Fabrication of chemical microarrays by efficient immobilization of hydrazide-linked substances on epoxide-coated glass surfaces

Article abstract of DOI:10.1002/anie.200462720

(Chemical Equation Presented) Fixed to the spot: A new, efficient, and simple immobilization technique for the construction of chemical microarrays has been developed. This technique is applicable to the site-selective attachment of diverse substances, in

Full text of DOI:10.1002/anie.200462720

Angewandte
Chemie
Microarray Immobilization
selective. Strategies that employ efficient and chemoselective
ligation processes would be generally applicable to the
fabrication of microarrays that possess covalently linked,
biologically interesting molecules. Herein we describe a novel
chemoselective immobilization process in which hydrazide-
containing compounds react with epoxides coated on glass
slides. This new technique has been applied to the efficient
construction of chemical microarrays, which have been used
to evaluate protein binding to peptides and small molecules.
Several criteria must be met in designing a general
method to prepare diverse chemical microarrays. Firstly, the
diverse substances containing the specific functional groups
used for selective reactions with the modified solid surfaces
must be easily prepared by solid-phase synthesis. Also,
functional groups that will selectively react with the small
molecules must be readily incorporated onto the solid
surfaces. Lastly, following their release from a solid support,
the diversely structured and functionalized small molecules
must undergo site-specific covalent attachment to the modi-
fied surfaces. Strategies employing highly chemoselective
ligation reactions fit these criteria. We have investigated a
novel technique for immobilization, which relies on the use of
reactions between hydrazide-containing small molecules and
epoxide-coated glass slides (Scheme 1). The hydrazide groups
are incorporated into the small molecules while they are
attached to a solid support and are used as a handle in their
Fabrication of Chemical Microarrays by Efficient
Immobilization of Hydrazide-Linked Substances
on Epoxide-Coated Glass Surfaces**
Myung-ryul Lee and Injae Shin*
Small molecules that regulate biological processes serve as
valuable tools in studies of the functions of biomolecules,
[
1]
especially proteins, as well as in the development of drugs.
An important component of efforts that target the discovery
of bioactive molecules is high-throughput screening. Tech-
nologies that rely on the use of DNA, protein, and carbohy-
drate microarrays have been widely employed to accelerate
the selection of lead compounds and as high-throughput
analytical tools in genomic, transcriptomic, proteomic, and
[
2–4]
glycomic research.
Microarray platforms enable the simul-
taneous assessment of a large number of samples that are
available in limited quantities.
Similarly, small-molecule microarrays have been used as
high-throughput methods to identify substances that selec-
[
5]
tively bind to proteins. Most of the small molecules of
interest in these efforts possess a number of different
functional groups, such as hydroxy (OH), amino (NH ),
2
[
6]
carboxy (CO H), and sulfanyl groups (SH). The major
solid-phase synthesis. The epoxide-derivatized glass slides
are easily created by immersing amine-coated glass slides into
a solution of poly(ethylene glycol) diglycidyl ether (3%
2
requirement of techniques used to fabricate small-molecule
microarrays is that immobilization of the diversely function-
alized compounds to the modified surfaces must be highly
[
7]
solution in 10 mm NaHCO , pH 8.3).
3
Scheme 1. Strategy for the fabrication of a chemical microarray based on the immobilization of hydrazide-containing compounds on epoxide-deriv-
atized glass slides.
[
*] M.-r. Lee, Prof. Dr. I. Shin
Department of Chemistry, Yonsei University
Seoul 120-749 (Korea)
Fax: (+82)2-364-7050
E-mail: injae@yonsei.ac.kr
A hydrazide-linked fucose probe (S- or L-Fuc-NHNH , S:
short, L: long; Scheme 2) was used to probe optimal
conditions (pH, time, and concentration) for the immobiliza-
2
tion process. Preparation of S- or L-Fuc-NHNH was initiated
2
by transforming alcohol groups on a Wang resin into p-
[
**] This work was supported by a grant of the Center for Integrated
Molecular Systems (KOSEF) and the Ministry of Science and
Technology.
