Epimeric monosaccharide-quinone hybrids on gold electrodes toward the electrochemical probing of specific Carbohydrate-Protein Recognitions

Article abstract of DOI:10.1021/ja110478j

Carbohydrates represent one of the most significant natural building blocks, which govern numerous critical biological and pathological processes through specific carbohydrate-receptor interactions on the cell surface. We present here a new class of elect

Full text of DOI:10.1021/ja110478j

ARTICLE
pubs.acs.org/JACS
Epimeric Monosaccharide-Quinone Hybrids on Gold Electrodes
toward the Electrochemical Probing of Specific Carbohydrate-
Protein Recognitions
Xiao-Peng He,†,‡ Xiu-Wen Wang,† Xiao-Ping Jin,† Hao Zhou,† Xiao-Xin Shi,‡ Guo-Rong Chen,† and
Yi-Tao Long*,†
†Key Laboratory for Advanced Materials and Institute of Fine Chemicals, and ‡School of Pharmacy, East China University of Science and
Technology, 130 Meilong Road, Shanghai, People’s Republic of China
S Supporting Information
b
ABSTRACT: Carbohydrates represent one of the most sig-
nificant natural building blocks, which govern numerous
critical biological and pathological processes through specific
carbohydrate-receptor interactions on the cell surface.
We present here a new class of electrochemical probes based
on gold surface-coated epimeric monosaccharide-quinone
hybrids toward the ingenious detection of specific epimeric
carbohydrate-protein interactions. Glucose and galactose,
which represent a pair of natural monosaccharide C4 epimers,
were used to closely and solidly conjugate with the 1,4-
dimethoxybenzene moiety via a single C-C glycosidic bond, followed by the introduction of a sulfhydryl anchor. The
functionalized aryl C-glycosides were sequentially coated on the gold electrode via the self-assembled monolayer (SAM)
technique. X-ray photoelectron spectroscopy (XPS) was used to confirm the SAM formation, by which different binding energies
(BE) between the glucosyl and the galactosyl SAMs on the surface, probably rendered by their epimeric identity, were observed.
The subsequent electrochemical deprotection process readily furnished the surface-confined quinone/hydroquinone redox
couple, leading to the formation of electrochemically active epimeric monosaccharide-quinone SAMs on the gold electrode.
Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) used for the detection of specific sugar-lectin interactions
indicated that the addition of specific lectin to the corresponding monosaccharide-quinone surface, i.e., concanavalin A (Con A)
to the glucosyl SAM and peanut agglutinin (PNA) to the galactosyl SAM, resulted in an obvious decrease in peak current,
whereas the addition of nonspecific lectins to the same SAMs gave very minor current variations. Such data suggested
our uniquely constructed gold surface coated by sugar-quinone hybrids to be applicable as electrochemical probes for the
detection of specific sugar-protein interactions, presumably leading to a new electrochemistry platform toward the study of
carbohydrate-mediated intercellular recognitions.
arbohydrates are ubiquitously distributed in all living organ-
isms that exist in nature, governing a wide spectrum of
fluorophore- or biotin-labeled substrates toward the probing of
carbohydrate-receptor recognitions.6-11
C
pivotal biological and pathological events including cell-cell
recognition, signal transduction, autoimmune stimulation, tumor
metastasis, etc. by generating multivalent interactions with cor-
responding carbohydrate receptors on cells.1-5 Meanwhile, since
the diverse “sugar chains” that are pervasive on the cell surface are
concomitantly varied by cellular malignancy, development, or
differentiation, the real-time and accurate probing of specific
carbohydrate-receptor interactions that are associated with
these dynamic cellular events became crucial for early-state
disease diagnosis. However, natural carbohydrates as well as
their receptors lack measurable signals due to the absence of
“reporters” such as a fluorophore. To tackle this issue, numerous
analytical methods including NMR or fluorescence spectroscopy,
isothermal calorimetry, and quartz crystal microbalance (QCM)
and surface plasmon resonance (SPR) techniques have been
developed in the past decade, which were mainly based on
Indeed, carbohydrates are extraordinarily complex owing to their
tremendous structural and especially configurational diversity. For
example, glucose and galactose represent a pair of the simplest
monosaccharide epimers that differ only in the C4 stereocontrol,
whereas their biological characteristics are entirely different. Nature
is exquisitely tuned to sense such a small configurational difference
through specific molecular recognition of carbohydrate recognition
domains(CRD), whereas artificial chemical probes that can reliably
and quickly capture such nuances are still challenging.
Electrochemistry is known as a swift and sensitive analytical
technology with facile and inexpensive instrumentations, which
has been successfully employed in a multitude of wonderful
biochemical studies including ion-channel recording,12a-c DNA
Received: November 22, 2010
Published: February 22, 2011
r
2011 American Chemical Society
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Figure 1. (A) Synthetic route of compounds 1 and 2. (B) Fabrication of the electroactive sugar-quinone gold surface by the SAM technique,
electrochemical deprotection, and the sensing for lectins.
recognition,12d,13a,14a single-nucleotide polymorphism (SNP) study,13b
cancer biomarking,14b etc. Nevertheless, efforts that are devoted to the
probing of specific carbohydrate-receptor interactions on the basis of
electrochemically active sensors remain scant.15 We have previously
prepared a series of glucose-quinone or galactose-quinone hybrids
with a short and solid C-glycosidic linkage that is resistant to acidic as
well as enzymatic cleavage.16 With these ideal features, we sought to
investigate whether such quinonyl epimeric C-glycosides would
potentially be utilized in electrochemical study.
technique. X-ray photoelectron spectroscopy (XPS) was sequen-
tially conducted for confirming the SAM formation, followed by
electrochemical deprotection,21 which eventually generated the
surface-confined hydroquinone/quinone redox couple.
