-4, H-5, H-6, H-7, H-8, H-9,
4.84 (d, 1H, J = 5.4 Hz, OH), 4.73 (d, 1H J = 5.0 Hz, OH), 4.45 (t, H-10, H-11, H-12), 3.42 (m ∼ t, 4H, 2 NH2), 2.73–2.66 (m, 8 H,
2H, J = 5.3 Hz, H-25), 4.42 (t, 1H, J = 6.0 Hz, OH), 4.26 (d, 2H, H-1, H-28), 2.08 (t, 4H, J = 7.5 Hz, H-19), 1.61 (quint, 4H, J =
J = 5.6 Hz, H-28), 3.81 (mc, 1H, H-2man), 3.72 (t, 2H, J = 5.2 Hz, 7.3 Hz, H-27), 1.49 (mc, 4H, H-20), 1.34 (mc, 4H, H-26),
H-24), 3.65 (mc, 1H, H-3man), 3.63–3.56 (m, 3H, H-6aman
,
1.27–1.21 (m, 20H, H-21, H-22, H-23, H-24, H-25) ppm;
H-12), 3.56–3.45 (m, 24H, H-4man, H-6bman, H-13–H-23), 3.41 13C NMR (125 MHz, DMSO-d6): δ 172.5 (C-18), 145.4 (C-16),
(mc, 1H, H-5man), 2.81 (t, 2H, J = 7.2 Hz, H-38), 2.31 (s, 3H, 123.5 (C-15), 72.3–70.1 (C-2, C-3, C-4, C-5, C-6, C-7, C-8, C-9,
H-40), 2.06 (t, 2H, J = 7.4 Hz, H-30), 1.48 (mc, 4H, H-31, H-37), C-10, C-11, C-12), 69.3 (C-13), 49.8 (C-14), 41.4 (C-1), 38.4
1.31–1.19 (m, 10H, H-32–-36) ppm; 13C NMR (125 MHz, (C-28), 35.8 (C-19), 34.6 (C-17), 29.3, 29.2, 29.2, 29.1, 29.0, 29.0
DMSO-d6): δ 195.8 (C-39), 180.9 (C-11), 172.5 (C-29), 153.8 (C-21, C-22, C-23, C-24, C-25, C-26), 28.2 (C-27), 25.7 (C-20)
(C-7), 145.4 (C-27), 131.5 (C-9), 123.5 (C-8), 117.4 (C-9), 99.7 ppm; EA: calcd for C56H108N10O14S2·3.5 H2O (M = 1271.78 g
(C-1man), 75.4 (C-5man), 71.1 (C-3man), 70.6 (C-2man), 70.2–69.1 mol−1): C 52.85, H 9.11, N 11.01, S 5.04; found: C 53.10,
(CH2OCH2), 67.2 (C-4man), 61.5 (C-6man), 49.7 (C-25), 35.7 H 8.72, N 10.25, S 4.18; MALDI-ToF MS: [M + H]+ calcd for
(C-30), 34.5 (C-28), 31.0 (C-40), 29.56, 29.3, 29.2, 29.1, C56H109N10O14S2: 1209.7566; found: 1210.0132; [M + Na]+ calcd
28.9, 28.8, 28.6 (C-31–C-36), 25.7 (C-37) ppm; MALDI-ToF MS: for C56H108N10O14S2Na: 1231.7386; found: 1232.0632.
[M + H]+ m/z calcd for C43H73N6O14S2: 961.4626; found:
961.6045; [M + Na]+ calcd for C43H72N6O14S2Na: 983.4446;
found: 983.5957.
Bis-{N-[20-(p-(α-D-mannopyranosyloxy)-phenylthioureido-
hexaethylene glycolyl]-1H-[1,2,3]-triazole-4-yl-methyl]}-
11,11′-disulphanediyl diundecanoic acid diamide (8)
The diamine 7 (16 mg, 13 μmol) and mannoside 3 (8.4 mg,
Bis-[N-(propynyl)-11,11′-disulphanediyl diundecanoic acid
26 μmol, 2.0 eq.) were dissolved in a mixture of THF and
diamide (6)
DMSO (2.5 mL, 4 : 1) and treated with DIPEA (5.0 μL, 27 μmol,
To an ice-cold solution of dicarboxylic acid 5 (400 mg, 2.02 eq.). The reaction mixture was stirred for 48 h at 40 °C.
