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toward LecA, better than the meta-regioisomer 38b, whereas
the ortho-derivative 38a was mostly detrimental to the bind-
The influence of the aromatic moiety was also confirmed.
Glycoclusters 40 with a furanyl (d) or pyridinyl (g) ring exhibit-
ing properties. The same influence was observed for the EG M
ed the weakest binding with K values of around 200 nm,
2
d
3
9 and EG M 40 species in the Linker 1 series. The ortho-sub-
whereas those glycoclusters with thiophenyl (e) or meta-benzyl
3
stitution pattern (a) probably generates significant steric hin-
drance because the amino acids of LecA are close to the car-
bohydrate-recognition domain. This effect is probably less im-
portant for the meta-regioisomer (b), whereas while the para-
substituted derivative (c) is the least congested, thus providing
the best affinity observed.
(f) groups exhibited better affinities with K values of around
80 nm, but were still about seven- and threefold less potent,
d
respectively, than glycocluster 40j with a phenyl linker (K =
d
28 nm).
As the stabilisation involves a face-to-edge p–p interaction
between the ring and His50 of LecA, this interaction might be
improved in the case of the thiophenyl (e) moiety based on
the higher aromaticity due to the lower electronegativity of
the sulfur atom relative to the oxygen atom in the furanyl ring
(d). Furthermore, the addition of a methylene moiety on going
from the para-Ph (j) to the meta-Bn (f) groups was detrimental,
probably because the oxygen atom provides an enrichment in
electron density on the aromatic ring of 40j, which is benefi-
cial for a better face-to-edge p–p interaction with the His50
residue.
Finally, a comparison of Linker 2 in the series of d–g pro-
vides insight to the influence of the aromatic ring. In this case,
the order of influence within the glycoclusters was observed
to be thiophen (e)>meta-Bn (f)>furan (d)>pyridine (g). The
pyridine-based (g) Linker 2 was superior among these four
congeners only in the case of glycocluster 40, and then it was
only superior to the furan-based (d) Linker 2.
Measurements of dissociation constants (K ) on microarrays
d
[13]
Reymond and co-workers reported that an amine group
To gain a better understanding of the influence of Linker 2,
(as a lysine residue of the core moiety of the peptide-based
glycocluster) close to the aromatic moiety can interact with
the aspartic acid (Asp) residue Asp47 of LecA, thus leading to
stabilization of the interaction through coulombic forces. In
dissociation constants (K ) were measured by means of micro-
d
array to provide quantitative insight into the glycocluster-bind-
ing properties toward LecA (Table 2). The K values were deter-
d
our case, the free amine group of the TyrNH -based glycoclus-
2
ter 40h did not improve the binding relative to glycocluster
Table 2. Dissociation constants (K
ray.
d
) measured by carbohydrate microar-
4
0j (K =47 and 28 nm, respectively). In contrast, the introduc-
d
tion of a carboxylic acid group into the TyrCO H-based glyco-
2
2
Glycocluster
Linker 2
K
d
[nm]
Rank
R
cluster 40i led to the best binding with a K value of 19 nm,
d
thus being the best LecA ligand identified to date by using mi-
croarrays.
4
4
4
4
4
4
4
4
4
4
0a
0b
0c
0d
0e
0 f
0g
0h
0i
EGortho-Ph
EGmeta-Ph
EGpara-Ph
furanyl
thiophenyl
meta-Bn
304
52
39
202
77
81
10
5
3
8
6
6
8
4
1
2
0.963
0.993
0.996
0.991
0.997
0.985
0.986
0.995
0.994
[
[
[
[
a]
a]
a]
a]
Molecular simulations
pyridyl
197
47
TyrNH
TyrCO
para-Ph
2
Investigations were performed by using molecular-simulation
studies to rationalize the binding properties observed on the
microarrays and their implications in the binding of the galac-
tosides and their aglycons in the binding site of LecA. The stoi-
chiometry typically observed in such mannose-centered glyco-
2
H
19
28
[
b]
[c]
0j
–
[a] Could not be distinguished because the root-square deviation (RSD)
of the K
than 8%. [b] The K
d
[
measurements determined by means of microarrays was less
67]
[46]
d
value previously reported. [c] Does not apply.
[14]
clusters with LecA was measured as n=0.5. The calculations
were performed by considering that two branches of the same
glycocluster would interact simultaneously with two binding
sites of the same LecA tetramer.
mined only for the EG M (40) Linker 1 series, which had been
3
identified as the optimal linker for this position in the macro-
Glycoclusters 40c, 40h, and 40i (Figures 4–6) were investi-
gated by using molecular simulations and docking techniques.
Glycocluster 40c was selected to evaluate the influence of the
ethyleneglycol moiety introduced at the anomeric center upon
binding to LecA. Glycoclusters 40h and 40i provided the best
molecule. The quantitative K values (Table 2) were in agree-
d
ment with the qualitative data obtained by direct fluorescence
(Figure 3).
The preference in the substitution pattern at the phenyl ring
was confirmed to be para (c)>meta (b)@ortho (a). Indeed, the
Kd values decreased by nearly sixfold between the ortho and
meta isomers and by nearly eightfold between the ortho and
para isomers.
binding properties from the K measurements and were there-
d
fore further investigated. Because the high affinities observed
point toward chelate-binding modes as previously observed
[21,31,50]
for this lectin,
an empirical calculation of the potential
A comparison between 40c and 40j showed that the intro-
duction of an ethyleneglycol group between the galactose and
phenyl rings had only a limited and small negative effect on
energy of interaction DE was carried out for the six different
possible combinations for the simultaneous binding of the two
galactose epitopes displayed at the tip of two branches at-
tached at the core mannopyranoside ring (Table 3).
the affinity (K =39 and 28 nm, respectively).
d
Chem. Eur. J. 2016, 22, 1 – 11
7
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