Carbohydrate coatings via aryldiazonium chemistry for surface biomimicry

Article abstract of DOI:10.1021/cm4027896

Carbohydrates are extremely important biomolecules and their immobilization onto solid surfaces is of interest for the development of new biomimetic materials and of new methods for understanding processes in glycobiology. We have developed an efficient surface modification methodology for the functionalization of a range of materials with biologically active carbohydrates based on aryldiazonium chemistry. We describe the synthesis and characterization of carbohydrate reagents, which were subsequently employed for the one-step, solution-based modification of carbon, metals, and alloys with monosaccharides. We used a combination of spectroscopic and nanogravimetric methods to characterize the structure of the carbohydrate layers; we report an average surface coverage of 7.8 × 10^-10 mol cm^-2 under our experimental conditions. Concanavalin A, a mannose-binding lectin, and Peanut Agglutinin, a galactose-binding lectin, were found to bind from solution to their respective monosaccharide binding partners immobilized at the surface. This result suggests that the spontaneous chemisorption of aryldiazonium monosaccharide precursors leads to the formation of monosaccharide layers that retain the biological recognition specificity of the parent carbohydrate molecule. Finally, we carried out measurements using fluorescently labeled Bovine Serum Albumin (BSA) and found that these carbohydrate coatings reduce unspecific adsorption of this protein at carbon surfaces. These results suggest that aryldiazonium-derived carbohydrate coatings may offer a promising strategy for preventing undesirable protein accumulation onto surfaces.

Full text of DOI:10.1021/cm4027896

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Article
Carbohydrate Coatings via Aryldiazonium Chemistry for Surface Biomimicry
Dilushan R Jayasundara, Thomas Duff, Maria Daniela Angione, Jean
Bourke, Deirdre M Murphy, Eoin Martin Scanlan, and Paula E. Colavita
Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/cm4027896 • Publication Date (Web): 24 Sep 2013
Downloaded from http://pubs.acs.org on October 3, 2013
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Chemistry of Materials
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Carbohydrate Coatings via Aryldiazonium Chemistry
for Surface Biomimicry
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Dilushan R. Jayasundara, Thomas Duff, M. Daniela Angione, Jean Bourke, Deirdre M. Murphy, Eoin
M. Scanlan* and Paula E. Colavita*
School of Chemistry, University of Dublin Trinity College, College Green, Dublin 2, Ireland.
Centre for Research on Adaptive Nanostructures and Nanodevices (CRANN), University of Dublin
Trinity College, Dublin 2, Ireland
* Corresponding authors. Eꢀmail: eoin.scanlan@tcd.ie; colavitp@tcd.ie.
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Abstract
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Carbohydrates are extremely important biomolecules and their immobilization onto solid surfaces is
of interest for the development of new biomimetic materials and of new methods for understanding
processes in glycobiology. We have developed an efficient surface modification methodology for the
functionalization of a range of materials with biologicallyꢀactive carbohydrates based on aryldiazonium
chemistry. We describe the synthesis and characterization of carbohydrate reagents, which were
subsequently employed for the oneꢀstep, solutionꢀbased modification of carbon, metals and alloys with
monosaccharides. We used a combination of spectroscopic and nanogravimetric methods to characterize
the structure of the carbohydrate layers; we report an average surface coverage of 7.8×10ꢀ10 mol cmꢀ2
under our experimental conditions. Concanavalin A, a mannoseꢀbinding lectin, and Peanut Agglutinin, a
galactoseꢀbinding lectin, were found to bind from solution to their respective monosaccharide binding
partners immobilized at the surface. This result suggests that the spontaneous chemisorption of
aryldiazonium monosaccharide precursors leads to the formation of monosaccharide layers that retain
the biological recognition specificity of the parent carbohydrate molecule. Finally, we carried out
measurements using fluorescently labeled Bovine Serum Albumin (BSA) and found that these
carbohydrate coatings reduce unspecific adsorption of this protein at carbon surfaces. These results
suggest that aryldiazoniumꢀderived carbohydrate coatings may offer a promising strategy for preventing
undesirable protein accumulation onto surfaces.
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Keywords: carbohydrate, saccharide, diazonium, carbon, coatings.