Next, as illustrated in Figure 1B, the applicability of our uniquely
characterized monosaccharide-quinone SAMs was preliminarily
attempted toward the probing of specific carbohydrate-lectin22
interactions via electrochemistry. Interestingly, upon addition of
the specific lectin to corresponding monosaccharide-quinone
SAM, i.e., Con A to the glucosyl SAM and PNA to the galactosyl
SAM, a remarkably decreased peak current was observed in its
cyclic voltammetry (CV) plots, whereas the addition of the same
lectins in a reverse fashion gave much minor current variations.
Differential pulse voltammetry (DPV) successively enabled a more
detailed investigation on such glycosyl SAM-lectin interactions,
demonstrating that the current decrease of the sugar-quinone
SAMs is proportional to the lectin concentrations increased.
Furthermore, a panel of nonspecific lectins was assayed on both
glycosyl SAMs for further ascertaining their biospecificity via DPV,
which resulted in minor changes in peak current. These results have
supported the notion of proposing our uniquely characterized
sugar-quinone SAMs as novel electrochemical probes for the
detection of specific sugar-receptor interactions.
The self-assembled monolayer (SAM) technique17 has re-
cently absorbed considerable interest as a potent complement for
fabricating carbohydrate-coated surfaces that highly mimic the
natural morphology of sugar chains pervaded on the cell
surface,18 offering reliable and presumable structure relationship
toward multivalent carbohydrate-receptor interactions.19 In
view of such compelling merits, we were prompted to fabricate
gold surface-attached SAMs that are electrochemically measur-
able based on epimeric monosaccharide-quinone hybrids.
In order to attach sugar-quinones on the gold surface, we
initially synthesized aryl C-glycosides 1 and 2 (Figure 1A) with a
sulfhydryl precursor. From amino C-glucosyl-1,4-dimethoxyben-
zene and C-galactosyl-1,4-dimethoxybenzene,20 which were
synthesized by our previous synthetic schemes involving the
Friedel-Crafts alkylation16 and a nitration/reduction se-
quence,20 the peracetylated sulfhydryl aryl C-glycosides 4 and
5 were obtained via N-acylation. Successive deacetylation with
AcCl gave the desired glycosides 1 and 2 with a free sulfhydryl
anchor that can be coated on a gold electrode via the SAM
’ EXPERIMENTAL SECTION
Materials. Concanavalin A (Con A), (peanut agglutinin) PNA,
Pisum satiwum agglutinin (PSA), soybean agglutinin (SBA), Ulex
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europeaus agglutinin (UEA-I), wheat germ agglutinin (WGA), Datura
stramonium agglutinin (DSA), and Sambucus nigra agglutinin (SNA)
were purchased from Sigma-Aldrich or Shanghai Shrek Biotechnology
Co., Ltd. All reagents and materials are of analytical grade, and solvents
were purified by standard procedures. All solutions for analytical study
were prepared with deionized water obtained with a Milli-Q System
(Billerica, MA, U.S.A.).
General Procedure for the N-Acylation. To a solution of 6 or 7
(1 equiv)20 and 2-(acetylthio)acetic acid (3 equiv) in dry DMF (5 mL)
and CH2Cl2 (0.5 mL) were added 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC, 3 equiv) and 1-hydroxbenzotriazole
(HOBt, 2 equiv) at 0 °C, then the mixture was allowed to r.t., stirring for
6 h. Then, solvent was removed in vacuum and the residue washed
successively with aqueous HCl (1 N), saturated aqueous NaHCO3, and
brine and extracted with CH2Cl2. The combined organic layers were
dried over MgSO4, filtered, concentrated in vacuum to dryness, and then
purified by column chromatography.
N-(2,5-Dimethoxy-4-((2S,3R,4R,5R,6R)-3,4,5-trihydroxy-6-(hydroxy-
methyl)tetrahydro-2H-pyran-2-yl)phenyl)-2-mercaptoacetamide (2).
From compound 5, reversed-phase HPLC (tR = 9.4 min over 76 min of
85% methanol/water, purity 100%) afforded compound 1 as a reddish-
white solid (5 mg, 8%). 1H NMR (400 MHz, D2O): δ = 8.27 (s, 1H),
7.63 (s, 1H), 7.23 (s, 1H), 4.66 (d, J = 10.0 Hz, 1H), 4.01 (d, J = 2.8 Hz,
1H), 3.84 (t, J = 9.2 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 3.78-3.75 (m,
2H), 3.72 (dd, J = 2.4, 9.6 Hz, 2H), 3.68 (d, J = 10.0 Hz, 2H). HR-ESI-
MS: calcd for [M - H]- 388.1066, found 388.1065.
NMR, Optical Rotation, HPLC, and HRMS. 1H and 13C NMR
spectra of compounds 1, 2, 4, and 5 (shown in the Supporting
Information) were recorded on a Bruker AM-400 spectrometer in
CDCl3 or D2O solutions using tetramethylsilane as an internal standard.
