High-affinity multivalent wheat germ agglutinin ligands by one-pot click reaction

Article abstract of DOI:10.3762/bjoc.8.91

A series of six mono-, di-, and trivalent N,N'-diacetylchitobiose derivatives was conveniently prepared by employing a one-pot procedure for Cu(II)-catalyzed diazo transfer and Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) starting from commercially available amines. These glycoclusters were probed for their binding potencies to the plant lectin wheat germ agglutinin (WGA) from Triticum vulgaris by an enzyme-linked lectin assay (ELLA) employing covalently immobilized N-acetylglucosamine (GlcNAc) as a reference ligand. IC50 values were in the low micromolar/high nanomolar range, depending on the linker between the two disaccharides. Binding enhancements β up to 1000 for the divalent ligands and 2800 for a trivalent WGA ligand, compared to N,N'-diacetylchitobiose as the corresponding monovalent ligand, were observed. Molecular modeling studies, in which the chitobiose moieties were fitted into crystallographically determined binding sites of WGA, correlate the binding enhancements of the multivalent ligands with their ability to bind to the protein in a chelating mode. The best WGA ligand is a trivalent cluster with an IC50 value of 220 nM. Calculated per mol of contained chitobiose, this is the best WGA ligand known so far.

Full text of DOI:10.3762/bjoc.8.91

High-affinity multivalent wheat germ agglutinin
ligands by one-pot click reaction
Henning S. G. Beckmann, Heiko M. Möller and Valentin Wittmann*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 819–826.
Fachbereich Chemie and Konstanz Research School Chemical
Biology (KoRS-CB), Universität Konstanz, 78457 Konstanz, Germany
doi:10.3762/bjoc.8.91
Received: 23 March 2012
Accepted: 21 May 2012
Published: 01 June 2012
Email:
Valentin Wittmann* - mail@valentin-wittmann.de
* Corresponding author
This article is part of the Thematic Series "Synthesis in the
glycosciences II".
Keywords:
carbohydrates; click chemistry; cluster effect; lectins; multivalency
Guest Editor: T. K. Lindhorst
© 2012 Beckmann et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
A series of six mono-, di-, and trivalent N,N’-diacetylchitobiose derivatives was conveniently prepared by employing a one-pot
procedure for Cu(II)-catalyzed diazo transfer and Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) starting from commer-
cially available amines. These glycoclusters were probed for their binding potencies to the plant lectin wheat germ agglutinin
(WGA) from Triticum vulgaris by an enzyme-linked lectin assay (ELLA) employing covalently immobilized N-acetylglucosamine
(GlcNAc) as a reference ligand. IC50 values were in the low micromolar/high nanomolar range, depending on the linker between
the two disaccharides. Binding enhancements β up to 1000 for the divalent ligands and 2800 for a trivalent WGA ligand, compared
to N,N’-diacetylchitobiose as the corresponding monovalent ligand, were observed. Molecular modeling studies, in which the chito-
biose moieties were fitted into crystallographically determined binding sites of WGA, correlate the binding enhancements of the
multivalent ligands with their ability to bind to the protein in a chelating mode. The best WGA ligand is a trivalent cluster with an
IC50 value of 220 nM. Calculated per mol of contained chitobiose, this is the best WGA ligand known so far.
Introduction
The recognition of carbohydrate structures by carbohydrate many human diseases. However, carbohydrate–protein interac-
binding proteins (lectins) plays a fundamental role in numerous tions are often characterized by low binding affinities. A
intra- and intercellular events during development, inflamma- possible solution to compensate for these weak individual
tion, immune response, cancer metastasis, and pathogen–host receptor–ligand interactions is the multivalent presentation of
interactions [1,2]. Inhibition of such interactions by high- sugar epitopes on suitable scaffolds. This principle is not only
affinity ligands is of high medicinal interest for the treatment of used in nature but is also a valid strategy for the construction of
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Beilstein J. Org. Chem. 2012, 8, 819–826.
artificial lectin ligands [3-13]. Prime examples are the recently for the design of new multivalent ligands with improved
described ligands for the Shiga-like [14,15] and cholera toxins binding affinity.
[16,17] both belonging to the AB5 family of bacterial toxins.
