Preparation of carbohydrate arrays by using Diels-Alder reactions with inverse electron demand

Article abstract of DOI:10.1002/chem.201200382

Carbohydrate microarrays are an emerging tool for the high-throughput screening of carbohydrate-protein interactions that represent the basis of many biologically and medicinally relevant processes. The crucial step in the preparation of carbohydrate arrays is the attachment of carbohydrate probes to the surface. We examined the Diels-Alder reaction with inverse-electron-demand (DARinv) as an irreversible, chemoselective ligation reaction for that purpose. After having shown the efficiency of the DARinv in solution, we prepared a series of carbohydrate-dienophile conjugates that were printed onto tetrazine-modified glass slides. Binding experiments with fluorescently labeled lectins proved successful and homogeneous immobilization was achieved by the DARinv. For immobilization of nonfunctionalized reducing oligosaccharides we developed a bifunctional chemoselective linker that enabled the attachment of a dienophile tag to the oligosaccharides through oxime ligation. The conjugates obtained were successfully immobilized on glass slides. The presented strategies for the immobilization of both synthetic carbohydrate derivatives and unprotected reducing oligosaccharides facilitate the preparation of high-quality carbohydrate microarrays by means of the chemoselective DARinv. This concept can be readily adapted for the preparation of other biomolecule arrays. Copyright

Full text of DOI:10.1002/chem.201200382

FULL PAPER
DOI: 10.1002/chem.201200382
Preparation of Carbohydrate Arrays by using Diels–Alder Reactions with
Inverse-Electron-Demand
Henning S. G. Beckmann,[a] Andrea Niederwieser,[a] Manfred Wiessler,[b] and
Valentin Wittmann*[a]
Abstract: Carbohydrate microarrays
are an emerging tool for the high-
throughput screening of carbohydrate–
protein interactions that represent the
basis of many biologically and medici-
nally relevant processes. The crucial
step in the preparation of carbohydrate
arrays is the attachment of carbohy-
drate probes to the surface. We exam-
ined the Diels–Alder reaction with in-
verse-electron-demand (DARinv) as an
irreversible, chemoselective ligation re-
action for that purpose. After having
shown the efficiency of the DARinv in
solution, we prepared a series of carbo-
hydrate–dienophile conjugates that
were printed onto tetrazine-modified
glass slides. Binding experiments with
fluorescently labeled lectins proved
successful and homogeneous immobili-
zation was achieved by the DARinv.
For immobilization of nonfunctional-
ized reducing oligosaccharides we de-
veloped a bifunctional chemoselective
linker that enabled the attachment of
a dienophile tag to the oligosaccharides
through oxime ligation. The conjugates
obtained were successfully immobilized
on glass slides. The presented strategies
for the immobilization of both synthet-
ic carbohydrate derivatives and unpro-
tected reducing oligosaccharides facili-
tate the preparation of high-quality car-
bohydrate microarrays by means of the
chemoselective DARinv. This concept
can be readily adapted for the prepara-
tion of other biomolecule arrays.
Keywords: carbohydrates · Diels–
Alder reaction · high-throughput
screening · immobilization · micro-
arrays
Introduction
bilization of carbohydrates onto a surface have been report-
ed.[4] These methods include noncovalent strategies such as
the adhesion of polysaccharides[5] or neoglycolipids[6] on ni-
trocellulose surfaces, or the adhesion of fluorous-tagged gly-
cans[7] on fluorous surfaces. As an alternative, immobiliza-
tion strategies that anchor glycans permanently onto surfa-
ces by forming a covalent bond have been reported. Ap-
proaches for the covalent, site-nonspecific (i.e., randomly
oriented immobilization) include, for example, the immobili-
zation of carbohydrates onto photoreactive surfaces.[8] How-
ever, most methods aim for site-specific covalent immobili-
zation that presents the glycans in their natural orientation
towards their binding partners. To achieve this, the carbohy-
drate probes must be equipped with a suitable functional
group that can react with the solid support. Examples in-
clude the attachment of thiols[9] or amines[10] for immobiliza-
tion on electrophilic surfaces, the attachment of maleimide
residues[11] for immobilization on thiol-coated surfaces, the
attachment of dienes[12] for immobilization through classical
Diels–Alder reactions, and application of copper-catalyzed
azide–alkyne cycloaddition reactions.[13] These groups can be
easily installed when the carbohydrates are of synthetic
origin. In contrast, the site-specific covalent immobilization
of nonfunctionalized reducing oligosaccharides isolated
from biological samples remains a particular challenge. The
aldehyde functionality of the reducing end of unprotected
oligosaccharides can be used for chemoselective derivatiza-
tion by reductive amination,[14] by reaction with alkoxy-
Carbohydrate–protein interactions play a fundamental role
in various critical intra- and intercellular events, such as cell
differentiation, immune response, inflammation, cancer
metastasis, and pathogen adhesion.[1] Consequently, these in-
teractions are of high biological and medicinal relevance
and the development of efficient methods for their investi-
gation is a crucial objective in the field of modern glycobiol-
ogy.[2] Carbohydrate microarrays are an emerging technolo-
gy that enables the high-throughput screening of such carbo-
hydrate–protein interactions.[3] Many methods for the immo-
[a] Dr. H. S. G. Beckmann, Dipl.-Chem. A. Niederwieser,
Prof. Dr. V. Wittmann
Department of Chemistry and
Konstanz Research School Chemical Biology (KoRS-CB)
University of Konstanz
78457 Konstanz (Germany)
Fax : (+49)7531-88-4573
E-mail: mail@valentin-wittmann.de
[b] Prof. Dr. M. Wiessler
German Cancer Research Center
Division Medical Physics in Radiology
Biological Chemistry Group, INF 280
69120 Heidelberg (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201200382. It contains complete
1
experimental procedures, characterization data, and copies of the H
and 13C NMR spectra for all synthesized compounds as well as addi-
tional references.