921 μmol) in dry DMF (10 mL) DIPEA (625 μL, 3.68 mmol, 4.0 After removing the solvent in vacuo, the crude product was pur-
eq.) was added and the reaction mixture stirred for 5 min. ified by chromatography (CHCl3–MeOH 5 : 1) to obtain 8
Afterwards, HATU (1.05 g, 2.76 mmol, 3.0 eq.) was added and (14 mg, 8.0 μmol, 62%) as a colourless solid. [α]2D0 = +30.9
after an additional 30 min of stirring, propargylamine (236 μL, (c 0.23, DMSO); m.p. 78 °C; 1H NMR (600 MHz, DMSO-d6):
3.68 mmol, 4.0 eq.), dissolved in 3 mL dry DMF, was added δ 9.61 (br s, 2 H, NH), 8.26 (br s, 2H, NH), 7.86 (s, 2H, H-26),
dropwise. Then the reaction mixture was stirred for 6.5 h at 7.68 (br s, 2H, NH), 7.29, 7.01 (each d, each 4H, J = 8.8 Hz,
room temperature. The precipitated product was subsequently aryl-H), 5.33 (d, 2H, J = 1.3 Hz, H-1man), 5.01 (d, 2H, J = 4.3 Hz,
filtered and washed with ice-cold DMF to obtain 6 (375 mg, OH), 4.83 (d, 2H, J = 5.6 Hz, OH), 4.76 (d, 2H, J = 4.8 Hz, OH),
738 μmol, 80%) as a white solid. m.p.: 115 °C; 1H NMR 4.48 (d, 4H, J = 5.2 Hz, H-25), 4.45 (d, 2H, J = 6.0 Hz, OH), 4.26
(500 MHz, DMSO-d6): δ 8.08 (s, 2H, 2 NH), 3.83 (dd, 4H, J = 2.4 (d, 4H, J = 5.2 Hz, H-28), 3.81 (mc, 2H, H-2man), 3.79 (t, 4H, J =
Hz, 4 H-3), 2.98 (s, 2H, 2 H-1), 2.69 (t, 4H, J = 7.2 Hz, 4 H-14), 5.2 Hz, H-24), 3.66 (mc, 2H, H-3man), 3.64–3.55 (m, 6H,
2.07 (t, 4H, J = 7.4 Hz, 4 H-5), 1.61 (quint ∼ tt, 4H, J = 7.2 Hz, H-6aman, H-12), 3.55–3.46 (m, 48H, H-4man, H-6bman, H-13–
4 H-13), 1.48 (mc, 4H, 4 H-6), 1.34 (mc, 4H, 4 H-12), 1.31–1.21 H-23), 3.41 (mc, 2H, H-5man), 2.68 (t, 4H, J = 7.2 Hz, H-39), 2.07
(m, 20H, H-7, H-8, H-9, H-10, H-11) ppm; 13C NMR (125 MHz, (t, 4H, J = 7.4 Hz, H-30), 1.60 (quint, 4H, J = 7.2 Hz, H-38), 1.48
DMSO-d6): δ 172.3 (C-4), 81.8 (C-2), 72.9 (C-1), 38.6 (C-14), 35.6 (mc, 4H, H-31), 1.33 (mc, 4H, H-37), 1.26–1.20 (m, 20H, H-32–
(C-5), 29.3, 29.2, 29.1, 29.1, 29.0, 29.0 (C-7, C-8, C-9, C-10, C-11, H-36) ppm; 13C NMR (150 MHz, DMSO-d6): δ 180.7 (C-11),
C-13), 28.2 (C-3, C-12), 25.6 (C-6) ppm; EA: calcd for 172.0 (C-29), 153.4 (C-7), 144.9 (C-27), 133.4 (C-10), 125.0 (C-8),
C28H48N2O2S2 (M = 508.3157 g mol−1): C 66.09, H 9.51, N 5.51, 123.0 (C-26), 116.9 (C-9), 99.3 (C-1man), 74.9 (C-5man), 70.7
S 12.60; found: C 66.11, H 9.87, N 5.62, S 12.09.