Optical rotations were measured using a Perkin-Elmer 241 polarimeter
at room temperature and a 10 cm 1 mL cell. HPLC purification was
carried out on a Yilite P230II HPLC system. High-resolution mass
spectra (HRMS) were recorded on a Waters LCT Premier XE spectro-
meter instrument using standard conditions (ESI, 70 eV).
Tetra-O-acetyl-glucopyranosyl-S-(2-((2,5-dimethoxyphenyl)amino)-
2-oxoethyl)ethanethioate (4). From compound 6 (161 mg, 0.3 mmol)
and 2-(acetylthio)acetic acid (142 mg, 1.1 mmol), EDC (191 mg, 1.0
mmol), HOBt (93 mg, 0.7 mmol), column chromatography (EtOAc/
petroleum ether, 1:1) afforded compound 4 (161 mg, 80%) as a light
yellow solid; Rf1= 0.4 (EtOAc/petroleum ether, 1:1); [R]D = -0.9 (c =
0.1, CH2Cl2). H NMR (400 MHz, CDCl3): δ = 8.53 (s, 1H), 8.08
(s, 1H), 6.89 (s, 1H), 5.36 (t, J = 9.2 Hz, 1H), 5.26 (t, J = 10.0 Hz, 1H),
5.22 (t, J = 10.0 Hz, 1H), 4.96 (d, J = 10.0 Hz, 1H), 4.27 (dd, J = 4.8,
12.4 Hz, 1H), 4.13 (dd, J = 2.0, 12.4 Hz, 1H), 3.87 (s, 3H), 3.86-3.83
(m, 1H), 3.80 (s, 3H), 3.76-3.66 (m, 2H), 2.45, 2.07, 2.06, 2.00, 1.79
(5s, 15H). 13C NMR (100 MHz, CDCl3): δ = 195.2, 170.7, 170.2,
169.6, 169.3, 166.2, 151.5, 142.1, 128.6, 118.9, 109.6, 103.6, 76.1, 74.5,
73.0, 72.0, 68.9, 62.5, 56.4 (1), 56.4 (2), 34.4, 30.2, 20.8, 20.6, 20.4. HR-
ESI-MS (m/z): calcd for [M þ Na]þ 622.1570, found 622.1570.
Tetra-O-acetyl-galactopyranosyl-S-(2-((2,5-dimethoxyphenyl)amino)-
2-oxoethyl)ethanethioate (5). From compound 7 (181 mg, 0.4 mmol)
and 2-(acetylthio)acetic acid (169 mg, 1.3 mmol), EDC (225 mg, 1.2
mmol), HOBt (104 mg, 0.8 mmol), column chromatography (EtOAc/
petroleum ether, 1:1) afforded compound 5 (172 mg, 77%) as a yellow
solid; Rf = 0.3 (EtOAc/petroleum ether, 1:1); [R]D = þ76.0 (c = 0.1,
CH2Cl2). 1H NMR (400 MHz, CDCl3): δ = 8.55 (s, 1H), 8.09 (s, 1H),
6.95 (s, 1H), 5.52 (dd, J = 0.4, 3.2 Hz, 1H), 5.43 (t, J = 10.0 Hz, 1H), 5.21
(dd, J = 3.2, 10.0 Hz, 1H), 4.93 (d, J = 10.0 Hz, 1H), 4.20-4.06 (m, 3H),
3.89 (s, 3H), 3.81 (s, 3H), 3.76-3.66 (m, 2H), 2.45, 2.21, 2.03, 1.99,
1.80 (5s, 15H). 13C NMR (100 MHz, CDCl3): δ = 195.2, 170.4, 170.3,
170.1, 169.4, 166.2, 151.5, 142.1, 128.6, 119.3, 110.0, 103.6, 74.7, 73.5,
72.4, 69.4, 67.9, 61.6, 56.5, 56.3, 34.3, 30.1, 20.8, 20.7, 20.6, 20.5. HR-
ESI-MS (m/z): calcd for [M þ Na]þ 622.1570, found 622.1573.
General Procedure for the Deacetylation. To a solution of 4
or 5 (100.0 mg, 0.17 mmol) in MeOH (5 mL) was slowly added AcCl
(1.5 equiv.), stirring at reflux for 10 h. Then, the solvent was removed in
vacuum and the residue directly purified by column chromatography to
afford compound 1 and by reversed-phase high-performance liquid
chromatography (HPLC) to afford compound 2, respectively.
Monolayer Formation. The electrode surface was polished on an
emery paper and alumina slurry until a mirror-like surface was obtained,
followed by sonication in ethanolic solution at least for 2 min. Electro-
polishing was then conducted using consecutive cyclic voltammograms
in 0.5 mol/L sulfuric acid and potential scanning between 0.5 and 1.5 V
versus saturated calomel electrode (SCE) until a typical voltammogram
for gold was obtained. The SAMs were simply formed by immersing a
clean gold electrode in an ethanolic solution of 1 mM ethanethioates
(1, 2, or 3) for a period of 36 h.
XPS. XPS spectra were obtained on an Axis-165 X-ray photoelectron
spectrometer (Kratos Analytical) using a monochromatic Al KR X-ray
source (1486.7 eV). Survey spectra (0-1100 eV) were taken at constant
analyzer pass energy of 160 eV, and all high-resolution spectra for C1s,
N1s, O1s, S2p, and Au4f were acquired with a pass energy of 20 eV, a step
of 0.1 eV, and a dwell time of 200 ms. The takeoff angle between the film
surface and the photoelectron energy analyzer was 90°. The typical
operating pressure was around 5 ꢀ 10-10 Torr in the analysis chamber.
