Synthesis of a Difunctional Orthogonal Coupler for the Preparation of Carbohydrate-Functionalized sP(EO-stat-PO) Hydrogels

Article abstract of DOI:10.1002/mabi.201100041

The synthesis and characterization of a new difunctional coupler (4) based on trimethylolpropane (TMP) are described. The coupler is used to connect biologically active N-acetylglucosamine (GlcNAc) on amino-reactive microtiter plates and on star-shaped po

Full text of DOI:10.1002/mabi.201100041

Full Paper
Synthesis of a Difunctional Orthogonal Coupler
for the Preparation of Carbohydrate-
Functionalized sP(EO-stat-PO) Hydrogelsa
Thorsten Anders, Kathrin Adamiak, Helmut Keul,* Lothar Elling,
Martin Mo¨ller*
The synthesis and characterization of a new difunctional coupler (4) based on trimethylol-
propane (TMP) are described. The coupler is used to connect biologically active N-acetylglucos-
amine (GlcNAc) on amino-reactive microtiter plates and on star-shaped poly[(ethylene oxide)-
OH
OH
OH
OH
HO
HO
stat-(propylene oxide)] hydrogel layers in microtiter
plates. The biological activity of immobilized carbo-
hydrates is determined using an enzyme-linked lectin
assay.
AcHN
AcHN
O
O
N
N
N
N
N
N
O
O
OH
O
O
OH
O
O
HN
HN
O
O
NH
NH
NH
Introduction
O
NH2
O
NH2
H2N
NH2
NH2
NH
NH2
2NH
H2
H2
N
N
O
NH2
NH2
NH2
NH2
Post-synthetic modification of polymers and the functio-
nalization of surfaces are very important for scientific and
technical applications. Polymers or surfaces frequently do
not contain complementary functional groups for coupling
with functional molecules. Thus, to combine different
H2
N
O
4
NH2
HN
NH2
NH
O
H
N
H
N
H
N
O
N
HN
carbohydrate functionalized
sP(EO- -PO) hydrogel
stat
molecules, couplers with multiple reactive groups are
required. In our group, carbonate couplers have been
developed based on glycerol carbonate, to combine
monodisperse or polymeric building blocks (Scheme 1).[1–4]
An amino-functionalized building block A reacts via
nucleophilic substitution reaction with a difunctional
carbonate coupler, resulting in a functionalized carbonate
coupler. By a further addition reaction, a second building
block B is attached, resulting in formation of difunctional
mono- or polydisperse molecules. Because of differences in
reactivities between the two carbonate functional groups
towards amino nucleophiles, it is possible to perform these
reactions selectively. Recently, the reactivity of difunc-
tional carbonate couplers with different leaving groups
(LGs) was reported, and their coupling ability towards
amino acid methyl esters and glucosamine was deter-
mined;[5] LGswerepara-nitrophenol, phenol, imidazoleand
acetone oxime. It was shown that the substitution reaction
is much faster than the addition reaction; no conversion of
the five-membered ring carbonate was observed below
70 8C. Further examples of bifunctional couplers based on
five-membered cyclic carbonates were reported by He
et al.[4] If building block B is a polyamine, it is possible to
prepare multifunctional polymers for different applica-
tions; e.g., Pasquier applied this method for the preparation
of antimicrobial coatings.[1,6]
T. Anders, Dr. H. Keul, Prof. M. M¨oller
DWI an der RWTH Aachen e.V., and Institute of Technical and
Macromolecular Chemistry, RWTH Aachen, Forckenbeckstraße
50, D-52056 Aachen, Germany
E-mail: keul@dwi.rwth-aachen.de, moeller@dwi.rwth-aachen.de
K. Adamiak, Prof. L. Elling
Laboratory for Biomaterials, Institute of Biotechnology and
Helmholtz-Institute for Biomedical Engineering, RWTH Aachen,
Worringerweg 1, D-52056 Aachen, Germany
a Supporting Information for this article is available from the Wiley
Online Library or from the author.
A similar strategy designed to connect two building
blocks via a coupler was followed in the present study. To
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DOI: 10.1002/mabi.201100041 1201

T. Anders, K. Adamiak, H. Keul, L. Elling, M. Mo¨ller
www.mbs-journal.de
O
O
Carbohydrate functionalized linkers are
preparedviachemoenzymaticorchemical
catalyzed reactions. Enzymatic elonga-
tion, starting from functionalized mono-
saccharides, is an effective way to prepare
glycan structures for immobiliza-
tion.[16,23] In the present study, new
orthogonal couplers based on TMP were
developed for the direct immobilization of
carbohydrates on poly[(ethylene oxide)-
stat-(propylene oxide)] sP(EO-stat-PO)
coated microtiter plates.
NH2
+
H2N building block A
H2N
NH2
O
O
LG
H2N
O
NH2
NH2
difunctional carbonate coupler
- H LG
LG = leaving group
O
O
O
O
polyamine
H
N
building block A
+
functionalized
O
carbonate couplers
functionalized carbonate coupler
H2N building block B
To date, numerous research groups have
worked on the preparation and testing of
glycan structures, and most have used
amine or azide functionalized glycan struc-
tures for immobilization.[22] Incontrast, the
new coupler presented here has the poten-
tial to attach nearly any azide functiona-
lized glycan structure to an amino reactive
OH
H
N
H
N
building block B
building block A
O
O
multifunctional polyamines
O
O
difunctional mono- or polydispers molecules
Scheme 1. Coupling strategies for preparation of difunctional or multifunctional
molecules via carbonate couplers.[5]
accomplish this, an orthogonal difunctional coupler, a six-
membered ring carbonate with a propargyl ether side
chain, which is based on trimethylolpropane (TMP), was
synthesized, and is able to connect building blocks via
orthogonal reactions.[7]
surface; and can possibly be used to attach amine
functionalized glycan structures to azide reactive surfaces.