Besides GlcNAc, WGA also binds to chitooligosaccharides
The frequent observation that the binding affinity of a multiva- with even higher affinity. The association constant for the
lent ligand increases exponentially with the number of binding WGA–N,N’-diacetylchitobiose interaction, for example, has
sites has been termed the glycoside cluster effect [18,19]. Due been determined to be K ≈ 5 × 103 to 2 × 104 M–1. The corres-
to the exponential increase of binding affinities, the cluster ponding value for binding to GlcNAc is K ≈ 2 × 102 to 1.3 ×
effect often leads to the amplification of the binding selectivity. 103 M−1 [47]. This prompted us to design a series of multiva-
This was experimentally demonstrated, for example, by Mortell lent WGA ligands containing two or three N,N’-diacetylchito-
et al. while investigating glycopolymer ligands of biose moieties. To connect the chitobiose moieties we chose
concanavalin A (Con A) [20]. Whereas two diastereomeric several linkers of varying length and flexibility, which were,
(monovalent) C-glycosidic Con A ligands displayed only a nevertheless, all expected to allow simultaneous binding to
small difference in the free energies of binding to Con A, a adjacent binding sites in a chelating fashion, thus, leading to
sizable difference was measured between the corresponding especially effective ligands. In this report, we describe the
multivalent C-glycosides (calculated per monovalent ligand preparation of such a series of multivalent WGA ligands by a
within the glycopolymer). Such effects can be analyzed in the one-pot procedure for diazo transfer and azide–alkyne cyclo-
context of the chelate effect [21], and a number of theoretical addition [48] starting from commercially available di- and
models to treat multivalent receptor–ligand interactions have triamines and the propargyl glycoside of N,N’-diacetylchito-
been developed [22-27]. A simple conclusion following from biose. Binding potencies were determined by an enzyme-linked
these analyses is that multimerization of monovalent ligands lectin assay (ELLA), resulting in IC50 values in the low-micro-
with enhanced binding affinity can lead to multivalent ligands molar/high-nanomolar range. A trivalent ligand has a remark-
with disproportionally enhanced avidity. A prerequisite for an able IC50 value of 220 nM. Molecular dynamics calculations
effective multivalency effect, however, is that the linking spacer based on published X-ray crystal structures of WGA-ligand
between the individual epitopes has the correct geometry to complexes provide an explanation for the observed binding
allow a simultaneous multipoint association, i.e., a chelating affinities.
binding mode.
Results and Discussion
Wheat germ agglutinin (WGA), besides other plant lectins such Synthesis of glycoclusters
as Con A, has been intensively employed as a model lectin to The Cu(I)-catalyzed [49,50] Huisgen [3 + 2] cycloaddition [51]
study the influence of the structure of multivalent ligands on the of azides and alkynes (CuAAC) is a frequently used method for
binding affinity. WGA ligands of defined structure containing the covalent attachment of carbohydrate epitopes to azide- or
two to twelve GlcNAc residues obtained either by individual alkyne-presenting scaffolds [52-54]. Recently, we reported a
synthesis [28-36] or from screening of combinatorial libraries convenient one-pot procedure for diazo transfer and
[37,38] have been reported. WGA is a 36 kDa plant lectin azide–alkyne cycloaddition [48] giving access to multivalent
composed of two identical glycine- and cysteine-rich subunits triazole-linked structures starting from amines. For the syn-
[39] and is enriched in the seeds of Triticum vulgaris. It is thesis of triazole-linked glycoclusters, commercially available
specific for terminal N-acetylneuraminic acid and N-acetylglu- amines A1–A6 (Figure 1) comprising different spacer geome-
cosamine (GlcNAc) and has been shown to inhibit fungal tries were selected. These amines were employed in the sequen-
growth through interaction with fungal cell-wall components
[40-42] and to agglutinate transformed cells in vitro [43,44].
