Inhibition binding studies of glycodendrimer/lectin interactions using surface plasmon resonance

Article abstract of DOI:10.1016/j.tet.2010.05.038

Understanding protein/carbohydrate interactions is essential for elucidating biological pathways and cellular mechanisms but is often difficult due to the prevalence of multivalent interactions.Here, we evaluate the multivalent glycodendrimer framework as a means to describe the inhibition potency of multivalent mannose-functionalized dendrimers using surface plasmon resonance (SPR).Using highly robust, mannose-functionalized dithiol self-assembled monolayers on gold surfaces, we found that glycodendrimers were efficient inhibitors of protein/carbohydrate interactions.IC₅₀ values ranging from 260 nM to 13 nM were obtained for mannose-functionalized dendrimers with Concanavalin A.

Full text of DOI:10.1016/j.tet.2010.05.038

Tetrahedron 66 (2010) 5305e5310
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Inhibition binding studies of glycodendrimer/lectin interactions using
surface plasmon resonance
*
Kristian H. Schlick, Mary J. Cloninger
Department of Chemistry and Biochemistry, 103 Chemistry and Biochemistry Building, Montana State University, Bozeman, MT 59717, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Understanding protein/carbohydrate interactions is essential for elucidating biological pathways and
cellular mechanisms but is often difficult due to the prevalence of multivalent interactions. Here, we
evaluate the multivalent glycodendrimer framework as a means to describe the inhibition potency of
multivalent mannose-functionalized dendrimers using surface plasmon resonance (SPR). Using highly
robust, mannose-functionalized dithiol self-assembled monolayers on gold surfaces, we found that
glycodendrimers were efficient inhibitors of protein/carbohydrate interactions. IC50 values ranging from
260 nM to 13 nM were obtained for mannose-functionalized dendrimers with Concanavalin A.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 25 February 2010
Received in revised form 11 May 2010
Accepted 11 May 2010
Available online 20 May 2010
Keywords:
Glycodendrimers
Dendrimers
Surface plasmon resonance
Inhibition binding assay
Multivalency
1. Introduction
While many methods require the use of tags, such as radiolabels, SPR
eliminates the need for labeled reagents. In addition, when quali-
Protein/carbohydrate interactions are the initiating event in
a myriad of intercellular recognition events including cancer cell ag-
gregation, the metastatic spread of cancer, bacterial invasion, and
fungal and viral adhesion.1e4 Because monovalent carbohydrate/lec-
tin interactions are generally weak, nature often invokes multivalent
and cross-linking processes to increase the overall avidity and spec-
ificity of the association.5e8 The multivalent nature of the interaction
often leads to cross-linking and aggregate formation. Agglutination of
red blood cells, for example, is caused by lectin-red blood cell cross-
linking and is the basis of the hemagglutination inhibition assay.9
Traditionally, hemagglutination and precipitation techniques
have been used to determine relative interactions occurring between
proteins and carbohydrates.9,10 However, these assays are limited in
that they do not provide either thermodynamic or kinetic data about
the protein/carbohydrate interactions. Isothermal titration micro-
calorimetry (ITC)11 directly measures thermodynamic binding pa-
rameters but requires large amounts of both the ligand and the
receptor to obtain good signal. The requisite conditions of ITC have
the potential to promote aggregation conditions, especially when
using large, highly multivalent scaffolds, such as, dendrimers.
Surface plasmon resonance (SPR) technology provides a valuable
technique for accurately analyzing protein/carbohydrate in-
teractions. This method has gained a great deal of interest in recent
years, and has been used to analyze many ligand/ligate complexes.12
tative binding comparisons across a series of compounds are
needed, the need for extensive purification protocols is reduced
when specific ligand/receptor binding is involved. The ligand of in-
terest is passed through the system at a constant flowrate and non-
participating compounds are quickly washed away. SPR allows one
to monitor complex formations occurring in real time, offering in-
sights into the kinetics and mechanism of a reaction. The assay is
sensitive enough that lowaffinity interactions can be detected, since
measurements can be made in the presence of excess, unbound
protein. This is especially useful in protein/carbohydrate studies,
where the interactions are known to be weak.
The use of SPR to elucidate binding events has seen a significant
increase in recent years. Several noteworthy examples of the appli-
cation of SPR to the study of protein/carbohydrate interactions have
been reported; summaries of a few studies with direct relevance to
this work follow. Keusgen and co-workers, for example, recently
reported a lectin screen using immobilized oligosaccharides in SPR.