[
8]
nitrophenyl carbonates. The resulting resin was then treated
with hydrazine to yield the hydrazide-containing resin, which
was coupled with the Fmoc-protected L or S tether in the
presence of NEM, BOP, and HOBt. The amino groups
Supporting Information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2005, 44, 2881 –2884
DOI: 10.1002/anie.200462720
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Communications
Scheme 2. Synthesis of the hydrazide-linked fucose probes S- and L-
Fuc-NHNH . Py: pyridine, DIEA: ethyldiisopropylamine, DMF=N,N-
2
dimethylformamide, NEM=N-ethylmorpholine, BOP=1-benzotriazo-
lyloxytris(dimethylamino)phosphonium hexafluorophosphate,
HOBt=1-hydroxybenzotriazole, TES=triethylsilane, TFA=trifluoroace-
tic acid, Fmoc=9-fluorenylmethoxycarbonyl.
produced by treatment of the resin with piperidine were
treated with succinic anhydride to generate carboxylic acids,
which underwent an amide-bond-forming reaction with
aminoethyl a-fucopyranoside. Finally, the desired compounds
were released from the resin by treatment with 2% TES in
TFA.
Scheme 3. Synthesis of amine-linked biotin (L-B-NH ) and thiol-linked
GlcNAc (L-GlcNAc-SH) probes. PNP=p-nitrophenyl.
2
residues at a higher pH value, when the thiol moiety is
deprotonated (Figure 1a).
In order to find the proper immobilization conditions, a
solution of L-Fuc-NHNH was printed onto the epoxide-
2
derivatized glass slides and then the resulting slides were
probed with Cy5-labeled Aleuria aurantia lectin (Cy5-AA)
[
9]
for 1 h (Cy5: indodicarbocyanine). It was found that
substrate concentrations of 0.5–1 mm and times of 3–4 h at
pH 3–5 were appropriate for efficient immobilization reac-
[
8]
tions.
An important feature of the method we have developed
for constructing small-molecule microarrays is chemoselec-
tivity. This was demonstrated in studies of selective attach-
ment of hydrazides onto the epoxide-coated surfaces in the
presence of other potent nucleophilic groups, such as amines
and thiols. For these studies, amine-linked biotin (L-B-NH2)
and thiol-linked N-acetylglucosamine (L-GlcNAc-SH)
probes were synthesized on solid supports by using p-nitro-
phenol-activated Wang and 2-carboxyethanethiol 4-methoxy-
trityl polystyrene (PS-Trt-(4-OMe)) resins, respectively
Figure 1. a) Immobilization of L-Fuc-NHNH in the presence of one
2
equivalent of L-GlcNAc-SH (
&
probed with Cy5-AA, * probed with Cy3-
TV). b) Immobilization of L-Fuc-NHNH in the presence of L-B-NH2
2
(&
L-Fuc-NHNH only, * L-Fuc-NHNH :L-B-NH (1:1), L-Fuc-
~
2
2
2
NHNH :L-B-NH (1:4); all probed with Cy5-AA).
2
2
[
8]
(
Scheme 3).
A 1:1 mixture of L-Fuc-NHNH and L-GlcNAc-SH (1 mm
2
in 30% glycerol in 100 mm sodium phosphate, pH 3–10) was
applied to the epoxide-functionalized slides, which were then
treated with Cy5-AA and Cy3-labeled Triticum vulgaris lectin
Next, selective attachment of hydrazides to the epoxide-
coated slides in the presence of amines was examined.
Mixtures of L-Fuc-NHNH2 and L-B-NH2 (1:1 and 1:4 in
30% glycerol in 100 mm sodium phosphate, pH 3–10) were
applied to the epoxide-derivatized slides and then probed
with Cy5-AA. Since in some biologically interesting sub-
strates (especially peptides) several amino groups are present,
these immobilization reactions were run with mixtures
[
9]
(
wheat germ agglutinin; Cy3-TV; Cy3: indocarbocyanine).
Fluorescence analysis showed that the hydrazide substrate (L-
Fuc-NHNH ) was selectively immobilized in reactions con-
2
ducted at low pH values, while the thiol substrate (L-GlcNAc-
SH) underwent a more efficient reaction with the epoxide
2
882
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Angewandte
Chemie
containing an excess of L-B-NH2 of up to four molar
equivalents. As shown in Figure 1b, the fluorescence intensity
of spots containing L-Fuc-NHNH was barely changed when
2
L-B-NH2 was present, a result indicating that hydrazide-
linked fucose (L-Fuc-NHNH ) was selectively attached to the
2
surface over the range of pH 3–10 even when a large excess of
amine-containing biotin was used. The results of these
competition experiments demonstrate that hydrazides are
immobilized on the epoxide-coated surface more rapidly than
substrates with both amine and thiol functional groups.
As part of a practical application of this technique to the
fabrication of chemical microarrays, we synthesized hydra-
zide-linked substrates containing biotin (S- or L-B-NHNH ),
2
peptides (S- or L-GSH-NHNH , S- or L-strep-tag-NHNH2
2
and its analogues), and phenanthridinium derivatives
[
8]
(
Schemes 4 and 5). The strep-tag peptide sequence and its
Scheme 5. Synthesis of probes containing phenanthridinium deriva-
tives. Boc: tert-butoxycarbonyl, Tf: trifluoromethanesulfonyl.