sP(EO-stat-PO)s are known to be excellent materials for
surface modification, which avoid non-specific protein
adsorption.[24–27] The specific properties of sP(EO-stat-PO)-
coated surfaces depend mainly on layer thickness and
surface grafting density.[28] Through the application of
sP(EO-stat-PO) coatings, selective functionalization for
bioanalytical and biomedical applications is possible.[25]
Sauerzapfe et al.[23] described for the first time, the biofun-
ctionalization of sP(EO-stat-PO) coated surfaces with poly(N-
acetyllactosamine) (poly-LacNAc; [3Gal-b1,4GlcNAcb1,]n).[29]
Our aim was to prepare carbohydrate and glycan
building blocks for glycoarrays and glycopolymers, which
are becoming increasingly important in glycobiology and
glycobiotechnology.
Glycan chains of glycoproteins, glycolipids and glycosa-
minoglycans play important roles in biological recognition
processes.[8,9] Thus, the creation of macromolecules with
different architectures for carrying biologically active
glycans is essential for the development of applications
in biomedicine and biology.[10–13]
O
carbohydrate
N
carbohydrate
carbohydrate
NH2
The biological activity of such glycoconjugates is often
tested using enzyme-linked lectin assays (ELLA). Lectins are
carbohydrate binding proteins which interact non-cova-
lently with glycan epitopes in a reversible and highly
specific manner; making them invaluable tools in the
researchofcarbohydratesinsolution, atcellsurfacesandon
artificial solid surfaces.[14]
NH
O
O
O
O
carbohydrate
N
N
N3
Platforms for the display of carbohydrates which are
ready to interact in lectin-based assays are referred to as
carbohydrate arrays.[15] Covalent immobilization of carbo-
hydrates is accomplished by functionalization of sugars at
their reducing end, in combination with complementary
activated surfaces, as previously described.[15–18] Possible
techniquesinclude:amidebondformationbetweenamine-
containing sugars and an activated ester-derivatized
surface; the use of 1,3-dipolar cycloaddition between an
azide and an alkyne (the so called ‘click reaction’);[19–21]
and reaction of thiols with substituted maleimides
(Scheme 2).[22]
N
carbohydrate
S
carbohydrate
SH
O
O
O
O
N
N
Scheme 2. Examples of the most widespread techniques for
covalent immobilization of carbohydrates on arrays.[22]
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Synthesis of a Difunctional Orthogonal Coupler for the Preparation . . .
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microtiter plates from Nunc and CovaLink
NH-Module microtiter plates from Nunc were
used as received. Phosphate-buffered saline
(PBS)solutionwaspreparedby(0.05M Na2HPO4,
0.15 M NaCl, pH¼ 7.2) THF was dried by reflux-
ing over sodium and distillation. Further
compounds were technical grade and used as
received. Water sensitive reactions were per-
formed under inert gas atmosphere (nitrogen).
Synthesis
(5-Ethyl-2,2-dimethyl-1,3-dioxan-5-
yl)methanol (1)
Scheme 3. Immobilization concept for carbohydrates on an amino-reactive substrate via
a difunctional orthogonal coupler.
TMP (53.92 g, 396.5 mmol) was treated with
acetone according to Perrier.[31] Resulting analytical data were in
accordancewiththeliterature, and compound1 wasobtainedwith
quantitative conversion.
Inthepresentstudy, weaimedtoprepareandinvestigate
biologically active carbohydrate functionalized sP(EO-stat-
PO) hydrogels, using a coupling strategy developed for
orthogonal couplers. The coupling concept includes the
connection of a carbohydrate building block with a
protected amine, using a difunctional orthogonal coupler
(Scheme 3). Removal of the protection group gives the
carbohydrate coupler, which can then be immobilized to a
surface coated with isocyanate terminated sP(EO-stat-PO).
The binding efficiency of the resulting biofunctionalized
hydrogel was tested by ELLA, and compared to a surface
where the carbohydrate functionalized coupler was
directly immobilized on a commercially available amino
reactive microtiter plate.
5-Ethyl-2,2-dimethyl-5-[(prop-2-ynyloxy)methyl]-1,3-
dioxane (2)
A mixture of (5-ethyl-2,2-dimethyl-1,3-dioxan-5-yl)methanol (1) (1.0
equiv., 63.36g, 363.6mmol), propargyl bromide (2.5 equiv., 78mL,
107.64g, 904.8 mmol) and catalytic amounts of tetrabutylammo-
niumhydrogen sulfate (3.66 g, 10.8 mmol), were dissolved in toluene
(120mL), and a 50% sodium hydroxide solution (120mL) was added
dropwise under vigorous stirring. The emulsion was heated to 608C
for 4 h. After cooling to room temperature, the organic layer was
separated and concentrated. The remaining yellow oil was dissolved
in chloroform (100 mL) and extracted with distilled water (4ꢀ
100mL). The organic solution was concentrated. Yield: 57.39 g
(74.4%). For the labeling of atoms in compounds 2–7 see Scheme 4.