The goal of this work was to devise a system that would ultimately
allow for the identification of new lectins from natural sources.13
Gabius and co-workers immobilized glycoproteins for sensitive de-
tection of galectin-1 and Ricinus communis agglutinin in solutions
containing mixtures of proteins,14 and Nishimura and co-workers
used SPR to evaluate how effectively sialyl Lewis X functionalized b-
cyclodextrins inhibited the binding of E-selectin to a SLeXn-BSA
functionalized sensor chip.15 Suda and co-workers recentlycompared
the binding of model heparin-binding proteins to immobilized
monosaccharides and clusters of carbohydrates. They evaluated the
* Corresponding author. Tel.: þ1 406 994 3051; fax: þ1 406 994 5407; e-mail
address: mcloninger@chemistry.montana.edu (M.J. Cloninger).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.05.038

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K.H. Schlick, M.J. Cloninger / Tetrahedron 66 (2010) 5305e5310
effectofliganddensityandligandclusteringbymeasuringthebinding
of a binding domain of vWf tothe SPR chip. They found that clusters of
oligosaccharides were more effective ligands than monosaccharides,
even when comparable (per carbohydrate) surface loadings were
present.16 In addition to using SPR to compare loading densities,
Fieschi and co-workers used mannose-functionalized Boltorn
hyperbranched dendrimers to inhibit the binding of DC-SIGN to
a gp120-functionalized surface.17 Kiessling and co-workers used SPR
with mannose-functionalized surfaces to determine the concentra-
tions of neoglycopolymers that inhibited the binding of Concanavalin
A (Con A) to the surface. The neoglycopolymers varied in length, and
the most potent compounds were those that could bind to multiple
Con A binding sites as well as cross-link the Con A lectins.18 Riguera
and co-workers compared SPR results for mannose-functionalized
dendrimers binding to surface bound Con Awith the inhibition of Con
Abindingtomannose-functionalizedgoldsurfaces.19 Schengrundand
co-workers used SPR to study the binding of HIV-1 gp120 to potential
glycodendrimer inhibitors. They found that binding affinities of the
glycodendrimers with rgp120 (as measured by SPR) and the ability of
the glycodendrimers to inhibit HIV infectivity (as measured by viral
inhibition assays) correlated well for sulfated glycodendrimers.20
Imberty, Matthews, Vidal and co-workers used SPR inhibition assays
to test the potential of galactose-functionalized calyx[4]arenes to
block adhesion of PA-IL to galactosylated surfaces.21 These examples
indicate that applying SPR technology to the investigation of protein/
carbohydrate interactions shows great promise.
to spontaneously desorb from gold surfaces under ambient con-
ditions, resulting in patchy surfaces with significant areas of metal
exposed.26 The stability of the surface can be significantly im-
proved through the use of multivalent dithiol compounds.27e29
The carboxyl-terminated dithiol compounds were coupled to
amine-functionalized mannose derivatives30 through amide bond
formation. This allowed for a stable mannose surface to be pre-
sented on the gold chip, which could be reused for multiple assays
(Fig. 1).
2.2. Surface plasmon resonance assays
Concanavalin A is tetrameric plant lectin that is able to specifi-
cally bind to mannose residues.31 Serial dilutions of Con A were
injected, and the SPR response was monitored to determine the
affinity of Con A for the mannose-functionalized surface. The ob-
served KD for the binding of Con A to the mannose-functionalized
surface was estimated at 78 nM (Fig. 2).
Because many lectins of current interest for biological processes
are not surface bound, robust biosensors for measurement of
multivalent binding interactions in the solution phase are needed.
We determined that dithiol compounds are an ideal system with
which to build a self-assembled monolayer on a gold surface. The
dithiols form a bivalent attachment to the gold that is robust
enough to undergo many inhibition/binding measurements. Here,
we describe the effectiveness of different generations of mannose-
functionalized dendrimers to inhibit binding of Con A to a man-
nose-functionalized surface through SPR.
2. Results
2.1. Surface functionalization with carbohydrates
We created a self-assembled monolayer (SAM)22,23 of dithiols
on an unmodified gold surface.24,25 Alkanemonothiols are known
Figure 2. Top: doubly-referenced sensorgram of Con A binding to the mannose-
functionalized surface. Bottom: affinity profile of Con A to the mannose-functionalized
surface, fit using Scrubber 2.