AHCTUNGTRENNUNG
amines or hydrazides,[15] or by conversion into glycosyl
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amines followed by acylation.[11,16] Although the direct im-
mobilization of reducing carbohydrates onto aminooxy- and
hydrazide-coated slides has been reported,[17] a two-step pro-
cedure employing bifunctional linkers offers advantages.
Typically, glycans isolated from natural sources, such as gly-
coproteins, are obtained as complex mixtures due to the mi-
croheterogeneity of the glycoconjugates. These mixtures
have to be separated prior to immobilization, which usually
requires attachment of a label to allow detection in chroma-
tographic processes. A suitably designed bifunctional linker
that can be attached to the reducing end of the glycans may
first serve as a label for purification, then, in a second step,
the obtained carbohydrate conjugates could be covalently
immobilized through the second functionality of the linker.
For this purpose, common nucleophile-based immobilization
methods such as thiol-maleimide addition[11,16a] or the re-
ACHTUNGTRENNUNG
action of amines[14–15,16b] with epoxides or activated esters
have been reported. The use of an inert but chemoselective-
ly addressable functionality instead of amines or thiols
would be beneficial in terms of handling and purification of
such conjugates. In addition, it should be compatible with
biologically relevant amino sugars. To the best of our knowl-
edge, such an approach to the immobilization of nonfunc-
tionalized carbohydrates based on a chemoselective ligation
has not been reported.
Scheme 1. DARinv of tetrazine 2 with exo-norbornene derivative 1 in so-
lution. The resulting tautomeric dihydropyridazine adducts 3 were oxi-
dized to pyridazine 4 to facilitate characterization.
porting Information for synthetic details), the amide bond
of which can be employed for convenient attachment to mo-
lecular entities.
Recently, we[18] and others[19] recognized the potential of
An initial DARinv experiment in solution is shown in
Scheme 1. The reaction of tetrazine 2 with norbornenedicar-
boxylic imide 1 proceeded smoothly at room temperature.
As expected, the intermediate dihydropyridazine 3 was iso-
lated as a mixture of several interconverting tautomeric
forms. Although this is not an obstacle for the application of
the DARinv on solid support, in this case, the tautomers of
3 were oxidized to the homogeneous pyridazine 4, which
was isolated in an excellent yield over two steps and fully
characterized.
Diels–Alder
reactions
with
inverse-electron-demand
(DARinv) as promising bioorthogonal ligation reactions.
The irreversible reaction of tetrazines as electron-deficient
dienes with electron-rich dienophiles proceeds without the
need for any additives, such as cell-toxic metal ions, and is
driven by a high thermodynamic force. Subsequently, the
DARinv was used for labeling of various biomolecules and
affinity probes.[20] Due to its outstanding characteristics, we
anticipated that the DARinv would also be an ideal chemo-
selective reaction for the preparation of biomolecule arrays.
Here, we report efficient methods for the immobilization of
both synthetic carbohydrate derivatives and unprotected re-
ducing oligosaccharides by DARinv.