Various scan numbers were carried out for the different elements to
obtain the high signal-to-noise ratio. The binding energies were refer-
enced to the Au4f7/2 at 84.0 eV, and peaks were fitted using the publicly
available XPSPEAK v. 4.1. The Shirley function was used as a back-
ground and Gaussian-Lorentzian cross-products used to fit the in-
dividual peaks. The samples for XPS measurements were prepared from
the SAMs containing compounds 1, 2, or 3 on gold-coated silicon chips
(5 mm ꢀ 5 mm size, West Chester, PA U.S.A.). Before the chips were
incubated in deaerated solutions of the SAMs, the chips were carefully
precleaned by soaking in hot Piranha solution (H2SO4/H2O2 = 3:1) for
10 min (Caution! Piranha solution should be handled with extreme care
and should never be stored in a closed container. It is a very strong oxidant
and reacts violently with most organic materials) and then sonicated in
Millipore H2O for three times.
Electrochemical Deprotection, CV, and DPV. All electroche-
mical experiments were conducted with a computer-controlled CHI 660
electrochemical station (Chenhua Co. Ltd., Shanghai, China). Working
electrodes were 3 mm diameter disk gold (Au) electrodes, used in
conjunction with a Pt auxiliary electrode, and a Ag/AgCl wire pseudor-
eference electrode worked as the reference electrode. Electrochemical
deprotection of SAMs containing compounds 1-3 was realized by
scanning the potential between -0.3 and 0.9 V with a scan rate of 100
mV/s in 0.1 M H2SO4 after the formation of the corresponding SAMs
on the Au electrode. After electrochemical deprotection of SAMs 1 and 2
on the gold surface, the electrode was incubated for 5 min in the
presence of various lectins in PBS (pH = 7.0) to determine the sugar-
lectin interactions. All voltammetric experiments were performed after
deaeration for 5 min with nitrogen. DPV measurement was recorded
with increasing amounts of lectins ranging from 0 to 10 μM, driving
N-(2,5-Dimethoxy-4-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxy-
methyl)tetrahydro-2H-pyran-2-yl)phenyl)-2-mercaptoacetamide (1).
From compound 4, column chromatography (DCM/MeOH, 6:1)
afforded compound 1 as a reddish-brown solid (20 mg, 32%); Rf = 0.5
1
(DCM/MeOH, 4:1); [R]D = þ9.1 (c = 0.1, MeOH). H NMR (400
MHz, D2O): δ = 6.93 (s, 1H), 6.56 (s, 1H), 4.58 (d, J = 9.6 Hz, 1H), 3.78
(dd, J = 1.6, 13.6 Hz, 1H), 3.76 (s, 3H), 3.70 (s, 3H), 3.68 (dd, J = 2.8, 7.6
Hz, 1H), 3.65 (d, J = 9.2 Hz, 1H), 3.60-3.55 (m, 2H), 3.58 (q, J = 7.2
Hz, 2H), 3.49-3.46 (m, 2H). 13C NMR (100 MHz, D2O): δ = 164.9,
152.8, 142.2, 137.5, 115.8, 112.6, 102.0, 80.1, 77.6, 75.3, 73.0, 69.8, 60.8,
56.9, 56.8, 36.9. HR-ESI-MS: calcd for [M - 74 þ Na]þ 338.1216,
found 338.1237.
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Figure 2. High-resolution XPS spectra of S2p, N1s, O1s, and C1s for SAM 1 (A-1-A-4), SAM 2 (B-1-B-4), and SAM 3 (C-1-C-4) on a gold surface.
Open circles stand for experimental raw data, red solid lines are for the total fits, black lines are for the component-fitted peaks, and green lines are for the
baselines.
potential from -0.3 to 0.4 V with a pulse amplitude of 50 mV and a
sample pulse width of 100 ms.
respectively. Glucosyl and galactosyl derivatives which only
differ in their C4 configuration were prepared in this study since
they uniquely interact with corresponding lectins, i.e., glucose-
Con A and galactose-PNA. Such specific epimeric carbohy-
drate-protein interactions were presumed discriminable via
electrochemistry.
SAM Formation and XPS Characterization. With the sulfhy-
dryl C-glycosyl derivatives 1 and 2 in hand, we attempted to
fabricate the glycosylated gold surface by using the known
sulfhydryl 1,4-dimethoxybenzene (Figure 1A, 3)23 as a control.
The formation of SAMs containing, respectively, compounds 1-
3 on the gold surface was simply realized by immersing a clean
gold electrode in an ethanolic solution of 1 mM 1, 2, or 3 for a
period of 36 h.