The glycodendrimers that were used in this study are shown in
Figure 3a, and the biosensor strategy is shown in Figure 3b. The
values for the number of mannosides were determined from MW
(MALDI-TOF MS) and are the average number of mannosides per
dendrimer. For a discussion of the homogeneity of the dendrimers,
see Ref. 32. The affinity of the glycodendrimers for Con A was
evaluated by studying their ability to inhibit binding of Con A to the
mannose-functionalized surface. Serial dilutions of the inhibitor
were pre-mixed with a constant amount of Con A, and the resulting
equilibrium responses were used to determine IC50 values (Figs. 4
and 5). A key point is that glycodendrimer/lectin binding occurs
in solution rather than at the monolayer surface, better mimicking
many protein/carbohydrate interactions.
Figure 1. A schematic representation of mannose functionalization of a gold surface.

K.H. Schlick, M.J. Cloninger / Tetrahedron 66 (2010) 5305e5310
5307
used in SPR assays, competition experiments involving mannose-
functionalized dendrimers and Con A proved to be problematic
with this chip. The change in instrument response elicited by the
addition of mannose-functionalized dendrimer was small com-
pared to the binding of Con A to the dextran matrix in the absence
of dendrimer, even though Con A has a 3- to 4 fold higher affinity
for mannose than for glucose.35 The minimal response change
likely occurred because of the large excess of glucose residues on
the dextran-coated gold surface relative to the number of mannose
residues on the glycodendrimers.
Furthermore, using a dextran surface with large multivalent
glycodendrimers may induce sugar/sugar binding events, compli-
cating the analysis of the specifically targeted binding events. The
mannose-functionalized monolayers used for this research were
extremely robust because of the use of dithiols. The same chip was
used repeatedly for at least six months with high precision in the
acquisition of the data.
Figure 3. (a) Mannose-functionalized poly(amidoamine) (PAMAM) dendrimers. (b) A
schematic representation of the inhibition binding experiment.
3.2. Surface plasmon resonance assays
SPR assays in which Con A was bound to a mannose-function-
alized gold surface indicated that the observed KD for the binding of
Con A to the mannose-functionalized surface was 78 nM. This value
indicates a significantly stronger interaction compared to the
reported KA of 7.6ꢀ103 Mꢁ1 (KD of 130
mM) obtained for mono-
valent methyl-mannose from microcalorimetry.36 This disparity
almost certainly occurs because Con A is able to bind multiple
surface sugars, increasing the overall affinity of Con A for the self-
assembled monolayer.5
The gold surface used was functionalized with a rather high
percentage (25%) of a carboxyl-terminated dithiol compound that
was subsequently coupled to a tethered mannose derivative. This
places the mannosides close enough to undergo bivalent binding
with the tetrameric Con A, preventing an accurate determination of
monovalent association and dissociation rate constants but allow-
ing equilibrium constants of highly multivalent systems to be de-
termined. The affinity profile for Con A, shown in Figure 2, does not
significantly fit a 1:1 kinetic binding model (data not shown).
Heterogeneous binding, which is a more effective mimic of relevant
biological surfaces is suggested and allows us to study the ability of
the dendrimers to inhibit multivalent binding modes to a surface.
Because we compared the inhibitory propensity of different gen-
erations of glycodendrimers for Con A binding to the monolayer,
determination of the exact degree of mannose incorporation into
the monolayer was unnecessary. Although other researchers have
used attenuated total reflection FT/IR spectroscopy to determine
the degree of SPR chip functionalization,16 this was not required for
our studies because the same chip was used in all of the experi-
ments, eliminating variability due to different chips.
Figure 4. Inhibition of 2 mM Con A injected over the mannose-functionalized gold
surface, normalized to 100% response from Con A without addition of inhibitor. Top:
sensorgram of Con A binding to the mannose-functionalized surface, inhibited by
mannose-functionalized G4 dendrimer. Bottom: inhibition by mannose-functionalized
G4 dendrimer. Dotted lines show 95% confidence levels, fit using GraphPad Prism 4.