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1. Introduction
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Carbohydrates are extremely important biomolecules that are involved in a diverse array of biological
functions including fertilization, cellꢀcell communication and inflammatory responses.1ꢀ4 The surfaces
of eukaryotic cells and proteins are often heavily glycosylated and this ‘glycocalyx’ modulates the early
stages of many biological processes and interactions, usually through binding interactions between
carbohydrates and proteins.5, 6 Glycosylated surfaces have emerged as extremely important materials
and as tools for tailoring the response of organisms to solid surfaces, for improving our understanding
of carbohydrate–protein interactions and for studying fundamental processes in glycobiology.
Therefore, there has been great interest in developing new strategies for the preparation of functional
glycosylated surfaces. Glycosylation alters the surface characteristics and biology of materials and has
allowed for the development of new materials that can mimic the glycosylation pattern of cells.7 These
materials have many important applications in medical devices, diagnostics and sensors. Surface
glycosylation reactions have found applications in the fabrication of carbohydrate microarrays, which
have been used to screen for carbohydrate binding proteins and have identified high affinity ligands for
lectin binding, antigenic carbohydrate structures and compounds that inhibit carbohydrateꢀprotein
interactions.8ꢀ13
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Due to the emergence of glycosylated materials as an important class of biomaterial, there has been
significant focus on the development of new methodologies for the functionalization of surfaces and
materials.11, 12, 14 Sophisticated ligation methodologies and patterning techniques have been employed
for the preparation of glycosylated surfaces.15 The three most commonly employed methodologies for
direct immobilization of carbohydrates on metal surfaces are thiol chemistry,16 amide bond formation
and Copper catalyzed [3+2] cycloaddition reactions.11, 12, 14 Thiol chemistry on gold and silver surfaces
is well established as a methodology for preparing carbohydrateꢀbearing monolayers, however, the bond
that anchors the alkylthiol to the surface is labile and can easily be broken. Both the amide bond
formation and copper catalysed ‘click’ reaction necessitate prefunctionalization of the surface material
with a ‘chemical handle’ suitable for the ligation reaction with the glycan. With the aim of developing
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an efficient, oneꢀstep procedure for the direct glycosylation of untreated surfaces, we turned our
attention towards diazonium grafting chemistry as an alternative to traditional carbohydrate
immobilization methodologies. Diazonium chemistry has been widely employed for the
functionalization of carbon with various aromatic groups.17, 18 The methodology is advantageous in that
it forms strong stable covalent bonds with carbon surfaces and can also be used to directly functionalize
metals such as copper, gold and alloys.19ꢀ23 The methodology can be carried out in the presence of
deprotected carbohydrates without any degradation of the glycan structure and the grafting methodology
can be carried out in aqueous conditions which are ideal for solubilizing the hydrophilic sugar groups.
The diazotization conditions are extremely mild and diazonium chemistry has successfully been
employed for the functionalization of proteins specifically at tyrosine.24 These reactions can be carried
out without any denaturing of the protein structure. However, to the best of our knowledge, the use of
diazonium chemistry has not previously been employed for the preparation of glycosylated surfaces. A
oneꢀpot, general grafting methodology for carbohydrates onto surfaces offers exciting potential for the
preparation of a range of glycosylated materials with applications in glycobiology. Herein we describe
the results of our studies into the development of arylꢀdiazonium grafting of carbohydrates onto a range
of materials including metals and alloys.
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2. Experimental
Chemicals and Materials. Sulfuric acid (H2SO4, concentrated), hydrogen peroxide (H2O2, 30%),
Concanavalin A (FITC Con A, C7642), phosphate buffered saline buffer (PBS, pH 7.4, 0.01 M), ,
acetone (HPLC), and methanol (MeꢀOH, semiconductor grade), sodium nitrite (NaNO2), from Sigmaꢀ
Aldrich,were used without further purification, fluoroboric acid (HBF4), nitric acid (HNO3), acetonitrile
(CAN, HPLC grade). Lectins from Arachis hypogaea (peanut agglutinin, PNA), FITC conjugate, and
Concanavalin A from Canavalia ensiformis (Jack bean, ConA), FITC conjugate, Type IV, were
purchased as lyophilized powder from Sigma Aldrich. Bovine Serum Albumin–fluorescein
isothiocyanate conjugate was also purchased from Sigma Aldrich.