The XPS that represents a quantitative and surface-sensitive
analytical technique to verify the chemical composition of the
SAM samples26 was used to substantiate the SAM formation. In
order to prevent the excess contamination by carbon and oxygen
species, the gold surface has been carefully precleaned with fresh
Piranha solution prior to incubation in deaerated solutions
containing compounds 1, 2, and 3. The high-resolution XPS
spectra then permitted direct quantification of the chemical
’ RESULTS AND DISCUSSION
Synthesis of Sulfhydryl Aryl C-Glycosides. To fabricate
glycosylated SAMs on the gold surface, we chose to employ a
sulfide linker which has been shown to efficiently immobilize
various sugar moieties onto a gold surface.19,24 As shown in
Figure 1A, amino C-glucosyl-dimethoxybenzene 6 and amino
C-galactosyl-dimethoxybenzene 7 were first obtained via the
Lewis acid promoted Friedel-Crafts alkylation16 and a nitra-
tion/reduction sequence.20 Then, the N-acylation of compounds
6 and 7 with commercially available 2-(acetylthio)acetic acid in
the presence of EDC and HOBt afforded sulfides 4 and 5,
respectively. Notably, an amide linkage was chosen to couple
aryl C-glycosides with the sulfhydryl precursor due to its ability
to form hydrogen bonds within the monolayer matrix, thus
reinforcing the stability and rigidness of the surface-attached
SAM.19,25 Successive deacetylation with AcCl and purification by
column chromatography or reversed-phase HPLC yielded the
desired glycosides 1 and 2 containing a free sulfhydryl anchor,
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Table 1. Binding Energy (BE, eV) of O1s and C1s of Compound 1-, 2-, and 3-Coated SAMs in the XPS Spectra
O1s spectra
C1s spectra
BE (ratio)a
BE (ratio)
component
SAM 1
SAM 2
SAM 3
component
SAM 1
SAM 2
SAM 3
methoxyl-O
carbonyl-O
sugar-O
n/Ab
n/A
529.9 (2)
531.4 (1)
532.9 (16)
532.9 (16)
529.9 (2)
531.3 (1)
n/A
benzene ring-C
S-C
284.7 (6)
285.3 (1)
284.4 (2)
286.1 (6)
286.5 (1)
284.7 (6)
285.3 (1)
285.8 (2)
286.4 (6)
286.8 (1)
284.7 (6)
285.3 (1)
285.8 (2)
n/A
532.7
532.7
methoxyl-C
sugar-C
water-O
532.5 (7)
carbonyl-C
286.7 (1)
a The values in parentheses are ratios of peak areas calculated from given XPS spectrum. b n/A means “not available”.
species to provide detailed information for the composition of
the SAMs 1-3 on the gold surface.
that of the galactosyl SAM 2 (285.8 eV, Table 1, C1s, SAMs 1
and 2).
As shown in Figure 2A-C, the XPS peaks of S2p, N1s, C1s, and
O1s for each compound were fitted and deconvoluted to give the
chemical shift data of the components within the coated mole-
cules, respectively. Two dominant peaks located at 161.7 and
162.9 eV with an area ratio of 2:1 and a peak separation of 1.2 eV
were observed in the S2p spectra (Figure 2, parts A-1, B-1, and
C-1) of SAMs 1-3, which could be assigned to the S atom bound
on the gold surface.27 From their N1s spectra as shown in
Figure 2, parts A-2, B-2, and C-2, the single peak centered at
399.8 eV without difference in chemical shifts indicates the
presence of amide-N on the surface of each SAM.
A plausible explanation of the chemical shifts for the glucosyl
SAM 1 was then tentatively proposed. Since the common carbon
and oxygen atom peaks are almost identically located in their
corresponding spectra for the galactosyl SAM 2 and the control
SAM 3 that lacks the saccharide moiety, the varied binding
energy (BE) of the glucosyl SAM 1 could be probably caused by
its stronger intermolecular hydrogen-bonding interactions with
the water molecule. Apparently, the only difference between the
glucosyl and the galactosyl SAM lies in the C4 configuration with
the former owning a C4 equatorial OH group and the latter
bearing a C4 axial OH group. As a consequence, the C4 axial
hydroxyl bond that is closer to the methoxyl group of the aglycon
with a β-configuration might have performed as a shutter to
further prevent the generation of intermolecular hydrogen bonds
with the water molecule. In contrary, the equatorial bond of the
glucosyl moiety that is spatially more distant from this methoxyl-
O might result in a spatial gap for intermolecular hydrogen bonds
between water and the methoxyl-O on the benzene ring. The
electron cloud density of this oxygen atom would thus be
decreased, which could result in its chemical shift toward the
high binding energy field, whereas the electron cloud density of
the methoxyl-C covalently attached with this oxygen atom might
have been increased for charge balance, leading to its chemical
shift toward the low binding energy field.28 Notably, such
difference caused by epimeric identity between SAMs 1 and 2
is also reflected in their chemical reactivity during the electro-
chemical deprotection process described below.
The O1s spectrum of SAM 3 (Figure 2C-3) then gave three
obvious peaks centered at 529.9, 531.3, and 532.5 eV, attributable
to the methoxyl-O, carbonyl-O, and the oxygen atom of water
molecules adsorbed on the surface (Table 1, O1s, SAM 3) with a
peak area ratio of 2:1:7. In contrast, the O1s spectra of the SAMs 1
and 2 that contain epimeric monosaccharides showed a remark-
able difference. As shown in Figure 2A-3, for the SAM that
contains the galactoside 2, its methoxyl-O and carbonyl-O peaks
were similarly observed at 529.9 and 531.4 eV, respectively
(Table 1, O1s, SAM 2), with a peak area ratio of 2:1. Another
peak centered at 532.9 eV seems to contain both the sugar-O and
the water-O peaks, which are proportionally larger than that of
the water-O peak shown for the control SAM 3 (Figure 2C-3).