Immobilization of synthetically derived carbohydrate–dieno-
phile conjugates: Encouraged by this initial experiment in
solution, we prepared a range of carbohydrate–dienophile
conjugates as probes for array experiments (Scheme 2). Oli-
go(ethylene glycol)-tethered norbornenedicarboxylic imides
or terminal alkenes were attached to GlcNAc, mannose, and
lactose by glycosylation (see the Supporting Information for
synthetic details). Norbornane conjugates 7 and 11, which
were obtained by hydrogenation of the corresponding nor-
bornenes, cannot undergo Diels–Alder reactions. They were
designed as probes for unspecific immobilization of the car-
bohydrate conjugates. Compound 13 carries no carbohy-
drate epitope and was used as a probe for unspecific binding
of the lectins to the Diels–Alder adduct or the oligo(ethy-
lene glycol) tether.
Results and Discussion
DARinv in solution: We identified exo-norbornenedicarbox-
ylic imides (such as 1, cf. Scheme 1) as appropriate dieno-
philes. Norbornenes exhibit an exceptionally high reactivity
in the DARinv.[21] Additionally, norbornenedicarboxylic
imides are stable dienophiles that are easily accessible from
the corresponding, commercially available anhydride (see
the Supporting Information for details) and, due to their
symmetry, do not give rise to the formation of regioisomers.
Beside norbornene dienophiles, we assumed that simple ter-
minal alkenes might also be attractive dienophilic tags. Al-
though alkenes are expected to exhibit a lower DARinv re-
activity compared with norbornenes, they are smaller and
the reaction products are less complex. As electron-deficient
dienes, we chose tetrazine derivatives such as 2 (see the Sup-
For the preparation of the carbohydrate arrays, commer-
cially available amine-coated glass slides 14 were used
(Scheme 3). The slides were diene-functionalized in one step
by reaction with tetrazine active ester 15. Solutions of carbo-
hydrate–dienophile conjugates and controls 5–13 in water/
dimethyl sulfoxide (DMSO) (4:1) were spotted onto the tet-
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Preparation of Carbohydrate Arrays
FULL PAPER
Scheme 2. Carbohydrate–dienophile conjugates.
Lack of fluorescence at positions for which the hydrogenat-
ed conjugates 7 and 11 had been spotted indicates that im-
mobilization does not occur by simple adhesion but by cova-
lent immobilization of the carbohydrate–dienophile conju-
gates.
The measured average fluorescence intensities are shown
in Figure 1b. Spots of norbornene-tagged carbohydrates
generally exhibited higher fluorescence intensities than
spots of carbohydrates tagged with a terminal alkene (cf. 5
vs. 6 and 8 vs. 9). This is likely due to the lower dienophilic
reactivity of terminal alkenes compared with norbornenedi-
carboxylic imides. Much weaker fluorescence intensity was
observed for compound 10, which lacks the spacer between
the carbohydrate and the dienophile (cf. 9 vs. 10). This high-
lights the importance of a spacer for the efficient interaction
of lectins with carbohydrates on a solid support, as previous-
ly observed for other immobilization reactions.[4b,24] In gen-
eral, very homogeneous spots were obtained, as exemplified
by the cross section through a row of spots shown in Fig-
ure 1c. This is a very important prerequisite if quantitative
data such as dissociation constants or IC50 values are to be
determined.[25]
Scheme 3. Preparation of carbohydrate microarrays.
razine-functionalized slides 16 using an automated array
printer. For each concentration, nine identical replicates
were printed in a block (3ꢁ3). By carrying out the immobi-
lization in a humidity chamber under controlled conditions
under which the spots do not dry out, we were able to
obtain very homogeneous spots. Immobilized carbohydrates
were detected by incubating the slides 17 with a rhodamine-
conjugated lectin followed by fluorescence scanning. Em-
ployed lectins include wheat germ agglutinin (WGA), which
is specific for N-acetylglucosamine (GlcNAc) residues,[22]
concanavalin A (Con A), which is specific for mannose resi-
dues, and peanut agglutinin (PNA), which is specific for ter-
minal galactose residues, such as those occurring in the T-
antigen or in lactose.[23]
The fluorescence images are shown in Figure 1a. Selective
binding of the lectins to their specific carbohydrate epitopes
was observed (GlcNAc conjugates 5 and 6 for WGA, man-
nose conjugates 8–10 for Con A and lactose conjugate 12
for PNA). For spots in which linker 13 had been immobi-
lized (which carries no carbohydrate epitope), only fluores-
cence near the background level was measured. This result
indicates that the lectins do not bind to the DARinv adduct.