(C-3man), 70.1 (C-2man), 69.8–68.6 (CH2OCH2), 66.9 (C-4man),
4012 | Org. Biomol. Chem., 2013, 11, 4006–4015
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
61.0 (C-6man), 49.3 (C-25), 43.5 (C-12), 37.8 (C-39), 35.2 (C-30), indices were in all cases assumed to be 1.37, a value that rep-
34.1 (C-28), 28.9–28.5 (CH2), 27.7 (C-37), 25.2 (C-31) ppm; EA: resents a good compromise for molecules with OEG moieties.
calcd for C82H138N12O26S4·7.6 H2O·1.2 DMSO (M = 2065.50 g
mol−1): C 49.04, H 7.82, N 8.14, S 8.07; found: C 49.10,
Infrared measurements
H 7.24, N 7.65, S 7.99; MALDI-ToF MS: [M + H]+ calcd for
IR spectra of neat substances were recorded using a Nicolet
6700 Fourier transform IR spectrometer with a liquid nitrogen-
C82H139N12O26S4: 1837.3059; found: 1837.0693; [M + Na]+ calcd
for C82H138N12O26S4Na: 1859.2877; found: 1859.1165.
cooled narrow-band mercury cadmium telluride (MCT) detec-
tor equipped with an ATR unit and with a Perkin-Elmer 1600
Series FT-IR spectrometer equipped with an MKII Golden
Gate™ Single Reflection ATR A-531-G unit. The Nicolet
spectrometer was also employed to characterise the freshly pre-
pared SAMs and the SAMs after execution of the surface ‘click
reactions’ using an infrared reflection absorption spectroscopy
(IRRAS) sample holder. Its beam path was purged with dry and
CO2-free air throughout the measurements. IRRA spectra were
recorded with p-polarised IR radiation in grazing-incidence at
an angle of 80° with respect to the sample surface normal.
SAMs of perdeuterated dodecanethiol on Au were used as a
reference for the IRRAS measurements. All spectra were
acquired at a resolution of 4 cm−1. In the IRRAS experiments,
512 scans were performed to improve the signal to noise ratio,
followed by smooth baseline correction.
Gold substrate preparation
Silicon wafers (100) coated with 10 nm of chromium and
200 nm of gold (111) were cut into small pieces of about 15 ×
20 mm2 size. For ellipsometry measurements, four small
regions on the gold substrates were marked with circles using
a sharp tip. The substrates were then rinsed with pure ethanol
and dried in a nitrogen stream, before chemisorbed contami-
nants were removed using hydrogen plasma (hydrogen
pressure: 0.5 Pa, 25 W, 2 min).27
Self-assembled monolayer preparation
Self-assembled monolayers were prepared by immersion of
gold substrates into solutions of the respective organodisul-
phides or thioacetates. Dimethylformamide p.a. was used as a
solvent for all solutions except for molecule 1, which was de-
posited from solution in tetrahydrofuran. Concentrations were
0.02 mM in the case of molecule 7 and 0.03 mM in the case of
molecule 3. If the solutions were not completely clear, they
were filtered through a syringe filter (polytetrafluoroethylene,
0.2 μm pore diameter). The concentration of 1 amounted to
0.2 mM; an equimolar amount of NH4OH was added to
promote the cleavage of the S-acetyl group. All solvents were
used without further purification. Immersion times varied
between 15 and 22 h. After removal from the solutions, the
samples were thoroughly rinsed with the respective solvent
used for the deposition, then rinsed with ethanol, and blown
dry in an argon stream.
Bacteria adhesion experiments
Fluorescent GFP-tagged E. coli (pPKL1162) with resistance to
ampicillin and chloramphenicol were used for bacteria
adhesion tests on gold substrate surfaces functionalised with
molecules 1, 7, and 8. Suspensions of the bacteria in CASO
broth with an optical density of 0.5 (which corresponds to ca. 6
× 108 bacteria per cm3) were used. For each kind of functiona-
lised surface, five specimens were used. Equivalent numbers
of bare gold surfaces were used as references. Prior to bacteria
adhesion, all samples were sterilised by thorough rinsing with
ethanol and heating to 45 °C for one hour. Adsorption experi-
ments were carried out for 2 hours at 37 °C in a shaker at 200
rpm, using 20 mL of the bacteria suspension per sample.
Afterwards, the samples were dipped into Falcon tubes with
20 mL PBS solution and shaken at 200 rpm for one minute.
Subsequently they were immersed in a beaker with sterile PBS
(phosphate buffered saline) solution, and after removal from
the solution and drip drying, they were mounted onto micro-
scope slides.
