Synthesis of sugar arrays in microtiter plate

Article abstract of DOI:10.1021/ja020887u

1,3-Dipolar cycloadditions between azides and alkynes were exploited to attach oligosaccharides to a C₁₄ hydrocarbon chain that noncovalently binds to the microtiter well surface. Synthesis of sugar arrays was performed on a micromolar scale in situ in the microtiter plate. As a model study, the β-galactosyllipid 5 was displayed on a 4-μmol scale. Formation of product was confirmed via ESI-MS, and the yield was determined via chemical and biological assays. Several complex carbohydrates (6-16) were also displayed in microtiter plates and successfully screened with various lectins. Moreover, sialyl Lewis x (17) was synthesized via the enzymatic fucosylation of a precursor displayed in the plate. Studies on inhibition of this biotransformation have been carried out, and the IC₅₀ value found for the known inhibitor 20 was consistent with previous studies in solution.

Full text of DOI:10.1021/ja020887u

Published on Web 11/14/2002
Synthesis of Sugar Arrays in Microtiter Plate
Fabio Fazio,† Marian C. Bryan,† Ola Blixt,‡ James C. Paulson,‡ and
Chi-Huey Wong*,†
Contribution from the Department of Chemistry and Skaggs Institute for Chemical Biology, and
Department of Molecular Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037
Received June 27, 2002
Abstract: 1,3-Dipolar cycloadditions between azides and alkynes were exploited to attach oligosaccharides
to a C14 hydrocarbon chain that noncovalently binds to the microtiter well surface. Synthesis of sugar arrays
was performed on a micromolar scale in situ in the microtiter plate. As a model study, the â-galactosyllipid
5 was displayed on a 4-µmol scale. Formation of product was confirmed via ESI-MS, and the yield was
determined via chemical and biological assays. Several complex carbohydrates (6-16) were also displayed
in microtiter plates and successfully screened with various lectins. Moreover, sialyl Lewis x (17) was
synthesized via the enzymatic fucosylation of a precursor displayed in the plate. Studies on inhibition of
this biotransformation have been carried out, and the IC50 value found for the known inhibitor 20 was
consistent with previous studies in solution.
Introduction
slides can be used to immobilize microspots of carbo-
hydrate polymers without covalent conjugation. Mrksich et al.6
Cells universally carry a sugar coating formed by glycopro-
teins and glycolipids, which are involved in highly specific
recognition events between cells and proteins, hormones,
antibodies, and toxins. Understanding the mechanism of these
processes may lead to the development of new anti-infective,
anticancer, and anti-inflammatory strategies.1-3
Due to the increasing need to decipher the information
contained in complex carbohydrates, simple and readily acces-
sible methods for high-throughput analysis must be developed.
Microarrays have been reported to be one of the most frequently
used approaches because large libraries of compounds can be
quickly screened and only small quantities of material are
required,4 an important consideration due to the low availability
of some complex carbohydrates. Few approaches have been
developed, thus far, for the fabrication of carbohydrate microar-
rays. Wang et al.5 have found that nitrocellulose-coated glass
exploited the Diels-Alder mediated immobilization of carbo-
hydrate-cyclopentadiene conjugates to a monolayer that pre-
sents benzoquinone groups displayed on a gold surface.
Recently, Shin et al.7 reported on the attachment of maleimide-
linked carbohydrates to a glass slide coated by thiol groups,
and Feizi et al.8 described microarrays of oligosaccharides
displayed as neoglycolipids on nitrocellulose.
Our goal is to develop a simple and efficient system to attach
complex sugars to a microtiter plate to screen for their specific
interactions with proteins.
One major difficulty with regard to this subject is to identify
the appropriate functional groups and the generally useful
chemistry for attaching saccharides to various types of surfaces,
either covalently or noncovalently. We have chosen cyclo-
addition reactions, as they are often simple, reagent-free, and
very selective, but we have avoided the use of olefins, as
sustaining the reductive conditions often used in the final
deprotection of synthetic oligosaccharides is difficult in the
olefin group. In our effort to develop a simple, noncovalent
method for the preparation of saccharide arrays in microtiter
plates, we have found that a saturated hydrocarbon that is 13
to 15 carbons in length can stick to the surface of polystyrene
microtiter plates, and the glycolipids displayed are stable under
repeated aqueous washings and are functional in biological
screens.9 Because the preparation of glycolipids for microarrays
* To whom correspondence should be addressed. E-mail: wong@
scripps.edu
† Department of Chemistry and Skaggs Institute for Chemical Biology,
The Scripps Research Institute.