Immobilization of unfunctionalized carbohydrates: After
the preparation of carbohydrate arrays using synthetically
prepared carbohydrate–dienophile conjugates had been es-
tablished, we investigated methods for the specific immobili-
zation of unprotected carbohydrates as they are typically ob-
tained from biological samples. Such methods involve the
specific attachment of a dienophile handle to the unprotect-
ed carbohydrate, followed by immobilization of the formed
conjugate on a tetrazine chip. We selected oxime ligation as
a powerful tool for the selective functionalization of unpro-
tected carbohydrates that proceeds without additives.[26] As
dienophile, we chose the reactive exo-norbornenedicarbox-
ylic imide, which was connected to the oxyamine by an oli-
go(ethylene glycol) tether, resulting in bifunctional linker 18
(Scheme 4, see the Supporting Information for details).
N,N’-Diacetylchitobiose 19 and mannotriose 20 were incu-
bated with linker 18 in acetate buffer at 378C (Scheme 4).
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V. Wittmann et al.
of inertness, it is superior to
previously reported amine or
thiol tags. Further develop-
ments could include the incor-
poration of a fluorescent tag
and/or an isotopic label into the
bifunctional linker providing
additional means for high-sensi-
tivity purifications as well as for
mass spectrometry analyses.
Conclusion
We have established the effi-
cient immobilization of synthet-
ic as well as unmodified mono-
and oligosaccharides on tetra-
zine-derivatized glass slides
using
a Diels–Alder reaction
with inverse-electron-demand
(DARinv). Two different dieno-
philic tags, terminal alkenes and
ring-strained norbornene deriv-
atives were incorporated into
synthetically derived carbohy-
drate–dienophile
conjugates,
the latter of which proved to be
more efficient for immobiliza-
tion. By using various probes,
we demonstrated that the car-
bohydrate–dineophile
conju-
gates were covalently immobi-
lized through the DARinv. Bi-
functional linker 18 enables the
convenient attachment of a di-
enophile to unprotected, reduc-
ing oligosaccharides through
chemoselective oxime ligation.
Figure 1. a) Fluorescence images of carbohydrate arrays after incubation with rhodamine-labeled WGA,
Con A, and PNA. b) Fluorescence intensities obtained from these fluorescence images. c) Cross section
through a row of spots of the array incubated with WGA.
After 24 h, HPLC analysis indicated a yield of 76% for con-
jugate 21 and 90% for conjugate 22. Dilution series of these
reaction mixtures were directly spotted onto tetrazine chips
as described above. Again, the successful immobilization of
both oligosaccharides was verified by incubation with rhoda-
mine-labeled lectins and subsequent fluorescence scanning.
After incubation with WGA, spots of the tagged N,N’-diace-
tylchitobiose 21 exhibited fluorescence as well as spots of
the tagged mannotriose 22 after incubation with Con A
(Figure 2). These results indicate that bifunctional linker 18
is a valuable tool for dienophile-tagging and subsequent im-
mobilization of unfunctionalized oligosaccharides. This two-
step process is experimentally straightforward and does not
require any additives or special equipment. Because most
oligosaccharide samples from biological sources are ob-
tained as complex mixtures, a separation is often necessary
prior to immobilization on the array. Due to the UV absorb-
ance of the imide, 18 facilitates HPLC separations. In terms
The resulting conjugates can be directly immobilized on tet-
razine-derivatized glass slides as we demonstrated in proof-
of-principle experiments. The methods reported herein facil-
itate the preparation of high-quality carbohydrate microar-
rays and are expected to be of great value in the emerging
field of glycomics. Additionally, this chemoselective immobi-
lization procedure can be readily adapted for the prepara-
tion of other biomolecule arrays.
Experimental Section
General procedure for carbohydrate functionalization using bifunctional
linker 18: A stock solution of bifunctional linker 18 (19 mm) was pre-
pared in acetate buffer (100 mm, pH 4.0) and the pH was readjusted to
pH 4. An aliquot of the stock solution of 18 (typically 200 mL, 1 equiv)
was added to the reducing carbohydrate (1.5 equiv) and the mixture was
held for 24 h at 378C. Subsequently, the mixture was analyzed by HPLC
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Scheme 4. a) Application of bifunctional linker 18 for immobilization of reducing oligosaccharides. b) HPLC chromatograms (UV detection at 220 nm)
after 24 h of oxime ligation and calculated yields of ligation products.
and HRMS and the turnover was estimated by comparing the peak areas
(220 nm) of 18 in the mixture and in the stock solution.
was cooled to 108C. Every probe was spotted in nine replicates (3ꢁ3).
After spotting, the slides were stored at RT in a closed petri dish on
a tray over NaCl solution (0.5 m) for 24 h. Subsequently, the slides were
briefly rinsed with MeOH followed by shaking gently in MeOH (3ꢁ
5 min).