While the results obtained by SPR show that the glycoden-
drimers exhibit a definite advantage over the monovalent sugar,
the increased activity observed with higher generations of den-
drimers in hemagglutination inhibition assays with Con A is not
evident in SPR assays. In fact, the mannose-functionalized den-
drimers showed only small affinity differences (on a per mannose
basis), compared to remarkable activity increases observed in
hemagglutination assays.
In contrast to the hemagglutination assay, SPR offers a method
to more directly measure the interactions occurring between Con A
and the sugar. In the hemagglutination inhibition assay, red blood
cells are added as sources of surface sugars, which are bound by
Con A. Inhibition of this interaction by glycodendrimers provides an
entry-level comparison of our system to known systems, although
the assay is not able to provide information regarding binding af-
finity. Rather, hemagglutination indicates the ability of
Whereas a marked increase in activity with increasing glyco-
dendrimer generation was previously observed by hemagglutina-
tion assays,33,34 the IC50 values obtained by SPR suggest that the
ability to inhibit Con A binding to the surface is largely independent
of dendrimer generation. While the glycodendrimers offer a defi-
nite advantage over methyl-mannose, higher generation den-
drimers exhibit similar abilities to inhibit binding when compared
to lower generations. Binding data are summarized in Table 1.
3. Discussion
3.1. Surface functionalization with carbohydrates
Although a commercially available gold surface coated with
dextran, a polymer composed of glucose residues, is commonly

5308
K.H. Schlick, M.J. Cloninger / Tetrahedron 66 (2010) 5305e5310
Figure 5. Inhibition by mannose-functionalized PAMAM dendrimers of 2 mM Con A injected over the mannose-functionalized gold surface, normalized to 100% response from Con
A without addition of inhibitor. Dotted lines show 95% confidence levels, fit using GraphPad Prism 4. (a) Mannose-functionalized G(2)-PAMAM, (b) mannose-functionalized G(3)-
PAMAM, (c) mannose-functionalized G(4)-PAMAM, (d) mannose-functionalized G(5)-PAMAM, (e) methyl-mannoside.
Table 1
sugar interactions is prominently displayed at high concentrations
Comparison of hemagglutination inhibition assays with SPR results for mannose-
of methyl-mannose (Fig. 5e). At sufficiently high concentrations of
functionalized dendrimers with Concanavalin A
methyl-mannose, Con A is prevented from binding to the surface.
Hemagglutination inhibition
assay resultsa,b
SPR inhibition assay resultsa
Even higher concentrations of methyl-mannose, however, afford an
increased response through nonspecific sugar/sugar interactions.
This suggests significant sugar/sugar interactions; a pre-organiza-
tion step between sugars may be a factor in cellular recognition, as
opposed to relying solely on the specificity of a protein for a sugar
residue.39e41
Because of the heterogeneous nature of the binding events,
kinetic data cannot be accurately determined from this experiment.
Several binding events are occurring, including multivalent binding
motifs, which make it difficult to deconvolute individual binding
events from the overall system. However, information can be
obtained from equilibrium data, particularly about the efficiency of
the tested inhibitors. Examining the response as a function of added
glycodendrimer reveals the efficiency at which the glycoden-
drimers are able to inhibit Con A from binding to the surface of the
self-assembled monolayer.
Dendrimer generation
(number of sugars)
Relative activity/
mannose (MIC)c
1 (6400 M)
w1 (9700 M)
IC50 values/
dendrimer)d
IC50 values/
mannoside)d
Methyl mannose (1)
G2 (16)
G3 (29)
G4 (55)
G5 (95)
m
240 mM
m
260 nM
130 nM
63 nM
13 nM
4.2
3.8
3.5
1.2
mM
20 (280
200 (30
300 (20
m
m
m
M)
M)
M)
m
m
m
M
M
M
a
Each reported value represents at least three assays.
Values are taken from Ref. 33 (G2) and 34 (G3eG5).
Minimum Inhibitory Concentration per mannoside for 0.7
b
c
m
M Con A.
d
2 mM Con A.
a glycodendrimer to drive aggregation processes, and not the
strength of protein/carbohydrate binding.37,38 As a result, SPR
provides a more accurate measurement of protein/carbohydrate
affinity, independent of aggregation and precipitation events.