However, only one broadened peak centered at 532.7 eV was
fittable in Figure 2A-3 for the SAM containing the glucoside 1,
which could probably ascribe to the chemical shift of its
methoxyl-O peak to the higher binding energy field, leading to
the superposition of the methoxyl-O, carbonyl-O, sugar-O, and
the water-O peaks (Table 1, O1s, SAM 1).
Consequently, the XPS analysis confirmed that compounds
1-3 have been successfully chemisorbed on the gold electrode
surface via self-assembly.
Interestingly, an obvious peak shift was also observed in the
C1s spectrum of SAM 1. By first comparing the C1s spectra
between SAMs 2 (Figure 2B-4) and 3 (Figure 2C-4), their
benzene ring-C, S-C, methoxyl-C, and carbonyl-C peaks were
similarly centered at 284.7, 285.3, 285.8, and 286.8 eV (286.7 eV
for SAM 3), whereas an additional peak appeared at 286.4 eV
exclusively for the galactosyl SAM 2, assignable to the sugar-C
(Table 1, C1s, SAMs 2 and 3). The peak ratios of both SAMs
(2 and 3) are in the correct proportion, listed in the parentheses
of Table 1, C1s, SAMs 2 and 3. In stark contrast, despite that the
benzene-C (284.7 eV), S-C (285.3 eV), sugar-C (286.1 eV),
and carbonyl-C (286.5 eV) peaks of the glucosyl SAM 1
(Figure 2A-4) were positioned likewise, an apparent chemical
shift of its methoxyl-C peak (284.4 eV) to the lower energy
binding field by 1.4 eV was clearly observed comparing with
Electrochemical Deprotection. Electrochemical deprotec-
tion of the XPS-confirmed methylated (hydroxy)quinone SAMs
1-3 was successively proceeded to generate the hydroquinone/
quinone redox couple.21b In Figure 3, CVs recorded at 100 mV/s
between -0.3 and 0.9 V versus Ag/AgCl in 0.1 M H2SO4 are
shownfor each SAM. The CV ofthecontrol SAM(3, Figure3C-1)
showed no obvious redox peak in the first scan segment,
whereas an oxidation peak at Epa = 0.357 V and a reduction
peak at Epc = 0.283 V were observed with successive scans.
Further increase in scan number led to consecutive increase of
peak currents but slight shifts of peak potentials. Eventually, the
redox peak current tended to stabilize on the electrode surface
after 10 continuous scans with the anodic and cathodic peaks
being symmetric and broad and a full width at half-height (fwhh)
equal to 0.1 V.
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Figure 3. Cyclic voltammograms (CVs) of SAMs on gold electrodes created from the practically deprotected forms of compounds 1 (A), 2 (B), and
3 (C). The CVs were recorded with 10 continuing scans at a scan rate of 100 mV/s in 0.1 M H2SO4 as illustrated in panels A-1, B-1, and C-1. The CVs
were recorded in 0.1 M H2SO4 at scan rates of 0.020, 0.040, 0.060, 0.080, and 0.100 V/s as illustrated in panels A-2, B-2, and C-2. Peak currents of SAMs
1, 2, and 3 on gold electrodes, Ip, as a function of scan rate, v, is illustrated in panels A-3, B-3, and C-3. All first scans were initiated in the positive direction
from -0.3 V.
For the SAMs 1 (Figure 3A-1) and 2 (Figure 3B-1) that
contain epimeric monosaccharides, new irreversible oxidation
peaks were apparently observed at Epa = 0.617 V (SAM 1) and
0.641 V (SAM 2) in the initial scan with the rapid generation of
new redox couples centered at Ef (formal potentials, calculated
by taking the average of the anodic and cathodic peak potentials) =
0.333 V (SAM 1) and 0.340 V (SAM 2), respectively, only after
the second scan. With increased scans, both SAMs displayed
slightly increased peak currents, suggesting a much easier depro-
tection process comparing to that of the control SAM 3. This
demonstrates that the monosaccharide moiety closely conju-
gated with the dimethoxybenzene may accelerate the electro-
chemical deprotection process. In addition, the glucosyl SAM 1 is
comparatively easier to be deprotected compared with the
galactosyl SAM 2 as its irreversible oxidation peak could be more
facilely generated by the initial scan (Figure 3, part A-1 vs part
B-1). This could be similarly caused by the axial C4 hydroxyl
group of the galactoside that is spatially closer to the β-aglycon,
impeding the formation of the hydroxyquinone/quinone redox
couple in a certain extent.29 The peak separation between the
oxidation and reduction peaks of both SAMs 1 (Figure 3A-1) and
2 (Figure 3A-2) is small, suggesting that the electron-transfer
kinetics between the glycosyl quinone and the electrode is a fast
process. Furthermore, both redox peaks in the CVs of SAMs 1
and 2 are symmetric and broad with fwhh’s equal to 0.125 V
(SAM 1) and 0.126 V (SAM 2), respectively.