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Deionized water was used for all aqueous solutions. All glassware, quartz crystals and cells were
cleaned with piranha solution before use (3:1, H2SO4 to H2O2; WARNING: Piranha solution should be
handled with caution; it is a strong oxidant and reacts violently with organic materials. It also presents
an explosion danger. All work should be performed under a fume hood).
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Synthesis of the Diazonium Tetrafluoroborate Derivative. The respective diazonium salts were
freshly prepared on treatment of the relevant 4ꢀaminophenyl glycoside with a solution of sodium nitrite
and fluoroboric acid (Scheme 1). The 4ꢀaminophenyl glycosides (compounds 1-8) were dissolved in
20.0 mL of 1.25 mM HBF4 and kept in a beaker of ice. 5.0 mL of 5.0 mM NaNO2 that was also kept in
ice were then added dropwise over a period of 10 min to the precursor solution in order to form the
diazonium salt at a final concentration of 1.0 mM. Functionalization of carbon substrates with precursor
7 required the use of a small amount of acetonitrile in the aqueous solution (approximately 1:10 v/v) due
to the limited solubility of peracetylated rhamnose in water. Surface modification reactions were carried
out by immersing the substrate into thus prepared solutions of diazoniated precursors for 60 min, at
concentrations varying in the range 0.010ꢀ1.0 mM. Surface deprotection of peracetylated carbohydrates
chemisorbed at surfaces was carried out by immersing samples in a freshly made sodium methoxide
solution (Na0 and CH3OH) for 60 min followed by neutralization with dilute HCl.
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Substrate Preparation. Amorphous carbon (aꢀC) substrates were prepared by magnetron sputtering of
Ti followed by aꢀC as previously described;25 samples were stored under Ar and used as deposited in
functionalisation reactions. Metal and alloy substrates were cleaned prior to in situ surface
functionalisation with aryldiazonium salts. Copper foil (pipe grade) was cleaned in 0.1 M HNO3, rinsed
with copious amount of deionized water and dried in Ar. Brass foil substrates (machining grade) were
polished with 0.3 ꢁm alumina slurry, sonicated in methanol three times and dried in Ar. Both Cu and
brass substrates were used immediately after cleaning. Gold substrates were prepared via electroplating
using a commercial electroplating solution (Spa Plating Ltd.) and stainless steel foil electrodes; after Au
deposition, substrates were rinsed with deionized water and dried under Ar.
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Characterization Methods. Infrared Reflectance Absorption spectroscopy (IRRAS) was carried out on
Ti/aꢀC samples and on Au, Cu and brass samples using a Fourier Transform Infrared (FTIR)
Spectrometer (Tensor 27, Bruker) equipped with a Mercury Cadmium Telluride (MCT) detector, a
specular reflectance accessory (VeeMax II) and a ZnSe polarizer. Spectra were taken at 80° incidence
using pꢀpolarized light; 256 spectra were collected at 4 cmꢀ1 resolution using a bare substrate as a
background sample; IRRAS data was obtained in triplicates as a minimum.
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Quartz crystals with 10 MHz resonant frequency were used for Quartz Crystal Microbalance (QCM)
measurements crystals used in this study; crystal were coated with 100 nm thick vapour deposited gold
electrodes (International Crystal Manufacturing). Carbon coated crystals were obtained via DCꢀ
magnetron sputtering of Au electrodes with 10 nm thick Ti adhesive layer followed by a 50 nm aꢀC
film, as previously described.25 The QCM setup has been previously described; briefly, it consists of a
static Teflon reaction cell, a lever oscillator and a frequency counter connected to a computer for data
recording.23 The crystal was clamped in the static cell with Oꢀrings on both sides resulting in only one
face, with a geometric area of 0.205 cm2, being immersed in the liquid. The frequency of a dry aꢀC
coated crystal, stable over a period of 60 min at 20 °C before insertion into the teflon cell, is taken as
reference frequency. Surface functionalisation and rinsing of QCM crystals was carried out following
the same procedure as that of substrates for spectroscopic analysis.