1H NMR (CDCl3, 300 MHz, d): 4.16–4.12 (m, H8), 3.73–3.41
(m, H4, H5), 2.44–2.43 (m, H10), 1.44–1.31 (m, H7, H2), 0.89–0.82
Experimental Section
Materials
Isocyanate-terminated sP(EO-stat-PO) [12 kDa,
polydispersity index (PDI) < 1.2] was prepared
according to the literature.[24–28,30] 2-N-acetyla-
5
5
4
5
5
4
8 9
5
5
4
10
6
7
8
6
7
8
O
6
O
O
1
7
7
O
9
O
1
HO
HO
O
1
mino-2-deoxy-b-D-glucopyranosylazide (b-D-
O
2
3
4
3
3
3
GlcNAc-N3) and tert-butyl-N-(3-aminopropyl)-
carbamate were prepared according supporting
information. TMP from Acros Organics, ethyl
chloroformatefromFluka, sodiumascorbateand
copper sulfate pentahydrate from Applichem,
triethylamine (TEA, 99.5%) from Sigma-Aldrich,
propagyl bromide solution (80% in toluene) from
Acros Organics, phosphomolybdic acid hydrate
from Merck, tetrahydrofuran (THF, p.a.) from
KMF, chloroform (HPLC grade) from Fischer
Scientific, tetrabutyl ammonium hydrogen sul-
fate from Acros Organics, Celite545 from Roth,
silica gel 60 (<0.2mm) from Applichem, bioti-
nylated lectin from Griffonia simplicifolia from
Vector Laboratories Inc, streptavidin peroxidase
from Roche Diagnostics GmbH, o-phenylenedia-
mine dihydrochloride (OPD) substrate from
DakoCytomation, Nunc Immobilizer Amino
O
2
2
2
6
4
13
N
OH
N
O
O
5
2
16
O
11 13
12
OH
N
1
7
O
11
12
OH
O
N
H
N
H
O
15
1
2
14
10
6
14
9
15 AcHN
18
3
8
10
6
3
5
4
OH
5
H
H
20
O
22
21
OH O 16 N
N
9
O
8
17
19
7
O
O
6
13
N
N
OH
5
4
O
OH
N
1
7
8
10
O
11
12
OH
14
2
3
9
15 AcHN
18
7
H
OH O 16 N
NH2
17
19
O
Scheme 4. Labeling of atoms in compounds 4–7.
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T. Anders, K. Adamiak, H. Keul, L. Elling, M. Mo¨ller
www.mbs-journal.de
(m, H1). 13CNMR(CDCl3, 75 MHz,d): 98.1 (C6), 79.9 (C9), 74.4(C10),69.80
mixture of distilled water/tert-butyl alcohol (100 mL/40 mL).
Sodium ascorbate (0.2 equiv. 0.32 g, 1.6 mmol) and
Cu2SO4 ꢂ 5H2O (0.1 equiv., 0.20 g, 0.8 mmol) were added. The
reaction mixture was stirred at room temperature for 72 h. Then
tert-butyl alcohol was evaporated under reduced pressure. The
remaining solution was freeze dried and purified by column
chromatography (ethanol/ethyl acetate, 1:3, Rf ¼ 0.31). Yield: 3.76 g
(75.5%).
(C4), 65.2 (C5), 58.6 (C8), 36.6 (C3), 26.4 (C7), 24.1 (C2), 21.1 (C7), 7.04 (C1).
2-Ethyl-2-[(prop-2-ynyloxy)methyl]propan-1,3-diol (3)
Aqueous hydrochloric acid (18%, 300 mL) was added dropwise to a
solution of 2 (57.39 g, 270.3 mmol) dissolved in technical THF
(300 mL). The reaction mixture was stirred at room temperature for
3 d. After cooling to 0 8C, the reaction mixture was neutralized with
solid sodium hydroxide and sodium bicarbonate. THF was
evaporated under reduced pressure and the resulting solution
was extracted with chloroform (100 mL). The organic solution was
concentrated and the remaining orange colored oil was purified by
column chromatography (ethyl acetate/hexane 1:1, Rf ¼ 0.13).
Yield: 23.79 g (51.1%).
1H NMR (CDCl3, 300 MHz, d): 4.15 (d, H6, J6,8 ¼ 2.4 Hz), 3.67–3.50
(m, H4, H5), 3.41 (OH), 2.49 (s, H8), 1.35–1.33 (m, H2), 0.86 (t, H1,
J1,2 ¼ 7.5 Hz). 13C NMR (CDCl3, 75 MHz, d): 79.7 (C7), 74.9 (C8), 72.1
(C6), 65.5 (C5), 58.7 (C4), 42.9 (C3), 22.7 (C2), 7.5 (C1).
1H NMR (D2O, 400 MHz, d): 8.12 (s, H15), 5.74 (d, H1, J1,2 ¼ 9.7), 4.70
(br, DOH), 4.52 (s, H10), 4.16 (m, H2), 3.81 (m, H6, H11), 3.75–3.5
(m, H3, H4, H5, H6), 3.36(s, H12), 3.27(s, H13), 2.97(m, H17, H19), 1.71(s,
NAc), 1.53 (m, H18), 1.31 (s, H22), 1.23–1.21 (m, H8), 0.68 (m, H7).
13C NMR (D2O, 100 MHz, d): 173.9 (NAc), 158.7 (C16), 158.65 (C20),
144.2 (C14), 124.0 (C15), 86.3 (C1), 78.9 (C5), 73.6 (C3), 69.8 (C13), 69.3
(C4), 65.0 (C11), 63.2 (C10), 61.5 (C12), 57.4 (C6), 55.3 (C2), 42.4 (C9), 37.8
(C17), 37.4 (C19), 29.0 (C18), 27.7 (C22), 22.1 (C8), 21.7 (NAc), 6.5 (C7). IR
(KBr): n ¼ 3 419–3 100 (nOH), 2 939–2 865 (nCH, nCH2, nCH3), 2 096
(nN3), 1 642 (nC¼O, amide), 1 564 (nNH, amide), 1 465–1 261 (dCH,
dOH), 1 099–1 040 (nCO), 635 (C¼C) cmꢁ1
.