After electrochemical deprotection, the CVs of each SAM in
0.1 M H2SO4 as a function of scan rate were recorded for
characterizing their corresponding monolayer properties. The
diagrams outlined in Figure 3, parts A-3 (SAM 1), B-3 (SAM 2),
and 3C-3 (SAM 3), revealed that the currents of all redox peaks
increased while the scan rate increased with no significant shifts
in peak potentials. Furthermore, as illustrated in these diagrams,
both anodic and cathodic peak currents for hydroquinone/
quinone redox couples of each SAM scaled linearly with the
scan rates ranging from 0.020 to 1.000 V/s (Figure 3, parts A-2,3,
B-2,3, and C-2,3, only shows their CV characteristics with scan
rates from 0.020 to 0.100 V/s; for full characteristics with scan
rates from 0.020 to 1.000 V/s see Supporting Information Figure
S-1), indicating that the oxidation and reduction processes of the
quinones are surface-controlled.
To characterize the monolayer properties of each SAM, the
surface coverage (Γ, mol/cm2) was calculated by integrating the
area of the anodic and cathodic peaks. Taken into account the
electrochemical roughness factor (Rf = 1.42 ( 0.11), which was
assessed by CV between -0.1 and 1.5 V versus SCE at 100 mV/s
in freshly prepared 0.1 M H2SO4, the coverage (Γ) for SAM 1 is
assigned to 3.91(0.28) ꢀ 10-11 mol/cm2, and those for SAMs 2
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Figure 4. CVs of (A-1) SAM 1 upon addition of PNA (4 μM, red dot line) and Con A (4 μM, blue dot line) at a scan rate of 40 mV/s and (B-1) SAM 2
upon addition of Con A (4 μM, red dot line) and PNA (4 μM, blue dot line) at a scan rate of 40 mV/s. DPV response of (A-2) SAM 1 upon the addition
of various concentrations of Con A ranging from 0.1 to 10 μM and (B-2) SAM 2 upon the addition of various concentrations of PNA ranging from 0.1 to
10 μM. Monitoring the change in DPV current between background and sample as a function of lectin concentrations: (A-3) Con A (O) and PNA (0);
(B-3) Con A (0) and PNA (O).
and 3 are 4.66(0.31) ꢀ 10-11 mol/cm2 and 9.47(0.45) ꢀ 10-11
mol/cm2, respectively. These values are smaller than the
typical coverage found for the hydroquinone/quinone redox
couple withbridges containingalkane thiols (Γ = 3.2-5.7 ꢀ 10-10
mol/cm2),30 which could be caused by the existence of the bulky
benzo(hydro)quinone and sugar moieties that furnish excessive
spatial hindrance. Meanwhile, the surface coverage per molecule
on the surface of the three SAMs was assessed, which equals to
0.24 ( 0.02 nm-2 for glucosyl SAM 1, 0.28 ( 0.2 nm-2 for
galactosyl SAM 2, and 0.57 ( 0.3 nm-2 for sugar-free SAM 3.
The successively calculated footprint of one molecule on a
gold electrode for each SAM was assigned to 4.2 ( 0.3 nm2
for 1, 3.6 ( 0.2 nm2 for 2, and 1.7 ( 0.2 nm2 for 3.
both SAMs did not change upon the addition of the specific
lectin, indicating that the electrochemical kinetics was not
influenced. As it is reported that the peripheric enzymatic surface
adjacent to the CRD of natural lectins may interact with
hydrophobic groups,5 the decreased current could consequently
be ascribed to the binding of lectin to both monosaccharide and
quinonyl moieties.15 When the lectin binds simultaneously to
the sugar-quinone hybrid in the system, the corresponding
electron-transfer process of the SAM would be blocked, sequen-
tially leading to the decrease of peak current. Moreover, the very
small peak current decrease afforded by the glycosyl SAM-
nonspecific lectin mixture might be most likely caused by the
partial interaction of the quinone moiety with such lectin.
Next, for investigating the monosaccharide-quinone SAM-
lectin interactions in a more detailed way, we recorded DPVs of
SAMs 1 and 2 with increasing amounts of lectins ranging from 0
to 10 μM in 0.1 M PBS solution at pH = 7.0. Clearly, as shown in
Figure 4, parts A-2 and B-2, upon the addition of gradually
increased specific lectin, i.e., Con A to SAM 1 and PNA to SAM 2,
the corresponding peak current gradually decreased with no
obvious peak shift or variation in breadth. This further demon-
strates that the decrease of peak current of both SAMs is
concentration-dependent. Eventually, while the highest concen-
tration (10 μM) of Con A was added to SAM 1, the correspond-
ing peak current decreased by 68%, whereas the addition of the
same amount of PNA to SAM 2 led to the decrease of peak
current by 40%. As shown in Figure 4, parts A-3 (1) and B-3 (2),
the percentage of decreased current Id=(I0 - Is)/I0 was subse-
quently plotted versus the lectin concentrations ranging from 0.1
to 10 μM where the Id is fractional decrease in current, I0 is the
current with no added lectin, and Is is the current with added
lectin. The resulting linear relationship represents the specific
monosaccharide-lectin interactions, i.e., Con A to SAM 1 (r2 =
0.9984) and PNA to SAM 2 (r2 = 0.9990), whereas the addition
of nonspecific lectins to the same SAMs leads to very minor
Indeed, the electrochemical approach fulfilled in the present
case has overcome the synthetic inconvenience and greatly
consumed the excessive purification effort, directly and facilely
providing the desired electroactive monosaccharide-quinone
SAMs for further study.