Biomolecule-layer interactions. Lectin binding studies were carried out on aꢀC surfaces coated using
precursors 4 and 6, two precursors bearing galactose and mannose units, respectively. FITC labeled
Concanavalin A (FITCꢀCon A), a plant lectin that is known to bind αꢀDꢀmannoside structures and FITC
labeled Peanut Agglutinin (FITCꢀPNA), plant lectin that is known to bind to galactoside units were used
in this study. Manꢀcoated surfaces were incubated for 2 h in a 0.5 mg/ml solution of FITCꢀConA in
0.010 M PBS buffer at pH 7.4 with added 0.1 mM CaCl2 and MgCl2.26, 27 Galꢀcoated surfaces were
incubated for 2 h in a 0.5 mg/ml solution of FITCꢀPNA in the same buffer solutions. Surfaces were
rinsed with PBS buffer in order to remove excess unbound protein prior to imaging. Protein adsorption
studies were carried on aꢀC surfaces modified using precursor 4; the galactoseꢀcoated carbon surface
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was incubated for 2 h with 200 ꢀl of a solution of Bovine Serum Albumin fluoresceinꢀconjugate (FITCꢀ
BSA), 0.2 mg/ml in PBS at pH 7.4.28ꢀ30 The surface was washed with the PBS solution to remove the
unbound protein prior to imaging.
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Fluorescence images were acquired with an Olympus BX51 inverted fluorescent microscope using a
cellSense digital image processing software; no digital filters were used, only brightness and contrast
were adjusted in all images presented. The images were acquired with a cube filter set having excitation
filter at 470ꢀ495 nm, a dichroic filter at 505 nm and a barrier filter at 510ꢀ550 nm. Analysis of emission
intensity was carried out using image analysis software (Gwyddion) from 1600×1200 px images, each
covering a sample area of approximately 4 mm2. Intensity values were calculated by defining a mask
(314×263 px) in carbohydrateꢀmodified and unmodified regions of the carbon surface. The mean
emission intensities were ratioed in order to compare the amount of adsorbed fluorophore on both types
of sample regions; standard deviations were calculated over three samples.
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3. Results and Discussion
3.1 Carbohydrate synthesis and surface modification
Diazonium grafting has emerged as a highly efficient and general strategy for the functionalization of
carbon materials but has not previously been applied to the modification of surfaces with carbohydrates.
The reactive diazonium species were generated in situ on treatment of 4ꢀaminophenyl glycosides with a
solution of sodium nitrite and fluoroboric acid (Scheme 1). The reactive diazonium species reductively
adds to carbon, as well as metallic surfaces, through robust covalent bonds as shown in previous
work.18, 22, 25, 31
A series of both protected and fully unprotected 4ꢀaminophenyl glycosides were prepared through the
general synthetic pathway outlined in Scheme 2. In the first step the commercially available
peracetylated sugar was treated with a stoichiometric quantity of benzylamine to selectively hydrolyse
the anomeric acetate and release the free hemiacetal. The hemiacetal was treated with an excess of
trichloroacetonitrile in the presence of 1,8ꢀDiazabicycloundecꢀ7ꢀene (DBU), to furnish the
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trichloroacetimidate donor. Glycosylation reactions with 4ꢀnitrophenol were carried out in the presence
of a lewis acid (BF3OEt2), to furnish the 4ꢀnitrophenyl glycoside.
