Fluorous-based carbohydrate microarrays

Article abstract of DOI:10.1021/ja054811k

The success of microarrays, such as DNA chips, for biosample screening with minimal sample usage has led to a variety of technologies for assays on glass slides. Unfortunately, for small molecules, such as carbohydrates, these methods usually rely on cova

Full text of DOI:10.1021/ja054811k

Published on Web 09/02/2005
Fluorous-Based Carbohydrate Microarrays
Kwang-Seuk Ko, Firoz A. Jaipuri, and Nicola L. Pohl*
Department of Chemistry and the Plant Sciences Institute, Gilman Hall, Iowa State UniVersity,
Ames, Iowa 50011-3111
Received July 18, 2005; E-mail: npohl@iastate.edu
Scheme 1
The success of microarrays, such as DNA chips, for biosample
screening with minimal sample usage has led to a variety of
technologies for assays on glass slides.1 Unfortunately, for small
molecules, such as sugars, these methods usually rely on covalent
bond formation, which requires unique functional handles and
multiple chemical steps.2 Herein, we present a new simpler concept
in microarray formation that is based on noncovalent fluorous-based
interactions and demonstrate the strength of these interactions in
the direct formation of carbohydrate microarrays for biological
screening.
Scheme 2
Unlike comparable lipid tails interacting with hydrophobic solid
phases,3 a single C8F17 tail is sufficient to bind biomolecules, such
as peptides, to fluorinated solid supports.4 The use of such
fluorinated solid supports for affinity chromatography can substan-
tially simplify the purification of synthetic carbohydrate intermedi-
ates in a process amenable to automation (Figure 1).5 An additional
Sugars commonly found in plants were chosen as initial targets
to test the feasibility of a noncovalent fluorous-based array for
protein-binding studies (Scheme 1). Genome sequencing projects
have revealed that the number of genes related to carbohydrate
metabolism is far greater in plants, such as Arabidopsis thaliana,
than in animals or fungi.6 In addition to providing an understanding
of plant biology, the study of carbohydrate-protein interactions in
plants could lead to the discovery of new sugar-binding lectins for
use as glycobiology tools.7 The known trichloroacetimidates of
peracylated mannose,8 galactose,9 and N-acetylglucosamine10 were
reacted with the fluorous-tagged allyl alcohol, and subsequent
deacylation and hydrogenation of the double bond yielded the initial
array components 1-3.
Although neighboring-group participation could be used to
control the stereochemistry of glycosylation for mannose, galactose,
and N-acetylglucosamine, the synthesis of R-linked fucose required
a different approach. Fucose glycosyl donors are usually built with
nonparticipating benzyl protecting groups to provide predominantly
the R-configured glycosylation products. Unfortunately, such
electron-donating groups on a 6-deoxysugar also serve to make the
resulting glycosidic linkage more acid sensitive. Several strategies
alleviate this problem, for example, the use of more electron-
withdrawing halobenzyl groups11 or replacing the 3- and 4-OH
protecting groups with an ester2 that can potentially serve as a
distant participating group. The latter approach is appealing, but
requires an additional basic deprotection step. To avoid this extra
step, we decided to test if a benzyl carbonate protecting group could
serve the same purpose with the advantage of removal during the
hydrogenation step. The requisite glycosyl donor was synthesized
from the known allylated compound13 5 in three steps to provide
the desired trichloroacetimidate 6 (Scheme 2). This donor was
glycosylated with the fluorous-tagged alcohol using trimethylsilyl
triflate to yield only the axially linked product in 90% yield. Indeed,
the benzyl carbonate could serve to direct formation of the R-anomer
and be removed by hydrogenation.
Figure 1. Strategy for the synthesis of carbohydrates and direct formation
of fluorous-based carbohydrate microarrays.
potential advantage of a fluorous-based approach is the direct
formation of microarrays; the production of carbohydrate microar-
rays from compounds made on solid-phase still requires multiple
solution-phase deprotection and derivatization steps.2c
We began our studies with the design of a suitable fluorous tag