Scheme 4. Synthesis of hydrazide-linked probes containing biotin, glu-
tathione (GSH), or the streptavidin tag (strep tag) and its analogues.
analogues were directly assembled on solid supports by using
the conventional Fmoc strategy. Strep-tag analogues were
prepared by replacing the histidine residue with 1 of the other
Figure 2. Fluorescence images of chemical microarrays containing
fucose, GSH, the strep tag, and biotin, when probed with a) Cy5-AA,
b) GST followed by FITC-labeled anti-GST antibody, c) Cy3-strepavidin,
and d) Cy3-avidin. Fluorescence images of chemical microarrays con-
taining e) phenanthridinium derivatives probed with Cy5-AChE (1
COR=H, 2 COR=cyclohexylcarbonyl, 3 COR=allyloxycarbonyl, 4
COR=phenylacetyl) and f) strep tag and its analogues probed with
Cy3-strepavidin (single letter amino acid codes given for the residue
that replaced histidine in the strep tag, P!L: replacement of proline
with leucine in the strep tag, Q!L: replacement of glutamine with
leucine in the strep tag). Spot size: ꢀ100 mm, distance between cen-
ters of spots: ꢀ250 mm.
1
9 amino acids or by replacing the proline and glutamine
residues with leucine. GSH was coupled to the resin by using a
disulfide exchange reaction. Four phenanthridinium deriva-
tives for acetylcholinesterase (AChE) inhibitors were pre-
pared by solution-phase synthesis and subsequently coupled
to the carboxylic acid derivatized resin.
Eight S/L-tethered fucose-, glutathione-, biotin-, and
strep-tag-containing hydrazides (1 mm in 30% glycerol in
1
00 mm sodium phosphate, pH 5) were microspotted onto the
epoxide-derivatized glass slides and then incubated with Cy5-
AA, Cy3-streptavidin, Cy3-avidin, or glutathione S transfer-
ase (GST). GST incubation was followed by treatment with
fluorescein isothiocyanate (FITC) labeled anti-GSTantibody.
As shown in Figure 2a–d, fucose, glutathione, and the strep
tag were selectively recognized by AA, GST, and streptavidin,
respectively. The strep tag is known to bind to streptavidin
rather than avidin and, as expected, microspots containing
Angew. Chem. Int. Ed. 2005, 44, 2881 –2884
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Communications
[
10]
this substance were recognized only by streptavidin. Micro-
spots containing biotin display much stronger fluorescence
than strep-tag microspots because the binding affinity of
streptavidin to biotin is much greater than that of streptavidin
2001, 8, 105; e) S. R. Weinberger, E. A. Dalmasso, E. T. Fung,
Curr. Opin. Chem. Biol. 2001, 5, 86.
4] For reviews of carbohydrate microarrays, see: a) I. Shin, J. W.
Cho, D. W. Boo, Comb. Chem. High Throughput Screening 2004,
[
7, 565; b) I. Shin, S. Park, M.-R. Lee, Chem. Eur. J. 2005, in press;
[
11]
to the strep tag.
Interestingly, microspots produced by
c) T. Feizi, W. Chai, Nat. Rev. Mol. Cell Biol. 2004, 5, 582; d) T.
Feizi, F. Fazio, W. Chai, C.-H. Wong, Curr. Opin. Struct. Biol.
2003, 13, 637.
reaction of L-tethered substrates bound to the corresponding
proteins more tightly than those with S-tethered substrates.
The fluorescence intensities of microspots containing L-
tethered compounds are more than twice as strong as those
of microspots containing S-tethered ones.
Small-molecule microarrays with four phenanthridinium
derivatives were also fabricated by following the method
described above. Fluorescence analysis of slides treated with
Cy5-AChE showed that substituted phenanthridinium deriv-
atives bound to the proteins more strongly than their
[5] a) D. P. Walsh, Y. T. Chang, Comb. Chem. High Throughput
Screening 2004, 7, 565; b) A. N. Koehler, A. F. Shamji, S. L.
Schreiber, J. Am. Chem. Soc. 2003, 125, 8420; c) M. Kꢀhn, R.
Wacker, C. Peters, H. Schrꢀder, L. Soulꢁre, R. Breinbauer, C. M.
Niemeyer, H. Waldmann, Angew. Chem. 2003, 115, 6010; Angew.
Chem. Int. Ed. 2003, 42, 5830, and references therein; d) F. G.
Kuruvilla, A. F. Shamji, S. M. Sternson, P. J. Hergenrother, S. L.