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Stereochemistry of the glycoside bond was defined by neighbouring group participation of the acetyl
group at the 2ꢀOH position. For the 4C1 sugars, glucose (Glc) and galactose (Gal), only the beta anomers
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were formed, for mannose (Man) the expected alpha anomer was formed exclusively. The C4 sugar,
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rhamnose (Rha), furnished the alpha anomer. The nitro compounds were treated under reducing
conditions with sodium borohydride in the presence of palladium on charcoal to furnish the desired 4ꢀ
aminophenyl glycoside in good yields. Subsequent treatment of the protected amines under zémplen
conditions furnished the fully deprotected 4ꢀaminophenyl glycoside in quantitative yields. Scheme 3
shows the 4ꢀaminophenyl glycosides prepared using the general strategy. Following purification and
characterisation of the 4ꢀaminophenyl glycosides (see Supporting Information for details) the
compounds were used in the surface functionalization reactions. Infrared Reflectance Absorption
Spectroscopy (IRRAS) was used in order to confirm that surface functionalisation takes place after
immersion of the substrates in aqueous solutions of the in situ generated aryldiazonium salts. Figure 1
shows IRRAS spectra obtained after surface modification of amorphous carbon coatings using the
aryldiazonium derivative of precursor compounds 1, 3, 5 and 7, which bear Glc, Gal, Man and Rha
monosaccharides, respectively. All four IRRAS spectra show the characteristic infrared absorbances of
the parent peracetylated glycoside which are observed in the reference infrared spectra of bulk powders
(see Supporting Information). Absorbance peaks at 1757 cmꢀ1 and 1371 cmꢀ1 can be attributed to
carbonyl and methyl groups of the acetyl moiety, respectively. Strong absorbances at ~1230 cmꢀ1 and in
the region 1025ꢀ1090 cmꢀ1 are associated to C—O stretching modes arising from the carbohydrate ring
and the acetyl protecting groups.32 Peaks in the region 1500ꢀ1550 cmꢀ1 arise from C—C skeletal
vibrations of phenyl rings;32 in particular, it was possible to observe in all spectra the presence of a peak
at 1508 cmꢀ1 which can be attributed to the strong 19a stretching mode of phenyl rings.33
Control experiments carried out by immersing the substrates in aqueous solutions of 4ꢀaminophenyl
glycosides alone, without carrying out diazotization reactions, yielded no IRRAS signatures, thus
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indicating that formation of aryldiazonium cations is necessary in order to observe the formation of
monosaccharide adlayers. These results indicate that peracetylated carbohydrate coatings were obtained
in a single step procedure from aqueous solutions via diazotization of acetalꢀprotected precursors
followed by chemisorption to carbon substrates. Importantly, this functionalization methodology was
found to be general to all of the monosaccharideꢀderivatives tested.
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Carbohydrate coatings obtained with acetalꢀprotected precursors could be deprotected at the surface to
yield the corresponding native monosaccharides. Figure 2 shows a comparison of IRRAS spectra of a
peracetylatedꢀGal coating obtained from 3 after immersion in methanol (trace a) and the same coating
after deacetylation reactions via immersion in Na/methanol solutions (trace b). After sonication in
methanol, in the absence of sodium methoxide, the integrated intensity of IRRAS peaks is reduced by
approximately 50% with respect to peaks obtained after sonication in water alone (comparison of Figure
2ꢀtrace a vs. Figure 1ꢀtrace b). This observation is consistent with previous studies of aryldiazonium
layer composition and indicates that adlayers consist of both strongly chemisorbed molecules and of
more weakly bound species that can be removed via organic extraction.25 After immersion in
Na/methanol solutions, we observed the complete disappearance of carbonyl and methyl peaks
associated to acetyl protecting groups from the IRRAS spectra. Peaks associated to C—O stretching
modes remain however present after deprotection and rinsing. Although the IRRAS peak intensities
might depend on monosaccharide orientation at the surface, it is possible to conclude from this result
that the core carbohydrate ring system remains intact and covalently bound to the surface after
deacetylation.
Surface modifications carried out via diazotization of the fully deprotected precursor 4 (Galꢀbearing
precursor) yielded IRRAS spectra similar to those obtained after functionalization with 3 followed by
deacetylation. This is illustrated in trace c (Figure 2), which shows the IRRAS spectrum of carbon
substrates after immersion in solutions of the diazonium salt of compound 4. Peaks in the IRRAS
spectrum obtained after functionalization using compound 4 closely match those of the parent
compound obtained from bulk powders (see Supporting Information). A comparison of traces b and c in
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Figure 2 suggests that similar carbohydrate layers are obtained on reaction with fully hydroxylated
precursors (e.g. 4) as from peracetylated precursors after deprotection (e.g. via deprotection of 3 at the
surface). The relative ratios of C—O stretching peaks at 1234 and 1084 cmꢀ1 are however different in
traces b and c; this can arise from differences in packing or orientation at the surface as discussed
below. In summary, these results confirm that both protected and deprotected carbohydrates can be
bound to the surface using this methodology and that effective deprotection chemistry can be carried out
at surface bound monosaccharides.