Recently, we determined the structural basis of multivalent
binding to WGA by X-ray crystallography [36] and EPR spec-
troscopy [45]. Crystal structure analysis of a complex of WGA
and four molecules of a divalent ligand containing two GlcNAc
residues showed that each ligand bridged adjacent binding sites
with a distance of approx. 13–14 Å between the anomeric
oxygen atoms of the GlcNAc residues. This structure confirmed
for the first time that all eight sugar binding sites of the WGA
Figure 1: Amines used for the synthesis of glycoclusters.
dimer [46] are simultaneously functional, and provides the basis
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Beilstein J. Org. Chem. 2012, 8, 819–826.
tial one-pot procedure [48] for diazo transfer and CuAAC ride 1 was eluted from a silica gel column for a second time. In
(Table 1). First, the Cu(II)-catalyzed diazo transfer was comparison, the use of the less polar propargyl β-D-glucoside
performed at ambient temperature until complete conversion of instead of 1 in the one-pot procedure with amine A4 led to the
the amines to azides. Then, CuAAC was provoked without any corresponding divalent glycocluster in a yield of 86% [48].
workup procedure by the addition of tris(benzyltriazolyl- Finally, O-deacetylation of the glycoclusters B1–B6 under
methyl)amine [55] (TBTA), sodium ascorbate, and the Zemplén conditions resulted in WGA ligands C1–C6
propargyl glycoside 1 [56] of N,N’-diacetylchitobiose and (Scheme 2).
heating of the mixture to 80 °C by microwave irradiation, until
TLC showed complete consumption of the intermediate azides
(see Supporting Information File 1 for full experimental data).
Table 1: Synthesis of glycoclusters B1–B6 using the one-pot proce-
dure for diazo transfer and azide-alkyne cycloaddition.
Scheme 1: Synthesis of glycocluster B5 with isolation of the intermedi-
ate diazide 2.
During the synthesis of trivalent compound B6 the formation of
a side product with a Rf similar to that of B6 was observed. This
is remarkable because all other reactions proceeded without the
formation of side-products. The mass of this side product (m/z
[M + H]+ = 1500.7) corresponds to a divalent compound in
which one arm of the tertiary amine is missing. Since this side
Amine
Product
Yield (%)
product could not be isolated in pure form, we investigated the
reaction of A6 with phenylacetylene (3, Scheme 3). Also with
this alkyne two main products were obtained which were diffi-
cult to separate by chromatography. Apart from the expected
tris(triazole) 4, the secondary amine 5 was isolated and charac-
terized. This structure corresponds to the assumed side-product
obtained during the synthesis of B6. A contamination of the
starting material A6 with secondary amine di(2-amino-
ethyl)amine was excluded. ESI–MS measurements indicated
that side-product formation may already take place during the
A1
A2
A3
A4
A5
A6
B1
B2
B3
B4
B5
B6
45
30
37
28
48a
6
aIn this case the intermediate diazide 2 was isolated (cf. Scheme 1).
According to TLC all reactions (except for B6) proceeded with diazo transfer reaction of A6 because the mass of the corres-
complete conversion of the amines to the desired glycoconju- ponding intermediate di(2-azidoethyl)amine (m/z [M + H]+ =
gates. However, some loss of material during purification of the 156.2) was found. The mechanism of this side product forma-
acetylated chitobiose derivatives by flash chromatography on tion is not clear. We assume that the mechanism is due to the
silica gel led to the moderate yields indicated in Table 1. Mono- special structure of A6 because comparable side-product forma-
valent compounds resulting from partial reactions of the di- tion was not observed with any other amine used.
amines were not observed. To exclude that the observed yields
are a result of the one-pot procedure, the preparation of diva- Determination of binding potencies by ELLA
lent B5 was carried out in two separate steps (Scheme 1). Diazo Binding potencies of compounds C1–C6, GlcNAc, and N,N’-
transfer with A5 gave diazide 2 in a yield of 95%. Subsequent diacetylchitobiose were determined by an ELLA employing
CuAAC of isolated 2 with alkyne 1 delivered B5 after flash covalently immobilized GlcNAc as a reference ligand, as
chromatography in 51% yield with no observed side products. described recently [34]. GlcNAc-coated microtiter plates were
Severe loss of about 50% of the material during flash chroma- incubated with mixtures of horseradish-peroxidase-labeled
tography was also experienced when a pure sample of disaccha- WGA (HRP-WGA) and synthetic WGA ligands in varying
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Beilstein J. Org. Chem. 2012, 8, 819–826.