Although the mannose-functionalized surface is successfully
bound by Con A, additional binding events can be observed in the
inhibition sensorgrams. Non-specific binding arising from sugar/
Of particular interest is the increased steepness of the curve when
the inhibition data are fit to the four-parameter logistic equation
(Fig. 4, bottom). Whereas, inhibition of the Con A-surface sugar in-
teraction by methyl-mannose yields a rather shallow slope, inhibition

K.H. Schlick, M.J. Cloninger / Tetrahedron 66 (2010) 5305e5310
5309
by glycodendrimers yield markedly steeper slopes. By contrast, in-
5.1.1. Preparation of 2-(2-aminoethoxy)ethyl-
a-
D-mannopyrano-
hibitionbygalactose-functionalizeddendrimers couldnotbefittothe
equation and yielded similar responses throughout the dilution series
(data not shown). This suggests that addition of mannose-function-
alized dendrimer to the system induces a pronounced effect on Con A
binding, and that responses from the assay are more sensitive to
changes in dendrimer concentration than in methyl-mannose con-
centration. As the amount of glycodendrimer, that is, added is in-
creased, Con A sequestration (such that less Con A is available to be
bound to the self-assembled monolayer) is enhanced (Fig. 5). This is
indicative of the dendrimers’ ability to effectively bind Con A and to
isolate bound Con A from a sugar-functionalized surface, as expressed
by IC50 values (Fig. 5 and Table 1).
side. 2-(2-Azidoethoxy)ethyl-2,3,4,6-tetraacetyl-a-D-mannopyrano-
side (330 mg, 0.74 mmol) was dissolved in 10 mL of MeOH and
reduced over Pd/C and an excess of H2. The catalyst was removed and
the resulting mixture was filtered through a 0.45
mm filter. Removalof
the solvent invacuo yielded 217 mg (79% yield) of 2-(2-aminoethoxy)
ethyl-2,3,4,6-tetraacetyl-a-D-mannopyranoside, which was used
directly.
2-(2-Aminoethoxy)ethyl-2,3,4,6-tetraacetyl-
side (217 mg) was dissolved in 10 mL of 1:1 MeOH/H2O. The acetyl
groups were then removed with 1 M NaOMe in MeOH (522 L,
522 mol), stirring overnight. The solvent was removed under vac-
a-D-mannopyrano-
m
m
uum. The resulting sugar and acetic acid salts were dissolved in
Millipore water and were used without further purification.1H NMR
(DMSO-d6, 500 MHz, solvent peaks were visible but not recorded)
4. Conclusion
d
3.2e3.7 (m, 21H).13C NMR(DMSO-d6,125 MHz, solvent peaks were
visible but not recorded) d 30.1, 40.5, 61.7, 66.1, 67.5, 69.8, 70.7, 71.4,
Highly robust mannose-functionalized SAMs for inhibition
binding studies using SPR were formed on gold surfaces using
dithiols. SPR experiments with these dithiol SAMs demonstrated
that glycodendrimers efficiently inhibit protein/carbohydrate in-
teractions; the potencies reported here for glycodendrimers are
considerably higher than those obtained for monovalent mannose
(on a per mannose basis). However, the generation of the glyco-
dendrimer had less of an impact in these SPR studies than was
observed in previously reported hemagglutination inhibition as-
says.33,34 That the amount of inhibitor needed to achieve complete
inhibition of red blood cell aggregation is decidedly different from
the IC50 values reported here for SPR assays is readily rationalized
because of fundamental differences in what the assays measure. In
a hemagglutination inhibition assay, glycodendrimers probably
attenuate aggregation events between Con A and red blood cells by
efficiently sequestering the Con A lectins from the red blood cells. In
assays that do not rely on disruption of aggregation events, den-
drimers exhibit solely sugar/protein interactions, with less pro-
nounced increases in affinity due to glycoside clustering
effects.36e38 The SPR competition assays reported here reaffirm
that affinity enhancements for multivalent ligands are present.
These SPR competition assays are an effective tool for studying
solution phase binding events.
74.4, 100.4. These results agree well with previously published data
for this compound, which was synthesized via a slightly different
route and for which NMR data are reported in D2O.30
5.1.2. Preparation of carboxylated gold surface. 25% of a eCOOH
terminated aromatic dialkanethiol (SPT-0014, SensoPath Technol-
ogies) was mixed with 75% of a eOH terminated aromatic dia-
lkanedithiol (SPT-0013, SensoPath Technologies) to provide a 1 mM
dithiol solution in ethanol. A 1:10 dilution of this solution (v/v) in
Millipore water afforded a 0.1 mM solution that was injected over
a single flowcell area (flowcell 2) of a new gold chip several times at
1 mL/min for 60 min using Millipore water as running solution, until
no appreciable signal increase was observed. This procedure was
repeated using 100% eOH terminated aromatic dialkanethiol (SPT-
0013) over flowcell 1 on the same gold chip for a reference cell.