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The carbohydrate functionalization methodology was found to be also general with respect to the type
of substrate material used. Figure 3A shows IRRAS spectra obtained after functionalization of metals
and alloys via diazotization of the peracetylated Galꢀbearing precursor, compound 3. The IRRAS
signature of carbohydrate coatings at Au (trace a), Cu (trace b) and brass (trace c) substrates was found
to be similar; absorbance peaks match those of the parent compound as well as those observed for layers
obtained at carbon substrates (see Figure 1, trace a). Control experiments in aqueous solutions of 4ꢀ
aminophenyl glycosides alone in the absence of diazotization reactants, yielded no IRRAS signatures,
thus indicating that formation of aryldiazonium cations is necessary in order to observe the formation of
monosaccharide adlayers. Analogous results were obtained for precursors 1, 5 and 7 on Au, Cu and
brass substrates as shown in the Supporting Information.
We observed that in the case of metals and alloys there was a strong dependence of peak intensities on
the concentration of aryldiazonium precursor used for the functionalization reactions. Figure 3B shows
IRRAS spectra obtained from reactions of aryldiazonium cations of precursor 3 at 1.0 mM and 0.25 mM
concentrations at brass substrates after sonication in water and after sonication in organic solvents
(acetonitrile and methanol). Peak absorbances in IRRAS spectra are determined by a combination of
molecular surface density, molecular orientation and surface enhancement of the electric field
perpendicular to the surface.34 However, assuming an isotropic orientation of acetal groups within the
layer, net absorbances provide a good estimate of relative coverage at a given substrate.35, 36 Therefore, a
comparison of spectra obtained from 1.0 mM and 0.25 mM solutions indicates (traces a vs. c) that the
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surface density of adlayers formed at 1.0 mM concentrations is higher. Subsequent sonication of these
layers in organic solvents, however, drastically reduces the overall intensity of the spectrum (traces b
and d) by more than 90%, thus suggesting that a larger proportion of adsorbed species than at carbon
surfaces adsorbs via weak interactions. The fact that no physisorption was observed in the absence of
diazotization conditions suggests, however, that species more weakly bound to the surface still result
from reactions of aryldiazonium derivatives of monosaccharide precursor compounds.
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Quantitative information on the molecular surface density of carbohydrate films obtained at solid
surfaces from our oneꢀstep methodology was obtained via ex situ QCM measurements. Surface density
was determined by calculating the mass change at the quartz crystal from the change in resonance
frequency using the Sauerbrey equation:37
2 f02
ꢁf = −
ꢁm
(1)
A
ꢀρ
where, f0 is the resonance frequency of the fundamental mode of the QCM in air, A is the effective
surface area of the electrodes, and ꢂ and ρ are the density and shear modulus of quartz. The resonant
frequency of aꢀC coated crystals prior to surface modification was considered as the baseline in
determining frequency shifts and thereby the mass shifts for all of our experiments. Normalization of
mass shifts by the molecular weight of the chemisorbed compound after nitrogen elimination yielded
surface molar densities. The surface density of peracetylated monosaccharides obtained via spontaneous
aryldiazonium adsorption at carbon substrates from 1.0 mM 4ꢀaminophenyl glycoside solutions was
found to be (8.1 ± 0.5) ×10ꢀ10 mol cmꢀ2 for compound 1 (Glc), (8.0 ± 0.6) ×10ꢀ10 mol cmꢀ2 for compound
3 (Gal), (7.5 ± 0.4) ×10ꢀ10 mol cmꢀ2 for compound 5 (Man) and (7.6 ± 0.3) ×10ꢀ10 mol cmꢀ2 for
compound 7 (Rha). These values are lower than those reported for the spontaneous attachment of small
aryldiazonium salts at the same carbon substrate (e.g. pꢀnitrobenzene diazonium salt),25 as expected
based on the much larger geometrical crossꢀsection of compounds 1, 3, 5 and 7. In the case of
deprotected monosaccharides, measurements of surface densities yielded values of (12 ± 7) ×10ꢀ10 mol
cmꢀ2 for compound 4 (Gal) and (19 ± 2) ×10ꢀ10 mol cmꢀ2 for compound 8 (Rha). Previous studies of
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glycolipid monolayers of simple monoꢀ and disaccharides report that, when perpendicularly oriented in
cylindrical conformation, the surface area per molecule of saccharide headgroups is in the range 37ꢀ45
Å2,38, 39 so that 1 ML ≈ 4 ×10ꢀ10 mol cmꢀ2. The surface density obtained after deposition of fully
deprotected precursors 4 and 8 suggests that the spontaneous modification reaction leads to assembly of
1ꢀ4 ML at carbon surfaces. This coverage range compares well both with estimates of fully packed
monosaccharide layers and with quantitative determinations of limiting coverage of spontaneous
aryldiazonium reactions.22, 25 The molar surface density was found to be lower when the reaction was
carried out using fully acetylated precursor compounds; this is also consistent with the final molar
density being influenced by the magnitude of the molecular crossꢀsection.