Thiourea-bridging on surface
Gold substrate samples functionalised with molecule 7 (OEG-
amino-terminated SAM) were immersed into solutions of
molecule 3 (5 mM) and DIPEA (15 mM) in THF (p.a.). The sub-
strates remained in the solutions at ambient air and 40 °C in a
shaker for 15–60 h. Afterwards, the samples were taken out of
the solution, rinsed with ethanol, blown dry with an Ar or N2
stream and characterised by IRRAS and ellipsometry.
The numbers of bacteria per cm2 were determined using a
Horiba microscope fluorescence lifetime imaging system, com-
bined with an Olympus DX 5 optical microscope, running in
fluorescence microscopy mode and equipped with a Lumen
Dynamics X-Cite 120 Q mercury vapor short arc lamp. On each
sample, ten different locations with an area of 0.14 mm2 each
were randomly chosen for bacteria counting.
Ellipsometry
The thickness of the SAMs after preparation and after surface
‘click reactions’ was measured with a Sentech SE 400 ellipso-
meter equipped with a He Ne laser (wavelength 632.8 nm,
beam diameter 1–2 mm). The angle of incidence amounted to
70° with respect to the sample surface normal.
Complex refraction indices of the gold substrates were
measured at four labeled positions per substrate before the
Acknowledgements
latter were immersed into the disulphide or thioacetate solu- This work was financed by the DFG (SFB 677). Part of the work
tions. The extinction coefficients of the monolayers before and was supported by the Beilstein-Institut, Frankfurt/Main,
after ‘click reactions’ were assumed to be zero. The refractive Germany, within the research collaboration NanoBiC.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 4006–4015 | 4013

View Article Online
Paper
Organic & Biomolecular Chemistry
Y. Xia, L. E. Dike, D. E. Ingber and G. M. Whitesides, Proc.
Natl. Acad. Sci. U. S. A., 1996, 93, 10775–10778.
Notes and references
1 (a) A. Varki, Glycobiology, 1993, 3, 97–130; (b) R. A. Dwek,
Chem. Rev., 1996, 96, 683–720; (c) H.-J. Gabius,
H.-C. Siebert, S. André, J. Jiménez-Barbero and H. Rüdiger,
ChemBioChem, 2004, 5, 740–764; (d) J. C. Paulson, O. Blixt
and B. E. Collins, Nat. Chem. Biol., 2006, 2, 238–248.
2 (a) L. L. Kiessling and R. A. Splain, Annu. Rev. Biochem.,
2010, 79, 619–653; (b) C.-Y. Wu and C.-H. Wong, Chem.
Commun., 2011, 47, 6201–6207.
9 (a) M. Kleinert, N. Röckendorf and T. K. Lindhorst,
Eur. J. Org. Chem., 2004, 3931–3940; (b) M. I. Bethencourt,
L. Srisombat, P. Chinwangso and T. R. Lee, Langmuir, 2009,
25, 1265–1271.
10 (a) M. R. Lockett, M. F. Phillips, J. L. Jarecki, D. Peelen and
L. M. Smith, Langmuir, 2008, 24, 69–75; (b) E. Locatelli,
G. Ori, M. Fournelle, R. Lemor, M. Montorsi and
M. C. Franchini, Chem.–Eur. J., 2011, 17, 9052–9056.
3 (a) M. Mrksich, Chem. Soc. Rev., 2000, 29, 267–273; 11 (a) K. L. Prime and G. M. Whitesides, Science, 1991, 252,
(b) M. C. Bryan, O. Plettenburg, P. Sears, D. Rabuka,
S. Wacowich-Sgarbi and C.-H. Wong, Chem. Biol., 2002, 9,
713–720; (c) S. Fukui, T. Feizi, C. Galustian, A. M. Lawson
and W. Chai, Nat. Biotechnol., 2002, 20, 1011–1017;
(d) D. M. Ratner, E. W. Adams, J. Su, B. R. O’Keefe,
M. Mrksich and P. H. Seeberger, ChemBioChem, 2004, 5,
379–383; (e) T. Horlacher and P. H. Seeberger, Chem. Soc.