Scheme 2: Deacetylation of glycoconjugates B1–B6. (a) NaOMe, MeOH.
concentrations. After incubation, the plates were washed and
the remaining HRP-WGA bound to the microtiter plates was
quantified by a HRP-catalyzed color reaction. Dose-response
curves for inhibition of the binding of HRP–WGA to immobi-
lized GlcNAc are shown in Figure 2. From these curves the
concentrations at which the binding of HRP–WGA to GlcNAc
is reduced by 50% (IC50 values) were determined as a measure
of the potency of the synthesized inhibitors (Table 2). Also
shown in Table 2 are the relative inhibitory potencies (β values)
referenced to N,N’-diacetylchitobiose (β = 1).
With an 82 times lower IC50 value, N,N’-diacetylchitobiose is a
much better inhibitor than GlcNAc, which is in accordance with
the association constants determined by solution binding assays
[47]. The benzyl triazolyl appendage of C1 further enhances
binding by a factor of two, probably due to additional weak
hydrophobic interactions. However, introducing an additional
N,N’-diacetylchitobiose epitope leads to dramatically increased
affinities. The divalent chitobiose derivatives C2–C5 have IC50
values in the low-micromolar/high-nanomolar range, with some
Scheme 3: Formation of side-product 5 during the synthesis of 4.
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Figure 2: Dose-response curves for the inhibition of binding of HRP-labeled WGA to covalently immobilized GlcNAc by synthetic ligands C1–C6.
As expected, clustering of carbohydrate epitopes with higher
Table 2: Absolute and relative IC50 values of synthetic ligands C1–C6
for inhibition of the binding of HRP-labeled WGA to covalently immobi-
lized GlcNAc from dose-response curves shown in Figure 2.
WGA binding affinity not only leads to multivalent ligands with
higher absolute affinity but also to a higher binding enhance-
ment β relative to the respective monovalent compound. Earlier,
we reported WGA binding affinities of β-O-glycosidic divalent
GlcNAc derivatives with linker lengths comparable to those of
C2–C5 [34]. These GlcNAc derivatives displayed β values of
80–260 relative to GlcNAc, which are much lower than the β
values (330–1000, relative to N,N'-diacetylchitobiose) deter-
mined for C2–C5 (Table 2). The relative potency of C4 of 500
per chitobiose residue is even higher than that of tetra- to
octavalent GlcNAc clusters [28,31-34,37]. We are aware of
only one example of a divalent GlcNAc derivative with an
exceptional β value of 2350 [36]. In this case, however, the
Compound
IC50 (µM)
β
GlcNAc
51000
620
1/82
1
N,N'-diacetylchitobiose
monovalent C1
divalent C2
290
2.1
1.3
480
330
1000
860
2800
divalent C3
1.9
divalent C4
0.60
0.72
0.22
divalent C5
trivalent C6
variation due to different spacer properties. Their inhibitory GlcNAc moieties are α-O-glycosidically linked.
potencies relative to N,N'-diacetylchitobiose are 330–1000. It is
interesting to note the differences induced by differing linker For trivalent cluster C6 an IC50 value of 220 nM (660 nM per
geometries. Here, not the most hydrophobic linkers show contained chitobiose) was determined, which is 2800-fold lower
strongest binding but the ones that apparently promote multiva- than that of N,N'-diacetylchitobiose or 230000-fold lower than
lent binding most efficiently. Whereas the flexible linkers of C4 the IC50 value of GlcNAc. This is one of the best WGA ligands
and C5 lead to β values of 1000 and 860, respectively, the known. Masaka et al. reported a tetravalent N,N’-diacetylchito-
ligands C2 and C3 with the less flexible aromatic linkers have biose derivative with an IC50 value of 180 nM (720 nM per
significantly lower binding potencies (β values: 480 and 330). contained chitobiose) determined by a hemagglutination inhibi-
This observation points to the possibility that the aromatic tion assay [57]. Calculating the IC50 value per contained chito-
linkers cannot adopt a strain-free conformation if the ligand biose, trivalent C6 is the better ligand. However, since such
binds in a chelating mode to WGA. The best divalent WGA numbers are strongly dependent on the employed assay [58,59],
ligand is C4 (IC50 0.6 µM), which binds 1000 times stronger to they cannot be readily compared. Interestingly, the ligand
WGA than N,N’-diacetylchitobiose (500 times per chitobiose reported by Masaka et al. led to precipitation of WGA. In this
residue).