‡ Department of Molecular Biology, The Scripps Research Institute.
(1) (a) Sears, P.; Wong, C.-H. Cell. Mol. Life Sci. 1998, 54, 223. (b) Lis, H.;
Sharon, N. Eur. J. Biochem. 1993, 218, 1. (c) Varki, A. Glycobiology 1993,
3, 97. (d) Dove, A. Nat. Biotechnol. 2001, 19, 913. (e) Bertozzi, C. R.;
Kiessling, L. L. Science 2001, 291, 2357.
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Biochem. 1988, 57, 785.
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Science, 1991, 251, 767. (b) Whitesides, G. M.; Love, J. C. Sci. Am. 2001,
285, 38. (c) Cui, Y.; Wei, Q. Q.; Park, H. K.; Lieber, C. M. Science 2001,
293, 1289. (d) Pirrung, M. C. Angew. Chem., Int. Ed. 2002, 41, 1277. (e)
Kodadek, T. Chem. Biol. 2001, 8, 105. (f) Hergenrother, P. J.; Depew, K.
M., Schreiber, S. L. J. Am. Chem. Soc. 2000, 122, 7849. (g) Horan, S.;
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Advance online publication.
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Biotechnol. 2002, 20, 275.
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J. AM. CHEM. SOC. 2002, 124, 14397-14402
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A R T I C L E S
Fazio et al.
Scheme 1
Furthermore, alkynes with an electron-withdrawing neighboring
group favor 1,3-dipolar cycloaddition.13 To combine these
requirements, we prepared the alkyne 2 from propynoic acid
and tetradecylamine via DCC coupling.
Standard conditions for the construction of the triazole ring
involve refluxing the two components in an organic solvent
(often toluene). When run neat, this reaction afforded compound
3 in 89% yield and as expected, as a mixture of regioisomers
(4:1 mixture of the 1,4- and 1,5-regioisomers, Scheme 2).
However, the necessity of running this reaction at high tem-
perature even with the use of an electron-poor alkyne (at room
temperature, only traces of 3 could be detected after 7 d) does
not make it useful for synthesis in microtiter plates.
Reagents and Conditions: (a) 2-azidoethanol, BF3-Et2O, CH2Cl2, rt,
overnight; (b) C14H29NH2, DCC, CH2Cl2, 0 °C-rt, 2 h.
Recently, the copper(I)-catalyzed regiospecific 1,3-cyclo-
addition between terminal alkynes and azides was reported.
Sharpless et al.14 found that Cu(II)/sodium ascorbate catalyzes
the regiospecific formation of 1,4-triazoles from a broad range
of substrates. Meldal et al.15 utilized CuI/base as a catalyst and
a conjugated terminal alkyne as the dipolarophile linked to a
solid support. Both procedures form the triazole moiety re-
giospecifically (only the 1,4-regioisomer is formed), but more
interestingly for our propose, these reactions were run at room
temperature. We decided therefore to investigate the reactivity
of the acyl-protected azido-sugar 1 with alkyne 2. This
transformation was found to be extremely sensitive to the solvent
and base employed, as side reactions for the alkyne 2 can
compete with triazole formation.15 For instance, acetonitrile/
Et3N did not form 3, whereas simply changing the base from
Et3N to DIPEA formed 3 in 38% yield. We found that the
system alkyne/azide/DIPEA/CuI (1:1:1:0.1) in toluene afforded
regiospecifically the 1,4-triazole 3 in 85% yield (Scheme 2).
The optimized formation of triazole 3 thus represents a general
synthesis of glycolipid analogues.
can be difficult due to interference of the lipid moiety in isolation
and manipulation, we are looking for alternative methods for
the synthesis and attachment in situ of sugars to the microtiter
plate.
One reaction of interest is the 1,3-dipolar cycloaddition
between alkynes and azides, a well-known reaction for the
construction of [1,2,3]-triazole rings.10 The chemical property
of the azido group renders this approach suitable for reaction
in microtiter plates and applicable to carbohydrate chemistry.