5.1.3. Preparation of mannose-functionalized gold surface. The car-
boxylated gold surface was activated by injection of a mixture of
N-ethyl-N0-(diethylaminopropyl)-carbodiimide (EDC) and N-
hydroxysuccinamide (NHS) (30
min) dispensed by the Biacore instrument. 2-(2-Aminoethoxy)
ethyl- -mannopyranoside (10 mg/mL in 10 mM PBS, pH 7.2, in-
mL, 200 mM EDC, 50 mM NHS, 5 mL/
a-D
cludes acetic acid salts from the deprotection step) was injected
into flowcell 2 (12 min contact time) and allowed to react. This
procedure was repeated three times to allow for maximum func-
tionalization. Unreacted activated carboxyl groups were given ex-
tensive time to hydrolyze under running buffer; remaining
activated carboxyl groups were then capped by injection of etha-
Glycodendrimers are very potent frameworks for mediation of
protein/carbohydrate interactions. They are capable of both form-
ing and disrupting extensive cross-links. When designing thera-
peutic agents for human health, our results suggest that different
multimeric glycosides can exhibit similar activities for inhibiting
protein/carbohydrate interactions, but decidedly different activities
for interacting with large existing complexes are likely.
nolamine (20 mL, 1 mM pH 8.5).
5.1.4. Surface plasmon resonance. Concanavalin A (Con A) (100
in 10 mM PBS, pH 7.4) was injected over the mannose-functional-
ized surface at 10 L/min, allowing 300 s for dissociation, followed
by regeneration (5 L, 10 mg/mL methyl-mannose in 0.1 M HCl).
The titration range covered 10 Me31 pM by 2-fold dilutions. KD
was estimated using Scrubber 2.0.
For competition experiments with inhibitors, Con A (2
mL
5. Experimental section
5.1. General methods
m
m
m
Mannose-functionalized dendrimers were synthesized as pre-
viously described in Ref. 33. Solutions and running buffers were
degassed before use in SPR experiments. Unless otherwise noted,
10 mM PBS, pH 7.4 was used as running buffer for all SPR
experiments.
SPR experiments were performed using a Biacore 1000 upgrade
equipped with a Sensor Chip Au (Biacore AB, Uppsala, Sweden). The
instrument temperature was set to 25 ꢂC. Dialkanethiols (SPT-0013
and SPT-0014) were purchased from SensoPath Technologies
(Bozeman, MT) for functionalization of the gold surface. Conca-
navalin A was purchased from CalBioChem (Darmstadt, Germany).
All other chemical reagents were purchased from Fisher Scientific
(Waltham, MA).
mM
monomer) was mixed with an equal volume of inhibitor and the
solution was allowed to equilibrate for at least 1 h. The mixture
(70
for dissociation, followed by regeneration (5
mannose in 0.1 M HCl). The glycodendrimers were tested in trip-
licate, covering a range of 50 g/mLe39 ng/mL by 2-fold dilutions.
mL) was injected over the surface at 10
mL/min, allowing 300 s
mL, 10 mg/mL methyl-
m
For competition experiments with methyl-mannose, the assay
covered a range of 10 mg/mLe305 ng/mL by 2-fold serial dilutions.
Equilibrium responses were determined using Scrubber 2.0, then
analyzed in GraphPad Prism (version 4.0) using the four-parameter
logistic equation to determine IC50 values.

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K.H. Schlick, M.J. Cloninger / Tetrahedron 66 (2010) 5305e5310
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Binding responses were obtained using the aforementioned
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S.; Sobel, M. Bioconjugate Chem. 2006, 17, 1125e1135.
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conditions across a mannose-functionalized surface. The binding
responses were referenced by subtracting the response generated
from identical injection conditions over a flowcell containing only
eOH terminated dialkanethiols (flowcell 1), and double-referenced
by subtracting a response from buffer injections.
18. Mann, D. A.; Kanai, M.; Maly, D. J.; Kiessling, L. L. J. Am. Chem. Soc. 1998, 120,
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Acknowledgements
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Supplementary data
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