5-Ethyl-5-[(prop-2-ynyloxy)methyl]-1,3-dioxan-2-one
(propargyl carbonate coupler 4)
Removal of the Protection Group from 6: Preparation
of 7
1,3-Diol 3 (1.0 equiv., 23.79 g, 138.1 mmol) was dissolved in dry THF
(400 mL) and cooled to 0 8C. Ethyl chloroformate (2.0 equiv., 29.95 g,
276.0 mmol) and TEA (2.0 equiv., 27.92 g, 276.0 mmol) were added
dropwise. The reaction mixture was stirred at room temperature
for 48 h; TEA hydrochloride was filtered off and the filtrate was
concentrated. The remaining brown oil was dried in vacuum. Yield:
24.53 g (89.6%).
Boc-protected compound 6 (3.01 g, 4.87 mmol) was suspended
in distilled water (20 mL) and cooled to 0 8C. Aqueous HCl solution
(2 M, 20 mL) was added dropwise and the reaction mixture
was kept overnight at 4 8C. Then an aqueous sodium hydroxide
solution (1 equiv. with respect to HCl; 1.6 g, 0.04 mol NaOH
in 20 mL distilled water) was added, while the reaction mixture
was maintained at 0 8C. The neutralized solution was freeze
dried and the remaining solid was purified by column chromato-
graphy (ethyl acetate/ethanol 1:1, Rf ¼ 0.145). Yield: 2.07 g
(82.0%).
1H NMR (CDCl3, 400 MHz, d): 4.32 (AA’BB’, H5, JAA BB
’
’ ¼ 10.8 Hz),
4.17 (m, H5, H6), 3.52 (s, H4), 2.51 (s, H8), 1.54 (d, J ¼ 7.1 Hz, H2), 0.94
(t, J ¼ 7.5 Hz, H1). 13C NMR (CDCl3, 100 MHz, d): 148.5 (C9), 78.9 (C7),
75.3 (C8), 72.7 (C5), 67.8 (C6), 58.7 (C4), 35.3 (C3), 23.2 (C2), 7.3 (C1).
1H NMR (D2O, 400 MHz, d): 8.16 (s, H15), 5.78 (d, H1, J1,2 ¼ 9.7), 4.70
(br, DOH), 4.54 (s, H10), 4.19 (m, H2), 3.80 (m, H6, H11) 3.75–3.6
(m, H3, H5, H6), 3.58 (m, H4), 3.34 (s, H12), 3.25 (s, H13), 3.11 (m, H17),
2.94 (m, H19), 1.77 (m, H18), 1.73 (s, NAc), 1.31 (s, H22), 1.23–1.21
(m, H8), 0.68 (m, H7). 13C NMR (D2O, 100 MHz, d): 173.9 (NAc), 158.5
(C16), 158.1(C20), 144.3(C14), 123.6(C15), 86.4(C1), 80.7(C21), 79.9(C5),
73.5(C3), 69.8(C13), 69.3(C4), 64.8 (C11), 63.3 (C10), 61.6(C12), 57.4(C6),
55.3(C2), 42.4(C9), 37.8 (C17), 37.4 (C19), 29.0 (C18), 27.7(C22), 22.1(C8),
21.7 (NAc), 6.5 (C1).
2-Propargyloxymethyl-2-tert-butyloxycarbonylamino-
propylideneaminocarbonyloxymethyl-1-butanol (5)
A solution of propargyl carbonate coupler 4 (1.0 equiv., 16.03 g,
80.9 mmol) dissolved in chloroform (200 mL) was added dropwise
(over a period of 50 min) to a solution of tert-butyl-3-aminopro-
pylcarbamate (1.2 equiv., 16.90 g, 97.0 mmol) in chloroform
(200 mL). The reaction mixture was heated to reflux and stirred
for 96 h. After cooling to room temperature, the reaction mixture
was washed with distilled water (6 ꢀ 100 mL). The organic layer
wasdried oversodiumsulfateandconcentratedto yieldcompound
5 as a yellow oil. Yield: 25.91 g (86.0%).
The yields of all reactions are summarized in Table 1.
1HNMR(CDCl3, 400 MHz,d):5.50(br, NH),4.98(br, NH),4.14–4.04
(m, H7, 6), 3.43 (s, H6, H4), 3.22 (m, H11, H13), 2.46 (br, H9, OH),
1.62 (m, H12), 1.44 (m, H2, H16), 0.88 (t, H1, J1,2 ¼ 7.5 Hz). 13C NMR
(CDCl3, 100 MHz, d): 157.6 (C14), 156.5 (C10), 79.7 (C15), 79.3 (C8), 74.6
(C9), 70.7 (C5), 64.7 (C6), 63.6 (C4), 58.7 (C7), 43.2 (C3), 37.7 (C11), 37.1
(C13), 30.5 (C12), 28.4 (C16), 22.2 (C2), 7.4 (C1). IR (KBr): n ¼ 3 345
(nC CH, nOH), 2 970–2 882 (nCH2, nCH3), 2 115 (weak, nC C), 1 693
(nC¼O, amide), 1 525 (nNH, amide), 1 460–1 170 (dCH, dOH), 1 099–
Table 1. Yield of the reactions.
