Assembly of sugars on polystyrene plates: A new facile microarray fabrication technique

Article abstract of DOI:10.1016/j.tetlet.2004.01.159

The work presented herein is a new noncovalent glycoarray assembly method for microplates created by simply mixing together an isocyanate-containing C₁₄-hydrocarbon and an amine-containing carbohydrate. 2-Aminoethyl-β-D-galactopyranoside (1) was utilized in model studies and product formation was detected by both ESI-MS and lectin binding. The method has been further extended to array complex carbohydrates.

Full text of DOI:10.1016/j.tetlet.2004.01.159

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 2689–2692
Assembly of sugars on polystyrene plates: a new facile microarray
fabrication technique
Fabio Fazio, Marian C. Bryan, Hing-Ken Lee, Aileen Chang and Chi-Huey Wong*
Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, CA 92037, USA
Received 31 October 2003; revised 14 January 2004; accepted 14 January 2004
Abstract—The work presented herein is a new noncovalent glycoarray assembly method for microplates created by simply mixing
together an isocyanate-containing C14-hydrocarbon and an amine-containing carbohydrate. 2-Aminoethyl-b-
1) was utilized in model studies and product formation was detected by both ESI-MS and lectin binding. The method has been
further extended to array complex carbohydrates.
2004 Elsevier Ltd. All rights reserved.
D-galactopyranoside
(
ꢀ
Microarrays are becoming one of the most powerful
technologies for the identification and biological evalu-
ation of new drug candidates and targets. However, only
very recently have carbohydrate microarrays, or glyco-
arrays, appeared in the scientific literature as a promis-
ing high-throughput methodology for the study of cell–
applied the copper(I)-catalyzed 1,3-dipolar cycloaddi-
tion reaction between azides and alkynes to glycoarray
assembly. Our continuous efforts toward elucidation of
the function of complex carbohydrates in a rapid man-
ner prompted us to investigate alternative methods for
the preparation of carbohydrate arrays.
1
1–8
cell recognition processes mediated by carbohydrates.
Glycoarray development has been hindered first by
synthesis of carbohydrate libraries and second by
development of orthogonal attachment strategies that
are critical to their efficient display. With the advent of
high-throughput oligosaccharide synthesis, this leaves
the bottleneck with the development of the actual glyco-
Though complex oligosaccharides have been displayed
effectively through cyclic triazole formation and are
stable under the most organic reaction conditions,
1
oxygen, and water, we are interested in new coupling
strategies to increase applicability. In oligosaccharide
chemical synthesis, the terminal step is usually the re-
moval of protecting groups such as esters and ethers.
While the azido group sustains ester deprotection, the
reductive conditions commonly employed to remove
benzyl ethers (among the most common protecting
groups in carbohydrate chemistry) also reduce azides to
amines. Although amines can be easily reconverted into
azides and methods other than reductive cleavage are
available for the deprotection of benzyl groups, we
decided to directly exploit the amino group for the
9
;10
array technology.
Carbohydrates alone show very low retention to poly-
styrene plates due to their high polarity.
3;11
As such, a
variety of attachment strategies have been developed
including covalent attachment through Michael addi-
tion and Diels–Alder reaction and noncovalent attach-
ment through glycolipid application where the
saccharide is linked to a C -hydrocarbon to favor their
14
1;2;5;6
12–14
retention in polystyrene wells.
In order to directly
fabrication of a noncovalent carbohydrate array.
link library synthesis with oligosaccharide display,
techniques allowing for capture of an orthogonal group
to the surface for direct analysis with minimal interme-
diate treatment have to be developed. We have recently
While amide formation is surely an excellent ligation
method, we thought to exploit the reagent-free urea
formation between isocyanates and amines, a very well
15
known process in the gluing industry. Therefore,
amino-sugars were reacted with a C -isocyanate to
14
form the highly stable urea functionalized with a suit-
ably long hydrocarbon chain necessary for noncovalent
adsorption to the microtiter plate. This reaction was first
Keywords: Microarray; Oligosaccharides; Detection by ESI-MS.
*
Corresponding author. Tel.: +1-619-784-2487; fax: +1-619-784-2409;
e-mail: wong@scripps.edu
0
040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.01.159