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In summary, these results indicate that a oneꢀstep, solutionꢀbased, carbohydrate functionalization
methodology is successful at carbon, metal and alloy substrates; surface coverage can be determined
quantitatively and is comparable to typical surface densities observed at aryldiazonium adlayers.
3.2 Interaction of modified surfaces with biomolecules
Glycosylated surfaces find numerous applications, notably in glycoarrays,40 nanoparticle based
sensing41 and delivery,42, 43 and recently in new approaches of eliciting specific immune responses.44, 45
All of the above applications rely on biomoleculeꢀcarbohydrate recognition processes that must take
place at the solid/liquid interface. Carbohydrate conformation, orientation and crowding at the surface
are critical in determining whether these molecules can effectively elicit a specific biological response
once bound at the surface, with the specific choice of linker and surface density often affecting
biorecognition. Therefore, we decided to investigate whether monosaccharide layers prepared via
spontaneous chemisorption of aryldiazonium derivatives of 4ꢀaminophenyl glicosides display
biomoleculeꢀsubstrate interactions typical of carbohydrates, by carrying our surface binding
experiments using lectins.
Binding experiments with fluorescently labeled lectins were carried out via fluorescence microscopy.
Figure 4a shows a fluorescence microscopy image of a carbon coated surface, where only the lower half
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of the area shown was modified with αꢀmannose using compound 6 as a precursor as described in
section 3.1. The sample was incubated for 2 h in a 0.5 mg mLꢀ1 solution of Concanavalin A fluoresceinꢀ
conjugate (FITCꢀConA) in phosphate buffer (PBS); the specific interaction of mannose towards Con A
lectin has been widely explored and established.40 Figure 4a shows that, after rinsing in PBS, the area of
the sample that had undergone surface functionalization with αꢀmannose displays stronger FITC
emission than the area covered by bare amorphous carbon. The emission intensity was found to be 17 ±
1 times greater over regions of the carbon sample coated with mannose than over bare carbon, thus
indicating that ConA displays affinity towards surfaceꢀbound mannose groups. A similar result was
obtained by using galactoseꢀmodified surfaces prepared using compound 4 as a precursor, and Peanut
Agglutinin fluoresceinꢀconjugate (FITCꢀPNA), a lectin that displays binding specificity towards Gal
units.40 Also in this case, FITCꢀPNA was found to preferentially bind to the Galꢀmodified areas (see
Supporting Information). These experiments suggest that the spontaneous chemisorption of
aryldiazonium monosaccharide precursors leads to the formation of monosaccharide layers that retain
the biological recognition specificity of the parent carbohydrate molecule in solution.