respect it is worth mentioning that we never observed precipi-
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Beilstein J. Org. Chem. 2012, 8, 819–826.
tate formation during incubation of WGA with our synthetic (see Supporting Information File 1 for details). Subsequently,
ligands. This suggests that in our case intermolecular multiva- the linkers were energy minimized with the chitobiose moieties
lency (cross-linking) plays a negligible role and that the main fixed in their ideal positions. The models containing the
mechanism of affinity enhancement is chelating binding to the various linker structures were further energy minimized
same WGA dimer.
with the terminal GlcNAc residues kept in their optimal posi-
tions.
The 2.8-fold increased inhibition potency of C6 over the best
divalent ligand C4 indicates that C6 can reach only two WGA Our molecular modeling studies revealed that the para-disubsti-
binding sites simultaneously due to its geometrical properties, tuted aromatic linker of C3 cannot adopt a low-energy con-
which is fully in accordance with the structural investigations formation if the chitobiose residues are kept in their ideal posi-
described below. Otherwise, a significantly stronger binding tions in the binding sites of WGA, resulting in significant ring
enhancement would have been expected comparable to the strain of the triazole moieties as well as the central phenyl ring.
several-hundred-fold increase observed when moving from This ring strain can be reduced by slightly pulling the GlcNAc
mono- to divalent ligand structures.
residues directly attached to the linker out of the binding site,
but at the expense of a less efficient multivalent binding of the
two chitobiose entities. The chitobiose groups of the divalent
Molecular modeling
To provide a structure-based rationalization for the determined ligand C4, on the other hand, can maintain their ideal positions
binding potencies of C2–C6, we performed molecular modeling easily with the linker adopting an all-staggered low-energy con-
studies. Combining information from the crystal structures of formation (Figure 3). Ligand C2 allows for the positioning of
WGA3 in complex with N,N’-diacetylchitobiose (PDB ID: its chitobiose moieties ideally in both binding sites when
1K7U) [60] and WGA3 binding to a divalent ligand presenting adopting a fully extended conformation. Ligand C2, and even
two GlcNAc residues (PDB ID: 2X52) [36], two N,N’-diacetyl- more so ligand C3, possess very limited conformational
chitobiose residues were placed in a pair of adjacent primary freedom when adopting a chelating binding mode. Con-
binding sites of WGA and connected by the respective linker formational changes within the linkers of C2 and C3 lead to
Figure 3: Molecular model of divalent ligand C4 with its two chitobiose moieties occupying two adjacent binding sites of WGA. The linker is conforma-
tionally largely unrestricted and can adopt several low-energy conformations. WGA chain 1 is colored yellow, chain 2 is blue.
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Beilstein J. Org. Chem. 2012, 8, 819–826.
forces that pull one or the other GlcNAc/chitobiose out of its Acknowledgements
binding site. This situation may increase the entropic costs of This work was supported by the Deutsche Forschungsgemein-
chelating binding for these two ligands providing a further ex- schaft (FOR 434) and the University of Konstanz. H.S.G.B.
planation for their significantly lower binding affinity.
acknowledges a fellowship according to the Landesgraduierten-
förderungsgesetz.
With its linker length and flexibility, trivalent ligand C6 is in an
intermediate position between the relatively short-bridged and
rigid ligands C2 and C3 and the more flexibly connected
ligands C4 and C5. With six chemical bonds between the
triazole groups, C6 can present its N,N’-diacetylchitobiose
residues in rather similar distances as C2 or C3 having the same
linker length. However, C6 has two more rotatable bonds
between pairs of chitobiose moieties leading to significantly
increased conformational freedom. This is expected to facilitate
binding in a chelating fashion at relatively low entropic costs. It
is important to note that the third chitobiose unit of C6 cannot
reach a third carbohydrate binding site of the WGA dimer. The
closest distance to another binding site is approximately 24 Å
(measured between the anomeric oxygens of the inner GlcNAc
residues). The 2.8-fold increased potency relative to ligand C4
could possibly originate from the facilitated rebinding of mono-
valently bound trivalent ligand C6 due to the two-fold-higher
local concentration of chitobiose compared to the divalent
ligands.
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