2690
F. Fazio et al. / Tetrahedron Letters 45 (2004) 2689–2692
performed in solution on a relatively large scale
2 mmol) using 2-aminoethyl-b- -galactopyranoside (1)
as a model. Amino-sugar 1 was reacted with commer-
2 h reaction with tetradecyl isocyanate, ESI (Fig. 1D)
shows disappearance of the starting material and for-
(
D
þ
mation of the galactosyl-urea derivative 2 (463, M+H ;
þ
cially available tetradecyl isocyanate to form the urea
derivative 2 in 92% yield and high purity (Scheme 1).
485, M+Na ) together with formation of tetradecyl-
þ
16
amine (214, M+H ) derived from hydrolysis of excess of
tetradecyl isocyanate. When the reaction was complete,
traces of solvents were removed by placing the plate in a
dessicator under vacuum.
This transformation is clean, quick, and coupling
reagent-free. By simply mixing the two reactants
together, the urea derivative is obtained in very high
yield as a unique product. This reaction is not moisture
or air sensitive and product formation occurs at rt.
Therefore, these conditions are ideal for microscale
application. Indeed, we ran this reaction in the micro-
titer plate by mixing together the two reactants in
MeOH/isopropanol (1:1) and placing the plate in a
ventilated fume hood for evaporation of solvents.
In addition, it was important to confirm that this array
is stable under repeated aqueous washings. Indeed,
when wells were washed with milli-Q water (3 · 100 lL),
retention of galactosyl-urea 2 in the plate was observed
by both ESI-MS (Fig. 1E) and biological assay (Fig.
1B). A variety of lectins (carbohydrate-recognizing
proteins) have been previously applied to the analysis of
noncovalent arrays including Ricinus communis (Gal),
Tetragonolobus purpureas (Fuc), and Sambucus nigra
17
The course of the reaction was followed by electrospray
ionization-mass spectrometry (ESI-MS). Figure 1C
1
;2
(NeuAc). While the presence of galactose was again
being analyzed, a less toxic lectin than R. communis was
þ
shows ESI of the starting amine 1 (224, M+H ). After
HO
OH
HO
OH
O
C
N
( )
12
O
O
O
O
O
HO
NH2 MeOH/i-PrOH (1:1)
HO
NH
NH
( )12
OH
OH
rt, 2h, 92%
1
2
Scheme 1.
Figure 1. (A) Formation of compound 2 in the microtiter plate well (scale 2 mM). (B) Fluorescence analysis of lectin binding assay with S. japonica
for reaction in microtiter plates at different concentrations (0.2 lM–2 mM). ESI-MS of starting amine 1 (C) before reaction in plate, (D) after 2 h
reaction, and (E) after three washings with milli-Q water.

F. Fazio et al. / Tetrahedron Letters 45 (2004) 2689–2692
2691
OH
OH
OH
OH
OH
O
OH
O
O
O
O
HO
O
OH
O
AcNH
O
OH
OH
O
OH
O
HO
O
HO
3
HO
HO
HO
OH
HO
OR
OH
OH
OH
OH
HO
OH
HO
OH
HO
O
O
HO
HO
O
HO
HO
O
HO
O
HO
HO
O
O
HO
O
HO
HO
HO
OH
HO
O
O
HO
HO
HO
O
O
HO
O
O
HO
4
OR
O
5
OR
O
HO
HO
OH
HO
OH
HO
OH
HO
O
HO
O
HO
OH
O
HO
HO
OH HO
HO
6
OR
O
HO
HO
HO
O
HO
O
HO
HO
O
HO
O
HO
HO
O
O
HO
HO
O
HO
O
HO
HO
HO
O
O
HO
HO
O
HO
O
HO
O
HO
HO
O
O
HO
HO
O
7
OR
HO
HO
O
HO
O
HO
O
R= -(CH ) NH
2
5
2
OR
8
Figure 2. Oligosaccharides displayed in microtiter plate via urea formation with tetradecyl isocyanate.
desired. Therefore, a galactose-specific lectin from
throughput biological evaluation of carbohydrate–
9;10
protein and carbohydrate–carbohydrate interactions.
18
Sophora japonica was selected. Observation of the
fluorescein-labeled lectin over a range of concentrations
was readily detectable (from 0.2 lM to 2 mM). Also, the
lectin responded in a concentration dependent manner
to the galactose as would be expected. This indicates
that this method is indeed highly applicable to ELISA-
type lectin assays.
References and notes
1. Fazio, F.; Bryan, M. C.; Blixt, O.; Paulson, J. C.; Wong,
C.-H. J. Am. Chem. Soc. 2002, 124, 14397–14402.
2
. Bryan, M. C.; Plettenburg, O.; Sears, P.; Rabuka, D.;
Wacowich-Sgarbi, S.; Wong, C.-H. Chem. Biol. 2002, 9,
Various complex carbohydrates were also displayed in a
microtiter plate following this procedure. Cancer anti-
gen Globo-H (3) and gp120 oligomannose type sugars
7
13–720.
3
4
. Wang, D.; Liu, S.; Trummer, B. J.; Deng, C.; Wang, A.
Nature Biotech. 2002, 20, 275–281.
. Fukui, S.; Feitzi, T.; Galustian, C.; Lawson, A. M.;
Chai, W. Nature Biotech. 2002, 20, 1011–1017.
4
and product formation in all cases was confirmed with
–8 (Fig. 2) were displayed via the urea bond linkage
19–21
ESI-MS.
The formation of a large array of oligo-
saccharides following this newly developed method and
the biological screening with different lectins is currently
under investigation.
5. Park, S.; Shin, I. Angew. Chem., Int. Ed. 2002, 41, 3180–3182.
6. Houseman, B. T.; Mrksich, M. Chem. Biol. 2002, 9, 443–
454.
7
. Mellet, C. O.; Fernandez, J. M. G. ChemBioChem 2002, 3,
19–822.
8
In summary, this work shows a new and efficient
methodology for the noncovalent assembly of sacchar-
ides in microtiter plates. Lectin binding studies have
proven that this microarray is functional in biological
screening and therefore applicable in ELISA-type for-
mats. We believe that this new array, together with the
efficient methods available for synthesis of complex
oligosaccharides, can become useful for the high-
8
. Adams, E. W.; Ueberfeld, J.; Ratner, D. M.; OÕKeefe, B.
R.; Walt, D. R.; Seeberger, P. H. Angew. Chem., Int. Ed.
2003, 42, 5317–5320.
. Sears, P.; Wong, C.-H. Science 2001, 291, 2344–2350.
9
1
0. Seeberger, P. H.; Haase, W.-C. Chem. Rev. 2000, 100,
349–4393.
4
11. Lee, Y. C.; Lee, R. T. Recognition of Carbohydrates in
Biological Systems, Part A: General Procedures. In