Entry
Reactant
Product
Yield [%]
1
2
3
4
5
6
1
2
3
4
5
6
2
3
4
5
6
7
74.4a)
51.1
89.6
86.0
75.5
82.0
1 040 (nCO) cmꢁ1
.
Cycloaddition of b-D-GlcNAc-N3 to the Propargyl
Derivative 5: Preparation of 6
Propargyl derivative 5 (1.0 equiv., 3.00 g, 8.1 mmol) and azide sugar
b-D-GlcNAc-N3 (1.0 equiv., 1.98 g, 8.1 mmol) were dissolved in a
a)Raw product.
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substrate from DakoCytomation and stopped with 3 M HCl. The
resulting microtiter plates were measured at OD492 nm.
Immobilization and ELLA of b-D-GlcNAc-
Functionalized Coupler (7) on an Amino-Reactive
Microtiter Plate
For immobilization, a commercial Nunc Immobilizer microtiter
plate was used according to a previously described protocol.[32]
Solutions of b-D-GlcNAc functionalized coupler 7 (5.0 mM) in a
sodium carbonate buffer (0.6 M, pH ¼ 10.4) were prepared. Three
wells were filled with 0.1 mL of solution and incubated overnight.
An additional four blank samples (0.1 mL per well) were prepared,
for measuring background values. The next day, the resulting b-D-
GlcNAc functionalized microtiter wells were blocked with
50 ꢀ 10ꢁ3 m ethanolamine in PBS buffer (0.3 mL per well) for 1 h
at room temperature. Wells were then washed with PBS buffer
(3 ꢀ 0.4 mL per well), and a solution of commercial biotin-labeled
lectin II from Griffonia Simplicifolia (GSII) (10 mg GSII mLꢁ1 in PBS
with 10ꢁ3 M CaCl2, MnCl2 and MgCl2, respectively) was prepared
and added to the b-D-GlcNAc-functionalized wells (0.1 mL per well).
After incubation for 2 h at room temperature, the wells were
washed with PBS buffer (3 ꢀ 0.4 mL per well), and lectin binding
was detected by incubation with streptavidin peroxidase (1:1 000
dilutionof1 mg mLꢁ1).Colorimetricdetectionwasperformedusing
an OPD substrate from DakoCytomation, and stopped with 3 M HCl.
The resulting microtiter plates were measured at OD492 nm.
Chromatography
All reactions were monitored via thin layer chromatography, using
pre-coated Polygram R SIL G/UV254 from Macherey-Nagel. Spots
were detected by UV light (254 nm); and sugar spots were
visualized with 10% sulfuric acid in ethanol followed by heating
with a heat gun. Carbonyl or carboxylic acid groups were detected
using 8% phosphomolybdic acid in ethanol. Column chromato-
graphy was performed on a silica gel (Merck, grain size 0.063–
0.200 mm).
Spectroscopy
1Hand13CNMRspectrawererecordedusingaBrukerDPX300orAV
400 spectrometer at 300 or 400, and 75 or 100 MHz, respectively.
Deuterated chloroform (CDCl3) or deuterated water (D2O) were
used as solvents, and tetramethylsilane or ethanol were used as
internal standards. 1H peak assignments were made by first order
analysis of the spectra. 13C peak assignments were made by first
1H–
order analysis of the spectra, and supported by standard
13C correlation spectroscopy (COSY). Infrared (IR) spectroscopy was
performed using a Nexus 470 FT-IR from ThermoNicolet, with a
spectral resolution of 8 cmꢁ1 in KBr.
Immobilization and ELLA of b-D-GlcNAc-
Functionalized Coupler (7) on Microtiter Plates
Coated with an sP(EO-stat-PO) Hydrogel Film
Results and Discussion
sP(EO-stat-PO) hydrogel layers were prepared in CovaLink NH-
This work presents the synthesis of a difunctional
orthogonal coupler, which enables the combination of
two building blocks under mild reaction conditions. A
flexible immobilization concept is presented, where
common azido-functionalized carbohydrates were immo-
bilized onto amino reactive surfaces (Scheme 3).
Module microtiter plates according to
protocol.[33]
a recently published
Isocyanate terminated sP(EO-stat-PO) (200 mg) dissolved in dry
THF (2.0 mL) was diluted with distilled water (18.0 mL) and shaken
for 5 min, to ensure pre-crosslinking of the isocyanate terminated
sP(EO-stat-PO).[28] Subsequently, the solution was aliquoted to 50
microtiterwells (0.4 mL per well), and incubated for 10 min at room
temperature. Then 0.35 mL of the solution was removed and the
remaining solution (ca. 0.05 mL) was reacted for 1 h. The super-
natant was removed and the wells were used for direct substrate
immobilization.
This concept comprises three steps: (i) synthesis of the
difunctional orthogonal coupler; (ii) preparation of the
carbohydrate functionalized coupler and (iii) immobiliza-
tion on an amino-reactive substrate. The efficiency of
immobilization of the resulting carbohydrate-functiona-
lized substrate was tested using an ELLA.