1164–1167; (b) R. Chelmowski, S. D. Köster, A. Kerstan,
A. Prekelt, C. Grunwald, T. Winkler, N. Metzler-Nolte,
A. Terfort and C. Wöll, J. Am. Chem. Soc., 2008, 130,
14952–14953; (c) S. Herrwerth, W. Eck, S. Reinhardt and
M. Grunze, J. Am. Chem. Soc., 2003, 125, 9359–9366;
(d) K. L. Prime and G. M. Whitesides, J. Am. Chem. Soc.,
1993, 115, 10714–10721.
Rev., 2008, 37, 1414–1422; (f) N. Laurent, J. Voglmeir and 12 (a) W. I. Weis and K. Drickamer, Annu. Rev. Biochem., 1996,
S.
Flitsch,
Chem.
Commun.,
2008,
4400–4412;
65, 441–473; (b) H. Lis and N. Sharon, Chem. Rev., 1998, 98,
637–674.
(g) O. Oyelaran and J. C. Gildersleeve, Curr. Opin. Chem.
Biol., 2009, 13, 406–413; (h) C. Rillahan and J. C. Paulson, 13 M. Ambrosi, N. R. Cameron and B. G. Davis, Org. Biomol.
Annu. Rev. Biochem., 2011, 80, 797–823; (i) C. Grabosch, Chem., 2005, 3, 1593–1608.
K. Kolbe and T. K. Lindhorst, ChemBioChem, 2012, 13, 14 K. Ohlsen, T. A. Oelschlaeger, J. Hacker and A. Salam
1874–1879; ( j) J. Wehner, M. Weissenborn, M. Hartmann, Khan, Top. Curr. Chem., 2009, 288, 17–65.
C. J. Gray, R. Šardzik, C. E. Eyers, S. L. Flitsch and 15 A. Reisner, K. A. Krogfelt, B. M. Klein, E. L. Zechner and
T. K. Lindhorst, Org. Biomol. Chem., 2012, 10, 8919–8926; S. Molin, J. Bacteriol., 2006, 188, 3572–3581.
(k) S. Park, J. C. Gildersleeve, O. Blixt and I. Shin, Chem. 16 G. G. Anderson, K. W. Dodson, T. M. Hooton and
Soc. Rev., 2013, DOI: 10.1039/C2CS35401B. S. J. Hultgren, Trends Microbiol., 2004, 12, 424–430.
4 (a) B. T. Houseman and M. Mrksich, Chem. Biol., 2002, 9, 17 (a) X. Jiang, D. Abgottspon, S. Kleeb, S. Rabbani,
443–454; (b) M. Kleinert, T. Winkler, A. Terfort and
T. K. Lindhorst, Org. Biomol. Chem., 2008, 6, 2118–2132;
(c) L. Ban and M. Mrksich, Angew. Chem., 2008, 47,
3444–3447, (Angew. Chem., Int. Ed., 2008, 47, 3396–3399);
(d) N. Laurent, R. Haddoub, J. Voglmeir, S. C. C. Wong,
S. J. Gaskell and S. L. Flitsch, ChemBioChem, 2008, 9,
2592–2596; (e) F. Tantakitti, J. Burk-Rafel, F. Cheng,
M. Scharenberg, M. Wittwer, M. Haug, O. Schwardt and
B. Ernst, J. Med. Chem., 2012, 55, 4700–4713;
(b) M. Hartmann, H. Papavlassopoulos, V. Chandrasekaran,
C. Grabosch, F. Beiroth, T. K. Lindhorst and C. Röhl, FEBS
Lett., 2012, 586, 1459–1465; (c) C. Grabosch, M. Hartmann,
J. Schmidt-Lassen and T. K. Lindhorst, ChemBioChem,
2011, 12, 1066–1074.
R. Egnatchik, T. Owen, M. Hoffmann, D. N. Weiss and 18 M. Hartmann, A. K. Horst, P. Klemm and T. K. Lindhorst,
D. M. Ratner, Langmuir, 2012, 28, 6950–6959. Chem. Commun., 2010, 46, 330–332.
5 (a) J. C. Love, L. A. Estroff, J. K. Kriebel, R. G. Nuzzo and 19 M. Hartmann and T. K. Lindhorst, Eur. J. Org. Chem., 2011,
G. M. Whitesides, Chem. Rev., 2005, 105, 1103–1169; 3583–3609.
(b) Z.-L. Zhi, N. Laurent, A. K. Powell, R. Karamanska, 20 (a) D. Choudhury, A. Thompson, V. Stojanoff,
M. Fais, J. Voglmeir, A. Wright, J. M. Blackburn,
P. R. Crocker, D. A. Russell, S. Flitsch, R. A. Field and
J. E. Turnbull, ChemBioChem, 2008, 9, 1568–1575.
6 B. T. Houseman and M. Mrksich, Chem. Biol., 2002, 9,
443–454.