Electrochemical Probing of Specific Carbohydrate-Lectin
Interactions. With the accomplished electrochemical monosac-
charide-quinone SAMs, we sought to attempt preliminarily
its applicability toward the detection of specific epimeric carbo-
hydrate-protein interactions. Naturally occurring Con A and
PNA that are known to possess, respectively, specific glucose and
galactose recognition domains were used for this study.22
The CVs of monosaccharide-quinone SAMs 1 and 2 in the
absence (in black) or in the presence (in red or blue) of lectins
were first detected, shown in Figure 4. As exhibited in both parts
A-1 and B-1 of Figure 4, when a lectin that bears the specific CRD
was added to the corresponding SAM, i.e., Con A to the glucosyl
SAM 1 and PNA to the galactosyl SAM 2, the resulting peak
current decreased remarkably (in blue), whereas minor changes
in peak current (in red) were observed while the lectins were
added in a reverse fashion, i.e., PNA to 1 and Con A to 2.
Interestingly, the peak potentials as well as the peak breadth of
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’ CONCLUSIONS
In summary, we have successfully realized in this study the
fabrication of novel gold surface-coated epimeric monosaccharide-
quinone SAMs by taking advantage of simple synthetic progres-
sions of our previously synthesized aryl C-glycosides in conjunc-
tion with the subsequent SAM technique and electrochemical
deprotection. Their epimeric identity was concomitantly
reflected in both the XPS spectra and the electrochemical
deprotection process. The afforded electroactive SAMs contain-
ing monosaccharide epimers were then demonstrated efficacious
toward the sensitive probing of specific epimeric sugar-lectin
interactions (i.e., glucose-Con A, galactose-PNA) with weaker
or very minor responses to the addition of a panel of less specific
and nonspecific lectins via CV or DPV.
Indeed, since there develops increasing interest as well as
urgent need in the elaboration of carbohydrate-related biological
and pathological processes by probing the specific carbohydrate-
receptor recognitions, our newly fabricated sugar-quinone
SAMs might be desirable as an ingenious probe toward such
studies with the quick and sensitive electrochemistry. More-
over, on the basis of the presently established systematic
method for constructing surface-attached sugar-quinone
hybrids, numerous other electrochemically active sugar SAMs
containing diverse biologically important carbohydrate entities
could be developed and collected to further fabricate sugar
microarrays18 for efficiently monitoring specific carbohydrate-
receptor interactions that are naturally involved in the intricate
cellular processes, in the near future. This unique electroche-
mical platform would presumably facilitate the better under-
standing of the “glycomics” as well as the development of early-
state disease diagnosis.
Figure 5. Percentages (%) of peak current decrease of (A) SAM 1 and
(B) SAM 2 upon addition of 7 μM of various specific or nonspecific
lectins where I0 is the current with no added lectin and Is is the current
with added lectin.
changes in Id value with increasing concentrations. The detection
limit of both SAMs was calculated to be 75 nM (S/σb1 = 3, where
σb1 is the standard deviation of the peak current obtained in the
absence of lectin).
’ ASSOCIATED CONTENT
Additionally, in an attempt to further ascertain their biospe-
cificity, a panel of nonspecific lectins that are known to recognize
specifically other natural monosaccharide subunits including
PSA (mannose-specific), SBA (N-acetyl-galactosamine-specific),
UEA-I (fucose-specific), WGA (N-acetyl-glucosamine- and sialic
acid-specific), DSA (N-acetyl-glucosamine-specific), and SNA
(sialic acid-specific) was assayed on both glycosyl SAMs 1 and 2
via DPV. To our delight, with the addition of 7 μM Con A and
PNA to, respectively, the corresponding SAMs 1 and 2, remark-
able peak current decreases (44% for SAM 1 and 37% for SAM 2)
were similarly observed, shown in Figure 5 and Supporting
Information Figure S-2 (complete DPV plots of SAMs 1 and 2
upon addition of various lectins). In stark contrast, while the
selected nonspecific lectins such as PSA, UEA-1, WGA, DSA, and
SBA (to SAM 1 only) were added at the same concentration
(7 μM) to the SAMs, very minor peak current changes were
displayed, with the addition of SBA to SAM 2 as an exception.
Notably, it is known that SBA binds more specifically to N-acetyl-
D-galactosamine, whereas the galactose unit may also be recog-
nized by its CRD, however, with weaker binding affinity. Hence,
we would ascribe the relatively less significant peak current
decrease (24%) of the galactosyl SAM 2 in the presence of
SBA than that (37%) in the presence of PNA to its comparatively
lesser binding specificity to the former than to the latter.
These data have positively established our uniquely con-
structed sugar-quinone SAMs containing natural monosacchar-
ide epimers applicable toward the accurate probing of specific
sugar-protein interactions via electrochemistry.
S
Supporting Information. Figures S-1 and S-2, original
b
NMR spectra of compounds 1, 2, 4, and 5, and the HR-ESI-MS
spectrum of compound 2. This material is available free of charge
via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
ytlong@ecust.edu.cn
’ ACKNOWLEDGMENT
We thank the National Natural Science Foundation of China
(Grant No. 20876045, 91027035), Ministry of Health (Grant
2009 ZX 10004-301), Shanghai Science and Technology Com-
munity (No. 10410702700), and the Fundamental Research
Funds for the Central Universities (No. WK1013002). Y.-T.
Long is supported by the Program for Professor of Special
Appointment (Eastern Scholar) at Shanghai Institutions of
Higher Learning. X.-P. He also gratefully acknowledges the
French Embassy in China for a doctorate fellowship and his
French advisor Professor Juan Xie in ENS Cachan.
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