For substrate immobilization, solutions of b-D-GlcNAc functio-
nalized coupler 7 (5.0 mM) in a sodium carbonate buffer (0.6 M,
pH ¼ 10.4) were prepared. Four wells were filled with 0.1 mL of
solution and incubated overnight. An additional three blank
samples were prepared for measuring background values. The next
day the resulting b-D-GlcNAc-functionalized hydrogels in the
microtiterwellswereincubatedwithMilliQwater(0.3 mL per well)
for 1 h at room temperature. Wells were then washed with PBS
buffer solution (3 ꢀ 0.4 mL per well), and a solution of commercial
biotin-labeled lectin II from Griffonia Simplicifolia (GSII) (10 mg GSII
mLꢁ1 in PBS with 10ꢁ3 M CaCl2, MnCl2 and MgCl2, respectively) was
prepared and added to the b-D-GlcNAc-functionalized wells (0.1 mL
per well). After incubation for 2 h at room temperature, the wells
were washed (3 ꢀ 0.4 mL per well) and lectin binding was detected
by incubation with streptavidin peroxidase (1:1 000 dilution of
1 mg mLꢁ1). Colorimetric detection was performed using an OPD
Synthesis of the Difunctional Orthogonal Coupler (4)
Starting with TMP, the difunctional orthogonal coupler (4)
was prepared in four steps (Scheme 5). The two TMP
hydroxyl groups were protected as described by Perrier,[31]
leading to acetal 1 (for NMR data see Figure S1, Supporting
Information). Acetal 1 was used directly (as a raw product)
for the next step, in which the free hydroxyl group was
reacted with propargyl bromide to give the propargyl
ether 2.
Although compound 2 was first mentioned in the patent
literature, only low yields (38%) were reported; [34] whereas
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OH
(a)
OH
O
O
O
O
O
O
Supporting Information);[36] (ii) the cyclic
carbonate of orthogonal coupler 4 was
reacted with tert-butyl-N-(3-aminopro-
pyl)carbamate, resulting in the functio-
nalized coupler 5 [Scheme 6 step (a)].
This conversion was monitored using
1H and 13C NMR spectroscopy (Figure 1
and 2). The 1H NMR spectrum of 4 shows
signals typical of a six-membered cyclic
carbonate at d ¼ 4.32 and 4.17 (H5).[7,37]
After the ring-opening reaction, these
signals disappear and new signals at
d ¼ 4.04 (H6) and d ¼ 3.43 (H4), consistent
with the formation of the functionalized
coupler 5 appear. In the 13C NMR spec-
trum of coupler 4, signals typical of a six-
membered cyclic carbonate were
observed at d ¼ 148.5 (C9) and 72.7 (C5).
Conversion of coupler 4 to 5 shifts the
carbon atoms of the methylene groups
adjacent to the carbonate group in thesix-
membered ring: thus, the hydroxymethyl
group (C4) appears at d ¼ 63.6 and the
methylene group adjacent to the
urethane (C6) at d ¼ 64.7; and the newly
formed carbamate signal C10 appears at
d ¼ 156.5.
(c)
(d)
(b)
OH OH
O
O
OH OH
O
TMP
1
3
4
2
O
Scheme 5. Synthesis of difunctional orthogonal coupler (4) with different functional-
ities: (a) acetone, cat. p-TsOH, 48 h, room temperature; (b) propargyl bromide, toluene/
50% aqueous KOH (1:1), 60 8C, 4 h; (c) 18% HCl/THF (1:1), room temperature, 3 d; (d) ethyl
chloroformate, 1 equiv. TEA, THF, 0 8C addition, room temperature, 48 h.
Scheme 6. Synthesis of carbohydrate-functionalized coupler (7). (a) tert-butyl-3-
aminopropyl carbamate, chloroform, 80 8C, 3 d; (b) (1) AcCl, room temperature, 3 d;
(2) sodium azide, water/acetone (1:1), room temperature, 2,5 h; (3) sodium methoxide,
methanol, 0 8C, 2 h; (c) (1) copper sulfate, sodium ascorbate, water/tert-butyl alcohol
(1:0.4), room temperature, 72 h; (2) 4 M HCl, 0 8C, 2 h.
the reaction conditions outlined in the present study
produced higher yields (74.4%). Propargyl ether 2 was used
as raw product for deacetalization, in a mixture of half-
concentrated aqueous hydrochloric acid solution and THF
(1:1) at room temperature. The resulting diol (3) was
purified by column chromatography (for NMR data, see
Figure S2, Supporting Information). Cyclization of diol 3
to the difunctional orthogonal coupler 4 was performed
with ethyl chloroformate and TEA as
Forthesecondfunctionalizationstep,b-D-GlcNAc-N3 was
preparedin threesteps[Scheme6step(b)]. First, b-D-GlcNAc
was acetylated according to Horton,[38] functionalized with
sodium azide at the anomeric C-atom according to Ibatullin
and Shabalin[39] and selectively deacetylated according
to Zemple´n and Pacsu[40] (for details of the preparation of b-
D-GlcNAc-N3, see Supporting Information). Azide-functio-
nalized carbohydrates are commonly used for preparation
described.[7,35] Coupler 4 consists of a six-
6
5
4
6
7
8
O
9
O
O
1
(4)
membered ring carbonate with a propar-
gyl ether side group, and is similar to a
previously described orthogonal cou-
pler.[7] The difference between these
couplers is the functional group used for
linking the propargyl group to the six-
membered ring carbonate: an ether group
in coupler 4 versus a urethane group.
O
3
4
2
5
1
5
8
5
2
8
9
7
4
5
HO
O
16
3
H
N
H
N
12
4
5
14
O
O
O 15
7
10
O
1
2
6
16
11 13
(5)
Preparation of the Carbohydrate-
Functionalized Coupler: b-D-GlcNAc
Coupler 7
1
9
EtAc
2.0
6
EtAc
2
13
12
11
EtAc
OH
NH
NH
The b-D-GlcNAc-functionalized coupler 7
was prepared in two steps (Scheme 6): (i)
tert-butyl-N-(3-aminopropyl)carbamate
was prepared according to Mendrek (see
5.0
4.0
3.0
1.0
ppm (t1)
1
Figure 1. H NMR spectra of compounds 4 and 5 in CDCl3 (EtAc ¼ ethyl acetate).