Indeed, azides are stable toward a majority of organic synthesis
conditions, and syntheses of complex carbohydrates, routinely
performed in our laboratory, employ azido-sugars as an ideal
“masked form” of more reactive and more difficult to handle
amino-sugars. Moreover, enzymatic synthesis of complex
carbohydrates has also been performed using azido-sugars as
substrates,11 and, if necessary, conversion of amines to azides
using the metal-catalyzed diazotransfer reaction12 is relatively
straightforward, showing therefore the general utility of this
approach for microarray fabrication of carbohydrates.
With this knowledge, the triazole formation of free azido-
sugars was then undertaken, using 2-azidoethyl-â-D-galacto-
pyranoside (4) as a model obtained from 1 in 95% yield. The
thermal 1,3-dipolar cycloaddition between 4 and alkyne 2
afforded the triazole derivative 5 in 75% yield as a 5.6:1 mixture
of the 1,4- and 1,5-regioisomers.
Results and Discussion
Our first attempts to investigate this reaction were performed
in a model study using 2-azidoethyl-2,3,4,6-tetraacetyl-â-D-
galactopyranoside (1), which was prepared from â-D-galactose
penta-acetate in 76% yield (Scheme 1). The alkyne counterpart
was chosen as the side of attachment to the microtiter plate
and therefore needed to contain a long aliphatic chain (C14).
When the copper(I)-catalyzed cycloaddition (alkyne/azide/
DIPEA/CuI, 1:1:5:5) was employed, 5 was isolated in 80% yield
Scheme 2
Reaction Conditions for the Formation of 3; (a) Isolated Yields; (b) Mixture 4:1 of 1,4- and 1,5-regioisomers.
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Synthesis of Sugar Arrays
A R T I C L E S
Scheme 3
Reagents and Conditions: (a) DIPEA, CuI, MeOH, rt, 8 h.
as the single 1,4-regioisomer (Scheme 3). Altering the solvent
systems from MeOH to H2O/CH3CN (4:1) afforded 5 in
comparable yield.
This chemistry was then carried out on microscales to develop
new micro-fabrication methods for applications in the biological
screening of carbohydrates. To achieve this goal, two crucial
points had to be resolved. First, the purification procedures, such
as column chromatography, that were used in all previous cases
needed to be avoided in order for the reaction to be applicable
to high-throughput synthesis and screening. Second, due to the
low availability of many natural carbohydrates, the reactions
needed to be carried out on a very small scale (possibly
micromolar). Therefore, we developed two different approaches,
which exploit both the thermal and the copper(I)-catalyzed
formation of the triazole ring.
Thermal formation has the advantage of employing only the
alkyne and azide, which renders purification simple. The alkyne
was used in 5-fold excess. It possesses physical properties
completely different from those of the product. Therefore,
unreacted alkyne was simply washed away with hot Et2O, giving
5 in very high purity without further purifications at very low
concentration (4 µmol scale). The only problem associated with
this transformation is the lack of regiocontrol as described above.
However, when we ran this thermal conversion (80 °C, 24 h,
neat) in the presence of only traces of CuI, the triazole derivative
5 was obtained regioselectively and in high purity (Figure 1).
These results show that if terminal alkynes such as 2 are
employed, a complete regiochemical control can be achieved
in the thermal 1,3-dipolar cycloaddition reaction with azides.
The copper(I)-catalyzed reaction has the advantage of forming
the triazole at room temperature. When this reaction was run
in MeOH in the 100 µL microtiter plate well, the crude product
could be purified in situ, allowing the reaction to be carried
out on a micromolar scale (Figure 2).
Figure 1. 1H NMR of (A) purified 5 obtained by thermal 1,3-cycloaddition
(1,4/1,5-regioisomers 5.6:1). (B) crude 5 (4 µmol scale) obtained by thermal
1,3-dipolar cycloaddition in the presence of traces of CuI (single 1,4-
regioisomer).
product is retained in the well due to its hydrophobic interaction
with the well surface. Purified 5 was redissolved in MeOH and
analyzed by ESI-MS (Figure 3C).