2692
F. Fazio et al. / Tetrahedron Letters 45 (2004) 2689–2692
Methods in Enzymology; Abelson, J. N., Simons, M. I.,
Eds.; Academic: New York, 2003; Vol. 362, p 625.
2. Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett.
996, 37, 6029–6032.
3. Nyffeler, P. T.; Liang, C.-H.; Koeller, K. M.; Wong, C.-H.
J. Am. Chem. Soc. 2002, 124, 10773–10778.
4. Birch, A. J.; Rao, G. S. Adv. Org. Chem. 1972, 8, 1–
arrayed carbohydrates (2 mM solution in MeOH, 40 lL).
The plate was placed in a fume hood for 2 h to allow for
evaporation of solvent and then transferred for ca. 15 min
in a dessicator connected with a vacuum pump. Washings
with milli-Q water (3 · 100 lL) afforded the carbohydrate
microarray ready for biological screening.
1
1
1
1
1
18. McPherson, A.; Hankins, C. N.; Shannon, L. J. Biol.
Chem. 1987, 262, 1791–1794.
19. Burkhart, F.; Zhang, Z.; Wacowich-Sgarbi, S.; Wong, C.-H.
Angew. Chem., Int. Ed. 2001, 40, 1274–1277.
20. Calarese, D. A.; Scanlan, C. N.; Zwick, M. B.; Deechong-
kit, S.; Mimura, Y.; Kunert, R.; Zhu, P.; Wormald, M. R.;
Stanfield, R. L.; Roux, K. H.; Kelly, J. W.; Rudd, P. M.;
Dwek, R. A.; Katinger, H.; Burton, D. R.; Wilson, I. A.
Science 2003, 300, 2065–2071.
65.
5. Diphenylmethane diisocyanate (MDI) and Toluene diiso-
cyanate (TDI) are two common, widely used, isocyanate
based glues.
1
6. Compound 2: mp 151–152 ꢁC (from Et
2
O). 1H NMR
OD): 4.13 (d, 1H, J ¼ 7:45 Hz), 3.73 (m, 2H), 3.68 (d,
(
CD
3
1
1
H, J ¼ 7:45 Hz), 3.64 (m, 2H), 3.43 (m, 2H), 3.38 (dd,
H, J ¼ 10:08 Hz, J ¼ 3:50 Hz), 3.24 (m, 2H), 3.00 (t, 2H,
J ¼ 7:01 Hz), 1.37 (m, 2H), 1.20 (s, 22H), 0.81 (t, 3H,
21. ESI-MS of oligosaccharides 3–8, after urea formation in
microtiter plate (most abundant signals): compound 3:
J ¼ 7:01 Hz). 13C NMR (CD
3
OD): 161.27, 105.44, 76.81,
4.88, 72.57, 71.18, 70.33, 62.69, 41.41, 41.01, 33.08, 31.33,
þ
þ
7
3
C
C
58
H
H
105
N
3
O
O
31Na [M+Na] , 1362.6; compound 4:
17Cl
0.80, 30.77, 30.52, 30.48, 27.99, 23.73, 14.45. HR-
þ
38
72
N
2
17Na [M+Na] , 851.5,
5:
C
38
H
72
N
2
O
ꢀ
MALDI-FTMS: calcd for C23
85.3197; found, 485.3202.
H
46
N
2
O
7
Na [M+Na] ,
[M+Cl] ,
[M+Na] , 851.5, C38
pound 6: C44
863.3; compound
O
C
38
ꢀ
H
72
N
2
O
17Na
þ
4
H
73
N
2
17Cl [M)H] , 827.3; com-
ꢀ
1
7. Protocol for array formation: tetradecyl isocyanate (40 lL
of a freshly prepared 4 mM solution in isopropanol,
H
83
N
2
O22 [M)H] , 989.4, C44
[M+Cl] , 1025.3; compound 7: C50
1151.4; compound 8: C62H N O37 [M)H] , 1475.7.
113 2
H
82
N
2
O
22Cl
ꢀ
ꢀ
H
93
N
2
O
27 [M)H] ,
ꢀ
2
equiv) was added, in microtiter plate, to a solution of the