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After the cycloaddition reaction, the
Boc protection group of 6 was removed
using aqueous hydrochloric acid solution
5
5
5
4
6
7
8
O
9
O
O
1
4
1
O
8
3
(4)
6
at low temperature, leading to
7
2
2
[Scheme 6 step (c)]. In this step, the
reaction conditions, and in particular
temperature, are very important, because
of the sensitivity of the glucosamine to
acidic media: with increasing acidity and
increasingtemperaturetherateofdecom-
position increases. Successful conversion
(i.e., cycloaddition and deprotection) was
verified by means of 1H and 13C NMR
spectroscopy (Figure 4; see Figure S3,
3
7
9
8
9
7
16
4
5
7
HO
1
O
3
1
3
9
H
H
12
11 13
14
O
O
N
N
O 15
10
O
2
6
16
(5)
15
4
5
2
12
6
13
11
Supporting Information for the 1H–13
C
10
14
8
EtAc
EtAc
EtAc
EtAc
correlation spectrum). The formation of
the 1,2,3-triazole ring was verified by the
presence of: C14 chemical shift at
d ¼ 144.3; C15 shift at d ¼ 123.6 and of
H15 shift at d ¼ 8.16. This is in accordance
to the literature.[47] Deprotection was
150
100
50
ppm(t1)
Figure 2. 13C NMR spectra of compound 4 and 5 in CDCl3 (EtAc ¼ ethyl acetate).
of glycoconjugates, such as glycoarrays[22,41,42] and glyco-
polymers.[43–45] Here, b-D-GlcNAc-N3 was chosen as the
azide-functionalized carbohydrate because of the well-
known biological activity of b-D-GlcNAc as a ligand of
specific lectins and acceptor substrate for glycotrans-
ferases.[23,29,32,41,46]
verified by the disappearance of the tert-butyloxycarbonyl
group. In addition, small changes in the chemical shift of
the methylene group in a position to the reaction center
were observed: CH2ꢁNHꢁCOꢁOtBu at d ¼ 2.97 and
CH2ꢁNH2 at d ¼ 2.94.
The coupler conjugate 7 can be regarded as a b-D-GlcNAc
linker. Because of the length of the linker, the carbohydrate
should be more accessible to the lectins than in the absence
of aspacer between thesurface andtheb-D-GlcNAc. Thus, the
presence of 7 as a linker may influence the binding affinity
observed by ELLA.
The b-D-GlcNAc-coupler conjugate 6 was synthesized via
1,3-dipolar cycloaddition of b-D-GlcNAc-N3 to the functio-
nalized coupler 5. This cycloaddition can easily be followed
by IR spectroscopy, because of the characteristic signal of
the azido function at ꢃ2 100 cmꢁ1 (Figure 3). Reaction of the
azide with the propargyl group yields a 1,2,3-triazol,
resulting in a significant decrease in peak intensity at
Immobilization and ELLA Test of
2 100 cmꢁ1
.
b-D-GlcNAc-functionalized coupler 7
Immobilization of the b-D-GlcNAc functionalized coupler 7
on commercial Nunc Immobilizer microtiter plates was
performed according to a previously described protocol.[32]
Asolutionofb-D-GlcNAc functionalized coupler7insodium
carbonate buffer (5.0 ꢀ 10ꢁ3 M) was added to Nunc
Immobilizer well plates, incubated overnight and blocked
withethanolaminein PBSbuffer. Ethanolamine reacts with
excess functional groups in the wells. Wells were washed
with PBS buffer before addition of biotin-labeled lectin II.
Lectin binding was detected by incubation with strepta-
vidin peroxidase and colorimetric detection was performed
using OPD substrate.
3
GlcNAc-N3
GlcNAc-coupler (6)
2
1
0
Immobilization of b-D-GlcNAc functionalized coupler 7
on Nunc immobilizer microtiter plates coated with an
sP(EO-stat-PO) hydrogel layer first required preparation
of isocyanate terminated sP(EO-stat-PO).[24–28,30] Briefly,
these polymers were prepared from star shaped 6-armed
statistical copolymers of ethylene oxide/propylene oxide
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Wavelengh [cm-1]
Figure 3. IR spectrum of b-D-GlcNAc-N3 and b-D-GlcNAc coupler
(6).
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DOH
21
mixture was shaken for 5 min, to ensure
pre-crosslinking of the isocyanate termi-
nated sP(EO-stat-PO).[28] Terminal isocya-
nate groups of sP(EO-stat-PO) are par-
tially hydrolyzed, forming primary
amino groups, which react with remain-
ing isocyanate groups to form urea
bridges. This mixture was added to the
microtiterwellsandincubatedfor10 min
at room temperature. Simultaneously,
remaining isocyanate groups on the pre-
crosslinked sP(EO-stat-PO) react with
amino functions on the microtiter wells,
leading to immobilization of the hydro-
gel film. It is assumed that after 1 h of
crosslinking in the microtiter wells,
enough isocyanate groups remain for
binding b-D-GlcNAc-functionalized cou-
pler 7. Thus, sP(EO-stat-PO) coated micro-
titer plates were treated with a solution
of b-D-GlcNAc functionalized coupler 7 in
carbonate buffer (5.0 ꢀ 10ꢁ3 M). Conver-
EtOH
EtOH
10
15
11
5
34
7
1213
17
1
19
2
6
18
8
8.0
ppm (t1)
7.0
6.0
5.0
4.0
3.0
2.0
1.0
6
N
OH
N
13
5
4
O
N
1
OH
7
8
O
11
OH
EtOH
14
2
3
15
9
HN
20
21
8
21
EtOH
2
O
10
OH
12
O
H
18
7
16
N
H2N
9
5
3
1
19
17
19 17
4
O
20
18
15
12
10
11
6
14
13
16
150
100
50
ppm (t1)
Figure 4. 1H and 13C NMR spectra of compound 7 in D2O. EtOH ¼ ethanol internal
standard.