To determine the yield of this transformation, chemical and
biological assay methods were undertaken.9 Following the
Sulfuric Acid-Phenol (SAP)16 assay, an aqueous phenol solu-
tion was added to the wells, followed by concentrated H2SO4.
Absorbance of the phenol adduct was measured at 490 nm. The
average absorbance of reaction wells was divided by the average
absorbance of wells incubated with pure 5, which was synthe-
sized independently. This percentage was taken as the yield
(92%).
An additional biological assay was also undertaken exploiting
the recognition between the lectin Ricin B and â-D-galactose.17
Wells were incubated with TBS buffer containing bovine serum
albumin (BSA) to prevent protein binding to the well surface.
Ricin B chain from Ricinus communis (castor bean) was then
incubated in the well, followed by the specific anti-lectin for
Ricinus communis from rabbit. Wells were then incubated with
monoclonal anti-rabbit immunoglobulin conjugated with an
alkaline phosphatase. After the addition of p-nitrophenyl
phosphate, the absorbance was read at 405 nm. As before,
comparison with absorbance of pure 5 allowed yield determi-
nation (91%).
As an example, the reaction mixture (80 µL, Figure 2) was
incubated at room temperature. After 8 h, ESI-MS analysis
showed disappearance of starting material and formation of
product 5 (Figure 3B). The plate was then placed in a ventilated
fume hood for ∼3 h to allow evaporation of the solvent and
then washed with water to remove salts and the base. The
(10) Kuczkowski, R. L. In 1,3-Dipolar Cycloaddition Chemistry; Padwa, A.,
Ed.; Wiley-Interscience: New York, 1984; p 197.
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Chem. 2001, 66, 2442.
(12) Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett. 1996, 37, 6029.
(13) Hlasta, D. J.; Ackerman, J. H. J. Org. Chem. 1994, 59, 6184.
(14) (a) Rostovtsev, V. V.; Green, L. G.; Folkin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596. (b) Lewis, W. G.; Green, L. G.; Grynszpan,
F.; Radic, Z.; Carlier, P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 1053.
(16) (a) Saha, S. K.; Brewer, C. F. Carbohydr. Res. 1994, 254, 157. (b) Dubois,
M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F. Anal. Chem.
1956, 28, 350.
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A R T I C L E S
Fazio et al.
Chart 1. . Oligosaccharides Displayed in the Microtiter Plate via Copper(I)-catalyzed 1,3-dipolar Cycloaddition Reaction
This copper(I)-catalyzed triazole formation has been applied
to a series of oligosaccharides. The corresponding triazole
derivatives 6-16 (Chart 1) were synthesized in the microtiter
plate wells from the corresponding azides on a 0.6-1.1 µmol
scale which corresponds to a density of 4.1-7.6 µmol/cm2 (total
area per well 0.16 cm2) for the saccharide. Formation of products
and disappearance of starting materials was confirmed via ESI-
MS.
Lectin-binding assays were also performed on these oligosac-
charides bound to the well surface. Carbohydrates 6-12 display
various types and linkages of a terminal sialic acid. Fluorescein-
labeled Sambucus nigra lectin (SNA) was able to bind all sialic
acid-containing carbohydrates with preferential binding to the
R-2,6 linked over R-2,3 linked sialic acid, as expected.18
Carbohydrates 13-16 were recognized by Ricin B assays as
previously described.9
fucosylation of 7 and 9 with R-1,3-fucosyltransferase and GDP-
Fucose, followed by washings, was monitored with a fucose-
specific lectin from Tetragonolobus purpureas, and it was
observed that compound 7 but not 9 was fucosylated, consistent
with the previous study in solution19 (Figure 4). Conversion of
7 to sialyl Lewis x (17) was further confirmed using mass
spectrometry.
To establish that T. purpureas was indeed recognizing the
fucose and not binding nonspecifically to the microtiter plate,
varying levels of fucosyllipid 19, prepared from 2-azidoethyl-
R,â-L-fucopyranoside (18, Scheme 4), were attached to the
microtiter plate and analyzed against the lectin.