Schreiber, Nature 2002, 416, 653; e) K. S. Lam, M. Renil, Curr.
Opin. Chem. Biol. 2002, 6, 353.
[
12]
[6] a) F. Stieber, U. Grether, H. Waldmann, Chem. Eur. J. 2003, 9,
unsubstituted derivatives (Figure 2e).
Finally, we con-
3270; b) F. Stieber, R. Mazitschek, N. Soric, A. Giannis, H.
structed peptide microarrays with the L-tethered strep tag
and 21 of its analogues. The peptide microarrays, after
probing with Cy3-streptavidin, showed that streptavidin
only bound to the strep tag (Figure 2 f). This is consistent
with previous results showing that the amino acid sequence
Waldmann, Angew. Chem. 2002, 114, 4951; Angew. Chem. Int.
Ed. 2002, 41, 4757; c) C. Rosenbaum, H. Waldmann, Tetrahedron
Lett. 2001, 42, 5677.
[7] M. L. Lesaicherre, R. Y. Lue, G. Y. Chen, Q. Zhu, S. Q. Yao, J.
Am. Chem. Soc. 2002, 124, 8768.
8] See the Supporting Information for experimental details.
9] I. E. Liener, N. Sharon, I. J. Goldstein, The Lectins: Properties,
Functions, and Applications in Biology and Medicine, Academic
Press, New York, 1986.
10] T. G. M. Schmidt, A. Skerra, Protein Eng. 1993, 6, 109.
11] T. G. M. Schmidt, J. Koepke, R. Frank, A. Skerra, J. Mol. Biol.
1996, 255, 753.
[
13]
[
[
His-Pro-Gln is critical for streptavidin binding.
In conclusion, a new, efficient, and simple method for
fabricating chemical microarrays has been developed. The
technique employs selective immobilization reactions of
hydrazide-linked small molecules with epoxides on the solid
surfaces. The length of the tether between the hydrazide
groups and the active ligands governs protein binding; slides
containing longer tethers exhibit stronger binding of proteins
than those with shorter tethers. The immobilization technique
is suitable for covalently attaching diverse compounds,
including small molecules, carbohydrates, and peptides, to
glass surfaces. The utility of this method is shown by its
application to the fabrication of peptide and small-molecule
microarrays, which have been used to screen for selective
protein binding. We believe that the chemoselective ligation
reaction developed in this effort will find many applications in
the preparation of bioconjugates, such as neoglycopeptides,
peptide–nucleic acid conjugates, and tagged peptides.
[
[
[12] a) W. G. Lewis, L. G. Green, F. Grynszpan, Z. Radi c´ , P. R.
Carlier, P. Taylor, M. G. Finn, K. B. Sharpless, Angew. Chem.
2002, 114, 1095; Angew. Chem. Int. Ed. 2002, 41, 1053; b) Z.
Radi c´ , P. Taylor, J. Biol. Chem. 2001, 276, 4622.
[
13] a) J. J. Devlin, L. C. Panganiban, P. E. Devlin, Science 1990, 249,
404; b) K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M.
Kazmierski, R. J. Knapp, Nature 1991, 354, 82.
Received: November 25, 2004
Published online: April 12, 2005
Keywords: chemoselectivity · high-throughput screening ·
.
immobilization · microarrays · protein binding
[
1] For recent reviews, see: a) R. S. Lokey, Curr. Opin. Chem. Biol.
2003, 7, 91; b) C. A. MacRae, R. T. Peterson, Chem. Biol. 2003,
10, 901; c) A. C. Bishop, O. Buzko, K. M. Shokat, Trends Cell
Biol. 2001, 11, 167; d) B. R. Stockwell, Trends Biotechnol. 2000,
8, 449.
1
[
2] For recent reviews of DNA microarrays, see: a) S. V. Chittur,
Comb. Chem. High Throughput Screening 2004, 7, 531; b) D. J.
Villeneuve, A. M. Parissenti, Curr. Top. Med. Chem. 2004, 4,
1329; c) W. H. Koch, Nat. Rev. Drug Discovery 2004, 3, 749.
[
3] For recent reviews of protein microarrays, see: a) F. X. Zhou, J.
Bonin, P. F. Predki, Comb. Chem. High Throughput Screening
2004, 7, 539; b) H. Zhu, M. Snyder, Curr. Opin. Chem. Biol. 2003,
7, 55; c) D. S. Wilson, S. Nock, Angew. Chem. 2003, 115, 510;
Angew. Chem. Int. Ed. 2003, 42, 494; d) T. Kodadek, Chem. Biol.
2
884
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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