7 (a) M. Kind and C. Wöll, Prog. Surf. Sci., 2009, 84, 230–278;
(b) K. A. Barth, G. Coullerez, L. M. Nilsson, R. Castelli,
P. H. Seeberger, V. Vogel and M. Textor, Adv. Funct. Mater.,
2008, 18, 1459–1469; (c) Z. A. Gurard-Levin and
M. Mrksich, Annu. Rev. Anal. Chem., 2008, 1, 767–800.
8 (a) M. C. Fritz, G. Hähner, N. D. Spencer, R. Bürli and
A. Vasella, Langmuir, 1996, 12, 6074–6082; (b) D. J. Revell,
J. R. Knight, D. J. Blyth, A. H. Haines and D. A. Russell,
Langmuir, 1998, 14, 4517–4524; (c) M. Mrksich, C. S. Chen,
S. Langermann, J. Pinkner, S. J. Hultgren and S. D. Knight,
Science, 1999, 285, 1061–1066; (b) C.-S. Hung, J. Bouckaert,
D. Hung, J. Pinkner, C. Widberg, A. DeFusco, C. G. Auguste,
R. Strouse, S. Langermann, G. Waksman and S. J. Hultgren,
Mol. Microbiol., 2002, 44, 903–915; (c) J. Bouckaert,
J. Berglund, M. Schembri, E. De Genst, L. Cools, M. Wuhrer,
C.-S. Hung, J. Pinkner, R. Slättegärd, A. Zavialov,
D. Choudhury, S. Langermann, S. J. Hultgren, L. Wyns,
P. Klemm, S. Oscarson, S. D. Knight and H. De Greve, Mol.
Microbiol., 2005, 55, 441–455; (d) A. Wellens, C. Garofalo,
H. Nguyen, N. Van Gerven, R. Slättegärd, J. P. Hernalsteens,
L. Wyns, S. Oscarson, H. De Greve, S. Hultgren and
J. Bouckaert, PLoS One, 2008, 3, e2040; (e) Z. Han,
J. S. Pinkner, B. Ford, R. Obermann, W. Nolan, S. A. Wildman,
4014 | Org. Biomol. Chem., 2013, 11, 4006–4015
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
D. Hobbs, T. Ellenberger, C. K. Cusumano, S. J. Hultgren 24 (a) R. G. Snyder, H. L. Strauss and C. A. Elliger, J. Phys.
and J. W. Janetka, J. Med. Chem., 2010, 53, 4779–4792.
21 (a) C. Grabosch, M. Kleinert and T. K. Lindhorst, Synthesis,
2010, 828–836; (b) M. Kleinert, T. Winkler, A. Terfort and
T. K. Lindhorst, Org. Biomol. Chem., 2008, 6, 2118–2132.
22 (a) C. W. Tornøe, C. Christensen and M. Meldal, J. Org.
Chem., 2002, 67, 3057–3064; (b) V. V. Rostovtsev,
Chem., 1982, 86, 5145–5150; (b) R. A. MacPhail, H. L. Strauss,
R. G. Snyder and C. A. Elliger, J. Phys. Chem., 1984, 88,
334–341.
25 (a) H. Matsuura and T. Miyazawa, Bull. Chem. Soc. Jpn.,
1986, 41, 1798–1808; (b) H. Matsuura and T. Miyazawa,
J. Polym. Sci., 1969, 7, 1735–1744.
L. G. Green, V. V. Fokin and K. B. Sharpless, Angew. Chem., 26 P. Harder, M. Grunze, R. Dahint, G. M. Whitesides and
Int. Ed., 2002, 41, 2596–2599. P. E. Laibinis, J. Phys. Chem. B, 1998, 102, 426–436.
23 D. H. Buss and I. J. Goldstein, J. Chem. Soc., 1968, 12, 27 K. Raiber, A. Terfort, C. Benndorf, N. Krings and
1457–1461.
H. H. Strehblow, Surf. Sci., 2005, 595, 56–63.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 4006–4015 | 4015