Illustrated in Figure 5 is the absorbance curve for varying
concentrations of fucosyllipid 19. The background absorbance,
that is, the absorbance of wells that were blocked with bovine
To further explore the biological applications, enzymatic
transformations of compounds 7 and 9 attached to the microtiter
plate were also investigated. Fucosylation of the N-acetyl-
glucosamine moiety generates tetrasaccharides sialyl Lewis x
(from 7) or sialyl Lewis a (from 9). Sialyl Lewis x (17) is a
crucial mediator in inflammation and often found in the sera of
gastrointestinal, pancreatic, and breast cancer patients.19 The
Figure 3. ESI-MS analyses of (A) starting material 4; (B) reaction after
8 h; (C) purified 5.
Figure 2. Formation of 5 in microtiter plate well.
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Synthesis of Sugar Arrays
A R T I C L E S
Figure 4. Synthesis of Sialyl Lewis x (17) in microtiter plate via enzyme assisted fucosylation of precursor 7; (a) GDP-fucose, fucosyltransferase, 37 °C,
1 h.
Figure 5. Absorbance of peroxidase-conjugated T. purpureas lectin as a
function of fucosyllipid 19 concentrations.
Scheme 4
Figure 6. Observation of inhibition of the fucosylation of NeuAcR2,-
3Galâ1,4GlcNAc (7) to gernerate 17.
incubated with R1,3FucT, they could be washed and then
incubated with the T. purpureas lectin and observed as before.
In this assay, the FITC-labeled lectin was utilized and only 20
(a) 2, CuI, DIPEA, MeOH, rt, 8 h.
nmol of 7 was necessary to analyze fucosylation through
generation of 17. An IC50 value of 6.212 µM was determined
for this method, which correlates well with the previously
determined Ki ) 5 µM in solution20 (Figure 6).
serum albumin (BSA) but contained no 19, was measured at
0.056AU ((0.0007). Increasing concentrations of 19 where the
wells were blocked with BSA showed increasing absorbance
that plateaus at 3.553AU ((0.0000) at 100 mM. With the
establishment that varying concentrations of L-fucose bound to
Conclusion
This paper describes the use of Cu(I)-catalyzed triazole
formation for the synthesis and in situ attachment of saccharides
to the microtiter plate. The reaction was carried out at room
temperature in methanol in microtiter wells on micromole scale,
followed by evaporation and washing with buffer solution. The
glycolipids noncovalently displayed are stable under repeated
aqueous washings and are functional in biological screens. The
saccharide arrays also act as substrates for glycosyltransferases
and are suitable for high throughput specificity studies of sugar-
the surface of the microtiter plate could be observed and
quantitated, we applied this lectin-binding strategy to the analysis
of an inhibitor of a fucosyltransferase (FucT).
Prior to treatment of wells containing 7 with the enzyme,
R1,3FucT was incubated with compound 20 shown in Figure
6, which was previously found in our laboratory to be a potent
inhibitor of this enzymatic reaction.20 After the wells were
(18) Rogerieux, F.; Belaise, M.; Terzidis-Trabelsi, H.; Greffard, A.; Pilatte, Y.;
Lambre, C. Anal. Biochem. 1993, 211, 200.
(19) Ichikawa, Y.; Lin, Y.-C.; Dumas, D. P.; Shen, G. J.; Garcia-Junceda, E.;
Williams, M. A.; Bayer, R.; Ketcham, C.; Walker, E.; Paulson, J. C.; Wong,
C.-H. J. Am. Chem. Soc. 1992, 114, 9283.
(20) Mitchell, M. L.; Tian, F.; Lee, L. V.; Wong, C.-H. Angew. Chem., Int. Ed.
2002, 41, 3041.
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protein interaction, enzyme inhibitor screening, and diversity-
oriented synthesis.
We believe that with the strategy described here and the
methods available for rapid synthesis of oligosaccharides,21 the
preparation of saccharide arrays for high-throughput screening
and drug discovery is attainable.
Acknowledgment. We are grateful to Prof. K. B. Sharpless
for helpful discussion of the triazole chemistry. This research
was supported by the National Institutes of Health and Optimer
Pharmaceuticals, Co.
Supporting Information Available: Experimental procedures,
biological assays and spectroscopic data. This material is
available free of charge via the Internet at http://pubs.acs.org.
(21) (a) Sears, P.; Wong, C.-H. Science 2001, 291, 2344. (b) Plante, O. J.;
Palmacci, E. R.; Seeberger, P. H. Science 2001, 291, 1523.
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