for carbohydrate synthesis that could survive the necessary sequen-
tial reaction conditions and also be removed if desired. An allyl
group would be orthogonal to the trichloroacetimidate coupling
conditions and other deprotection conditions. Therefore, to create
a fluorous allyl protecting group as well as potential anchor for
microarray formation, a fluorous tail was synthesized in one step
by reaction of cis-1,4-butenediol with substoichiometric amounts
of 1H,1H,2H,2H-perfluorodecyl iodide to produce an alcohol for
use in glycosylations.
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10.1021/ja054811k CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Acknowledgment. We thank Dr. T. Nagashima at Fluorous
Technologies for a kind donation of fluorous silica gel, and H. Jin
at the ISU Microarray Facility. This material is based in part upon
work supported by the National Science Foundation under CAREER
Grant No. 0349139. N.L.P. is a Cottrell Scholar of Research
Corporation and an Alfred P. Sloan Research Fellow.
Supporting Information Available: Experimental details, including
1
copies of H NMR spectra, for the synthesis and production of the
carbohydrate microarrays and the complete ref 2h. This material is
available free of charge via the Internet at http://pubs.acs.org.
Figure 2. Fluorescence images of arrayed carbohydrates probed with FITC-
labeled lectins. Columns of 4 spots each of 2, 1, 0.5, and 0.1 mM
carbohydrates were incubated for 20 min with FITC-ConA (top) or FITC-
ECA with 1% TWEEN-20 detergent (bottom) with BSA.
References
After synthesis of the requisite fluorous-tagged sugars, the next
challenge was to find a suitable fluorinated surface. Solutions of
each sugar were spotted onto a commercially available glass
microscope slide coated with a Teflon/epoxy mixture employing a
standard robot used for DNA arraying. The spots were dried,
incubated with a solution of the fluorescein isothiocyanate-labeled
jack bean lectin concanavalin A (FITC-ConA) for 20 min, rinsed
repeatedly with assay buffer and distilled water, and then scanned
with a standard fluorescent slide scanner. The scan clearly showed
binding of FITC-ConA only to the mannose-containing spots. The
anomeric position could be distinguished as the â-linked GlcNAc
(2) was not recognized.
This lectin experiment demonstrated the ability of the C8F17 tail
to anchor the carbohydrates to the slide surface even after repeated
washes. However, the slide was also intrinsically and unevenly
fluorescent at 488 nM, a wavelength that is commonly used to detect
labeled analytes. Clearly, a new approach was necessary to obtain
an optically and fluorescently clear surface for the formation of
compound microarrays. To this end, a glass microscope slide was
reacted14 with a fluoroalkylsilane to provide a clear coating on which
water forms beads.
With the new microarray substrate in hand, we next probed the
scope of a fluorous-based microarray approach for compound
screening. Fluorous-tagged sugars were spotted on the coated slide
using an arraying robot, and then the slides were incubated with
FITC-labeled lectins (Figure 2). To test the reproducibility of the
process, the same concentration of sugar was spotted repeatedly.
In addition, several different concentrations of sugars were spotted.
To test the ability of the array to withstand detergents often included
in biological screens, the labeled plant lectin from the bush
Erythrina crystagalli (FITC-ECA) was used to probe the microar-
rays with Tween-20. The array withstood the 20 min incubation
time and repeated rinsing with this detergent-containing buffer.
A fluorous-based microarray method allows the facile formation
of a range of carbohydrate chips for the plant and other sciences
using synthetic sugars produced with the aid of fluorous-tagged
synthesis. Efforts are underway to automate portions of the solution-
phase fluorous-based synthetic process and to incorporate enzymatic
steps to expand the scope of carbohydrates that can be easily arrayed
for biological screening. Although not limited to carbohydrates,
the approach should be especially valuable for the production of
arrays containing compounds, such as glycosaminoglycan frag-
ments, that contain nucleophiles that complicate current defined
covalent attachment strategies.
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