Conjugate 21: Bifunctional linker 18 and N,N’-diacetylchitobiose 19 were
reacted according to the general procedure. RP-HPLC (20–40% B in
20 min): tR =8.91 min; HRMS (ESI-IT): m/z calcd for C33H52N4O16
:
761.3451 [M+H]+; found: 761.3437; calcd for C33H51N4O16 +Na+:
783.3271 [M+Na]+; found: 783.3255.
Incubation with fluorescence-labeled lectins: The carbohydrate arrays
were immersed in a solution of rhodamine–labeled lectins (1 mgmLÀ1) in
HEPES buffer (10 mm HEPES, 150 mm NaCl, pH 7.5, 0.1 mm CaCl2,
0.01 mm MnCl2 for WGA and Con A; 10 mm HEPES, 150 mm NaCl,
pH 7.5, 1 mm CaCl2, 1 mm MnCl2 for PNA) containing BSA (1% w/v).
Utilized lectins rhodamine–Con A, rhodamine–PNA, and rhodamine–
WGA were all purchased from Vector Laboratories (Burlingame, USA).
After gently shaking in the dark at RT for 1 h, the slides were gently
shaken in HEPES buffer with 0.1% Tween 20 (2ꢁ5 min) followed by
two very quick washing steps in water. Finally the slides were dried in
a stream of nitrogen.
Conjugate 22: Bifunctional linker 18 and 3,6-di-O-(a-d-mannopyrano-
syl)-d-mannopyranose 20 were reacted according to the general proce-
dure. RP-HPLC (20–40% B in 20 min): tR =6.09 min; HRMS (ESI-IT):
m/z calcd for C35H56N2O21 841.3448 [M+H]+; found: 841.3428; calcd for
C35H55N2O21 +Na: 863.3268 [M+Na]+; found: 863.3251.
Tetrazine-functionalization of glass slides: Aminopropylsilanilized glass
slides (Nexterion Slide A+ from Schott, Mainz, Germany) were im-
mersed in a solution of tetrazine active ester 15 (5 mm in anhydrous
DMSO containing 5% pyridine) for 72 h. Subsequently, the slides were
washed once with DMSO and twice with acetone for 5 min each. The
slides were dried in a stream of nitrogen and stored under nitrogen.
Fluorescence readout: Fluorescence of rhodamine–lectin conjugates
bound to the arrays was quantified by an array scanner (GenePix Person-
al 4100 A from Axon, now Molecular Devices, Sunnyvale, USA; excita-
tion 532 nm, detection filters 550–600 nm). Microarray image data were
analyzed by using GenePix 4.1 software. Median fluorescence of the spot
and of the surrounding background was determined for every spot. The
spot-fluorescence and the background-fluorescence were averaged over
the nine replicates. Given error values were calculated based on the stan-
dard deviations of the nine replicates.
Immobilization of carbohydrate probes: Dilution series of carbohydrate–
dienophile conjugates in water (or in acetate buffer in the case of func-
tionalized reducing carbohydrates) with 20% DMSO were spotted onto
tetrazine-functionalized glass slides (4 nL per spot) using an automated
array printer (Nano-Plotter 2 from GeSiM, Grosserkmannsdorf, Germa-
ny) that was equipped with a thermostat (Unistat Tango, Huber Kꢂltema-
schinenbau, Offenburg, Germany) and a Humidity Control II (Lucky
Reptile, Waldkirch, Germany) connected to a mist generator (Hobby
Hygro Plus, Dohse Aquaristik, Grafschaft-Gelsdorf, Germany). During
spotting, the relative humidity was adjusted to 68–70% and the slide tray
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Figure 2. Fluorescence images and intensities of carbohydrate arrays after
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Preparation of Carbohydrate Arrays
FULL PAPER
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Received: February 5, 2012
Published online: && &&, 2012
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V. Wittmann et al.
Served on a plate: Chemoselective
Carbohydrate Microarrays
immobilization of synthetic dieno-
phile–carbohydrate conjugates has
been achieved on tetrazine-derivatized
glass slides by employing Diels–Alder
reactions with inverse-electron-
demand. By using a bifunctional
linker, unmodified reducing oligosac-
charides can also be selectively tagged
with a dienophile and subsequently
immobilized (see figure).
H. S. G. Beckmann, A. Niederwieser,
M. Wiessler, V. Wittmann* . . &&&& — &&&&
Preparation of Carbohydrate Arrays
by using Diels–Alder Reactions with
Inverse-Electron-Demand
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www.chemeurj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!