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In addition to investigating lectin affinity towards surfaceꢀbound carbohydrates we were also interested
in assessing the potential of these aryldiazonim coatings as antifouling surfaces. It has been showed that
galactoseꢀrich surfaces display resistance towards nonꢀspecific protein binding.27, 46 Therefore, we
compared the level of nonꢀspecific protein adsorption at both Galꢀmodified and unmodified carbon
surfaces using Bovine Serum Albumin FITC conjugate (FITCꢀBSA). Figure 4b shows a fluorescence
microscopy image of a carbon coated surface, where only the lower half of the area shown was modified
with βꢀgalactose using compound 4 as a precursor as described in section 2.1. The entire sample was
incubated for 2 h in a 0.2 mg mLꢀ1 solution of FITCꢀBSA in PBS at pH 7.4,28ꢀ30 and then washed with
the PBS solution to remove unbound protein prior to imaging. Figure 4b clearly shows that the area of
the sample that had undergone modification with Gal units displays much lower FITC emission than the
unmodified area: the emission intensity from physisorbed FITCꢀBSA was found to be 4.1 ± 0.3 times
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greater at bare carbon surfaces compared to Galꢀmodified carbon surfaces. This result suggests that the
carbohydrate coating may be able to impart the ability to prevent undesirable albumin accumulation on
surfaces.
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In conclusion, we have demonstrated that aryldiazonium chemistry offers a viable approach for the
modification of surfaces with carbohydrates. We have shown that monosaccharides can be
spontaneously chemisorbed at carbon, metals and alloys under mild conditions. The resulting
carbohydrate layers retain the biological recognition properties of the parent monosaccharides used in
the functionalization. Furthermore, we show that these layers can potentially impart antifouling
properties onto surfaces. This functionalization methodology is mild and efficient and should find
general applications in glycoarray technology, antifouling coatings and as a general strategy for the
synthesis of a wide range of materials with biomimetic surfaces.
Acknowledgements. This publication has emanated from research conducted with the financial support
of Science Foundation Ireland under Grant Number 11/TIDA/B1983. DM acknowledges support
through Science foundation Ireland Grant No. 09/RFP/CAP2174; MDA acknowledges support through
Enterprise Ireland Grant No. CF/2012/2634. Supporting Information is available online from Wiley
InterScience.
Supporting Information Available. Detailed synthesis, purification and characterization of
glycosides, NMR and FTIR data of precursor compounds, IRRAS spectra of carbohydrate organic thin
films, Peanut Agglutinin binding experiments at Galꢀmodified surfaces. This material is available free
of charge via the Internet at http://pubs.acs.org.
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Scheme 1: Formation of glycosyl diazonium intermediate and subsequent net chemisorption reaction at
surfaces.
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Scheme 2. General synthetic strategy for the preparation of 4-aminophenyl glycosides.
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Scheme 3. Protected and unprotected 4-aminophenyl glycosides synthesized: Peracetylated 4-aminophenyl
Glucoside (1), Galactoside (3), Mannoside (5) and Rhamnoside (7) and the corresponding unprotected 4-
aminophenyl Glucoside (2), Galactoside (4), Mannoside (6) and Rhamnoside (8).
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Figure 1: IRRAS spectra of carbohydrate coated carbon substrates obtained via surface functionalization
from solutions of aryldiazonium salts of compounds 1 (trace a), 3 (trace b), 5 (trace c) and 7 (trace d) from
1.0 mM aqueous solutions.
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Figure 2: IRRAS spectra on carbon substrates carbohydrate layers. Trace a: peracetylated galactose layers
obtained via functionalization using precursor 3 at 1.0 mM concentration after exposure to methanol only.
Trace b: galactose layers obtained after deacetylation of the sample in trace a, via immersion in
Na/methanol solutions. Trace c: galactose layer obtained using Gal-bearing precursor 4 at 1.0 mM
concentration.
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Figure 3: (A) IRRAS spectra of Au (trace a), Cu (trace b) and brass (trace c) substrates coated with
peracetylated galactose via diazotization of compound 3 in aqueous solutions. (B) Effect of precursor
concentration and of rinsing protocols on surface coverage of adlayers of peracetylated galactose obtained
via diazotization of compound 3.
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Figure 4: Fluorescence microscopy images of carbon surfaces after binding experiments using fluorescently
labeled biomolecules; the lower half of the carbon surface has been modified with a monosaccharide,
whereas the upper half remains bare. (a) Binding experiment on Man-modified carbon surfaces using FITC-
ConA lectin (image width = 400 µm). (b) Adsorption experiment on Gal-modified carbon surfaces using
FITC-BSA protein (image width = 400 µm).
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Chemistry of Materials
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Tabel of Contents Graphic
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