multivalent interaction.6 Carbohydrate SAMs offer extensive
control over the ligands presenting pattern, density, and orienta-
tion, which are beneficial to elicit a clear structure-activity
relationship of these multivalent interactions. More importantly,
SAMs have been well applied to surface-based real-time, label-
free analysis methods such as surface plasmon resonance (SPR)
and quartz crystal microbalance (QCM). The combination of SAM
and SPR has been quite effective in elucidating the binding pattern,
affinity (either weak or strong), and specificity of lectins.
quantitatively with high fidelity. The literature shows that this
reaction has been applied successfully in the capture of saccha-
rides bound noncovalently or covalently onto microtiter plates.16
Collman and co-workers made azide-terminated SAMs on a gold
surface and attached oligodeoxyribonucleotide with reactive
acetylene groups to the SAMs.17 The result was a well-defined
structure of an oligonucleotide probe. Wong et al. also used this
click reaction to make oligosaccharide arrays for the screening
of complex carbohydrates.18 Consequently, we explored in this
report Cu(I)-catalyzed Huisgen 1, 3-dipolar cycloaddition reaction,
as a general strategy for fabricating sugar-SAMs. In particular,
we designed an activated alkyne linker on the gold surface that
could be readily coupled with azido sugars through Cu(I)-catalyzed
Huisgen 1,3-dipolar cycloaddition reaction. This cycloaddition
system is best for glycoarray fabrication since azido sugars are
readily accessible and have been used extensively in understand-
ing cellular processes through chemoselective ligation reaction
between an azide and a functionalized phosphine, termed the
Staudinger ligation by Bertozzi and others.19 This strategy
significantly reduces the synthetic labor for carbohydrate thiolates
and promises rapid and flexible construction of arrays of carbo-
hydrate SAMs for elucidating carbohydrate functions in biological
systems.20-22 The specificity of these carbohydrate SAMs and the
generality of this reaction were studied by analyzing the specific
binding of proteins to mannose (monosaccharide), lactose (di-
saccharide), and R-Gal (trisaccharide) SAMs.
Among several classes of SAMs,7 self-assembled monolayers
of alkanethiolates on gold currently hold the best model system8
since these monolayers form spontaneously by adsorption of
alkanethiols from their solutions onto clean gold surfaces. Two
general strategies have been used for making sugar SAMs on
gold. The first is based on the separate synthesis of the sugar
derivatives with a pendant alkanethiol group and subsequent
formation of SAMs (Figure 1a). The drawback of this strategy
comes from the tremendous synthetic effort for sugars anchored
with a pendant alkanethiol. Furthermore, as the complexity of
tethered sugars increases, there is no guarantee that the mol-
ecules will pack to form a structurally well-defined monolayer.4b
The second strategy is based on the direct chemical transfor-
mation of the functionalized SAMs (Figure 1b). This method
introduces sugar moieties onto the preformed functionalized
SAMs using selected chemical reactions.9 Obviously, the identi-
fication of a good reaction is the critical determinant for fabrication
of sugar SAMs. Previously, several interphase reactions have been
identified for anchoring sugar units, including Diels-Alder reac-
tion,10 thiol addition to activated maleimide,11 and disulfide
exchange.12 Here we describe using a “click chemistry” method
to fabricate sugar SAMs on gold substrate. By defining click, the
reaction should tolerate a variety of conditions and functionalities
and occur quantitatively with high fidelity. Those requirements
are characteristic of click chemistry, as defined by Sharpless.13
Since the properties of the monolayer depend on the terminal
functional group of the precursor alkanethiol, virtually any surface
can be prepared using well-developed click chemistry.
EXPERIMENTAL SECTION
Chemicals and Materials. Reagents were obtained from
commercial suppliers and used without further purification. All
glassware and syringes were dried in an oven overnight, allowed
to cool, and stored under a positive pressure of argon before use.
Dichloromethane was distilled and dried with CaH2. Compounds
were purified by flash chromatography (FC) on silica gel. Thin-
layer chromatography was run on SiO2 60F254 (Merck) and
visualized with UV, H2SO4, and KMnO4 reagents. 1H (250 and 500
MHz) and 13C (62.5 and 125 MHz) NMR spectra were measured
using Bruker NMR instrument. Concanavalin A (Con A) and
Erythrina cristagalli lectins (ECLs) were purchased from Sigma.
Anti-Gal antibody was purified by R-Gal affinity column from
human blood serum. All remaining materials for biological assays
were purchased from Sigma.
Among identified click reactions, i.e., cycloaddition, ring
opening to nonaldol type carbonyl chemistry, and oxidation
addition,14 the Cu(I)-catalyzed heterocycloaddition of terminal
azide to terminal alkyne has recently gained more interest since
this click reaction may take place in aqueous media.15 This reaction
also tolerates a variety of conditions and functionalities and occurs
N,N′-(Dithiodidecane-10,1-diyl)bispropiolamide (NDDA).
To a solution of 10,10′-dithiobisdecan-1-amine (0.9 g, 2.39 mmol)
and propiolic acid (0.4 g, 5.74 mmol) in dichloromethane (30 mL),
dicyclohexylcarbodiimide (1.3 g, 6.31 mmol) was added portion-
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K.; Vijayamohanan, K. Biosens. Bioelectron. 2002, 17, 1.
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1998, 120, 10575. (b) Vogel, J.; Bendas, G.; Bakowsky, U.; Hummel, G.;
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2002 Analytical Chemistry, Vol. 78, No. 6, March 15, 2006