sion of all iscocyanate groups was complete after incuba-
tion overnight under ambient conditions.[28]
(EO/PO) with a ratio of repeating units of 4:1, prepared via
anionic polymerization of the monomer mixture using
potassium sorbitol as an initiator. Each arm has a molar
mass of 2 kDa and the polydispersity of the star molecule is
small (PDI < 1.2). Resulting sP(EO-stat-PO) polymers were
functionalized with isophorone diisocyanate to yield
isocyanate terminated sP(EO-stat-PO)s.
The biological activity of the b-D-GlcNAc functionalized
microtiter plates was verified by ELLA, using biotin-labeled
GS II for b-D-GlcNAc specific binding and streptavidin
peroxidase for colorimetric detection. For both surfaces,
ELLA results clearly demonstrated a higher colorimetric
signal for the b-D-GlcNAc functionalized microtiter
plates than corresponding controls, verifying binding of
GSII to immobilized b-D-GlcNAc-functionalized coupler 7
(Figure 5).
sP(EO-stat-PO) hydrogel layers were prepared in Cova-
Link NH-Module microtiter plates according to a recently
published protocol.[33] The procedure used for preparation
of b-D-GlcNAc-functionalized sP(EO-stat-PO) coated micro-
titer plates is illustrated in Scheme 7.
Note that sP(EO-stat-PO)-coated microtiter plates had
highercolorimetric signalsthan controlplates, possiblydue
to layer thickness and coating inhomogeneity; however,
First, isocyanate terminated six-arm sP(EO-stat-PO) was
dissolved in THF and diluted with distilled water. Then, the
OH
OH
O
OH
OH
OH
NCO
HN
NCO
NCO
OH
OCN
NH
NCO
OCN
OCN
NCO
NCO
AcHN
O
O
O
AcHN
O
O
O
H2O/THF HN
N
H
N
H
HN
NCO
NCO
NCO
N
N
N
N
NCO
N
NCO
HN
NH2
NH2
O
NH
9:1
N
O
NCO
NCO
NCO
NCO
H2N
NCO
OCN
NH
NH2
N
H
OCN
OCN
NCO
NCO
OCN
OCN
NCO
-
CO2
OCN
NH2
H2N
NCO
NCO
OH
O
O
NCO
OH
O
O
HN
pre-crosslinking
isocyanate terminated
HN
6-arm sP(EO-stat-PO)
NH
NH
NH
NH2
O
O
NH2
NH2
NH2
NH2
NCO
NH2
NCO
NH2
NH2
NH2
NH2
NH2
H2N
H2N
HN
NH2
NH2
NH2
NH2
CNO
NH2
NH2
H2N
H2N
CNO
NH2
NH2
NH2
H
N
H
N
H
N
NH2
NCO
H2N
OCN
NH2
HN
NH
HN
HN
NH2
amino functional
microtiter plate
H
H
H
N
O
O
O
O
N
N
N
HN
7
H
N
H
N
H
N
N
N
GlcNAc functionalized
sP(EO-stat-PO) coated
sP(EO-stat-PO) coated
microtiter plate
microtiter plate
Scheme 7. Formation of dense sP(EO-stat-PO) films on amine-functionalized surfaces and immobilization of 7.
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azido-functionalized sugar derivatives on amino-reactive
substrates, without loss of biological activity. This concept
has the potential to lead to the development of new flexible
immobilization methods for common functionalized gly-
cans. By using sP(EO-stat-PO)-coated microtiter plates, we
generated a highly selective biofunctional hydrogel.
Acknowledgements: We gratefully acknowledge the DFG for
financial support (EL 135/8-1, MO 682/8-1). The contents of this
publication reflects the authors’ view. The authors thank Prof.
Groll for providing of the isocyanate-terminated six-arm sP(EO-
stat-PO).
Figure 5. Binding assay of b-D-GlcNAc-functionalized coupler 7
immobilized on amino-reactive microtiter plates and sP(EO-stat-
PO)-functionalized microtiter plates using lectin II from Griffonia
simplicifolia.
Received: January 28, 2011; Revised: April 15, 2011; Published
online: June 16, 2011; DOI: 10.1002/mabi.201100041
Keywords: biological applications of polymers; functionalization
of polymers; hydrogel coating; molecular recognition; orthogonal
couplers
comparing the different b-D-GlcNAc-functionalized micro-
titerplates, thehighercolorimetricsignalofthecommercial
amino-reactive microtiter plates was obvious. Conse-
quently, the amino-reactive microtiter plates had more
reactive groups availableforimmobilization of b-D-GlcNAc-
functionalized coupler 7 than the sP(EO-stat-PO) hydrogel-
coated microtiter plates. Otherwise, the blocking step in the
ELLA experiment would not be needed for sP(EO-stat-PO)
hydrogel-coated microtiter plates. As mentioned in the
introduction, the outstanding advantage of the sP(EO-stat-
PO) hydrogel are its non-specific protein adsorption
properties.[24–27] Thus the concept presented here enables
the preparation of highly selective biological surface
functionalization.
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