New H-bonding patterns in biphenyl-based synthetic lectins; Pyrrolediamine bridges enhance glucose-selectivity

Article abstract of DOI:10.1039/c2ob25900a

Synthetic lectins are molecules designed for the challenging task of biomimetic carbohydrate recognition in water. Previous work has explored a family of such systems based on bi/terphenyl units as hydrophobic surfaces and isophthalamide spacers to provide polar binding groups. Here we report a related receptor which employs a new spacer, 2,5-bis-(aminomethyl)-pyrrole, with an alternative (A-D-A) set of H-bonding valencies. The modified spacer leads to significant changes in binding selectivity, including a preference for glucose over all other tested substrates.

Full text of DOI:10.1039/c2ob25900a

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COMMUNICATION
New H-bonding patterns in biphenyl-based synthetic lectins; pyrrolediamine
bridges enhance glucose-selectivity†
Gururaj Joshia,b and Anthony P. Davis*a
Received 10th May 2012, Accepted 13th June 2012
DOI: 10.1039/c2ob25900a
surfaces, complementary to the axial CH groups in the carbo-
hydrates, while the isophthalamides contain H-bond donor/
acceptor units positioned to interact with polar groups in the sub-
strates. Given the challenging nature of the problem, these recep-
tors have performed remarkably well. For example prototype 1
binds β glycosides of N-acetylglucosamine (GlcNAc) 3 with Ka
up to 1000 M−1 and selectivities ≥20 : 1 vs. other monosacchar-
ides. Meanwhile alkoxy-substituted 2 prefers glucose 4, binding
this important substrate with Ka = 60 M−1, and selectivity
∼20 : 1 vs. axially-substituted carbohydrates.
Synthetic lectins are molecules designed for the challenging
task of biomimetic carbohydrate recognition in water. Pre-
vious work has explored a family of such systems based on
bi/terphenyl units as hydrophobic surfaces and isophthala-
mide spacers to provide polar binding groups. Here we
report a related receptor which employs a new spacer,
2,5-bis-(aminomethyl)-pyrrole, with an alternative (A-D-A)
set of H-bonding valencies. The modified spacer leads to signifi-
cant changes in binding selectivity, including a preference for
glucose over all other tested substrates.
The design of biomimetic receptors for carbohydrates in water
remains an important challenge.1 On the one hand, saccharides
are problematic targets. Typically they are coated with hydroxyl
groups and are therefore highly hydrophilic. They are also
“hydromimetic” in their resemblance to clusters of water mo-
lecules, and are therefore especially difficult to distinguish from
competing solvent. Indeed, the affinities of lectins (carbohydrate-
binding proteins) for their substrates are notoriously weak on the
general scale of biomolecular interactions.2 On the other hand,
despite the low affinities, natural carbohydrate recognition is
highly important.3 Protein–carbohydrate interactions play roles
in crucial biological processes such as fertilization,3c–g neuronal
development,3d–h hormonal activities,4 tumour metastasis,5
immune surveillance6 and inflammatory responses.7 Synthetic
receptors can throw light on the principles which underlie lectin
binding, could potentially complement these proteins as research
tools for glycobiology, and may eventually find applications in
medicine.1c,8
To date, our exploration of this system has been restricted to
changes in the parallel hydrophobic surfaces. Thus we have
extended the surfaces (from biphenyl to terphenyl) to bind disac-
charides,9b,f and have added substituents to moderate π-electron
density and (as in 2) tune selectivities.9d,e Throughout this work
the polar interactions, provided by the isophthalamide units,
have remained unaltered. The strength, nature and disposition of
the hydrogen-bonding units could have major effects on the
affinities and selectivities of the receptors, so variation of the
spacers is an important objective. We now describe the first
example of a biphenyl-based synthetic lectin with alternative
polar spacer units, and also the first in which all spacers are not
the same. The new design has significantly altered selectivity,
being the first to show preference for glucose against all other
tested carbohydrates.
Over the past few years we have explored a family of synthetic
lectins based on pairs of biphenyl and terphenyl units linked to
form cages using isophthalamide spacers.9 Exemplified by proto-
type 1 these molecules are designed for complementarity to the
β-glucosyl family of carbohydrates, i.e. those with all-equatorial
substitution patterns. The bi/terphenyl units provide hydrophobic
aSchool of Chemistry, University of Bristol, Cantock’s Close, Bristol BS1
1TS, UK. E-mail: Anthony.Davis@bristol.ac.uk;
Fax: +44 (0) 117 9298611; Tel: +44 (0) 117 9546334
In this first attempt at spacer variation, we chose to retain the
isophthalamides at one end of the receptor while introducing an
alternative unit at the other. The second spacer unit needed to be
roughly the same size as the isophthalamides (to maintain the
shape of the cage) but complementary in other respects. The
bDepartment of Chemistry and Biochemistry, University of Texas at
Austin, Austin, Texas 78712, USA
†Electronic supplementary information (ESI) available: Experimental
details, spectra for compound characterisation and binding studies, and
fitting curves for binding analyses. See DOI: 10.1039/c2ob25900a
5760 | Org. Biomol. Chem., 2012, 10, 5760–5763
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group of Roelens had previously reported the cage 5 as a power-
ful and selective receptor for lipophilic β-glucosides in chloro-
form.10 As illustrated in Fig. 1, the 2,5-bis-(aminomethyl)-
pyrrole (BAMP) spacers in 5 are similar in length to isophthaloyl
units but very different in terms of H-bonding characteristics.
While the isophthalamides can furnish the binding site with two
H-bond donor units (Fig. 1a) or a donor–acceptor combination
(Fig. 1b),11 the BAMP linkers contribute a centrally located
donor flanked by two acceptors (Fig. 1c) or (after protonation) a
further two donors (Fig. 1d). In water at pH 7 the BAMP unit
should be bis-protonated, but the deprotonated form could
readily be accessed and studied under basic conditions. Thus
BAMP seemed well-suited to serve as a complementary spacer
in this research. The spacers would need to support the isophtha-
lamides in maintaining water solubility, so carboxylate groups in
the pyrrole 3 and 4 positions were added to the design.
Macrotricycle 6 thus became the focus of this work.
Receptor 6 was prepared following the route shown in
Scheme 1. Suzuki–Miyaura coupling of iodo-bis-azide 79f with
boronate 812 gave an orthogonally protected biphenyl derivative,
which was treated with TFA to remove Boc groups. The resulting
diamine 9 was allowed to react with bis-pentafluorophenyl ester
109a under high dilution, to give the corresponding [2 + 2]
Fig. 1 H-bonding characteristics of the isophthalamide and BAMP
spacers.
Scheme 1 (i) DMSO, Pd(dppf)2Cl2, NaOAc, 80 °C, 6 h, 77%; (ii) DCM, TFA, 5 h, 97%; (iii) 10, THF, DIPEA, high dilution (1.2 mM), 30 h, 55%;
(iv) THF/MeOH sat. NH3, Pd/C, H2 (1 atm), 2 h, 94%; (v) 12, CHCl3–MeOH 9 : 1, overnight; (vi) MeOH, NaBH4, 2 h; (vii) THF, (Boc)2O, 55 °C,
overnight, 34% over three steps; (viii) DCM, TFA, 5 h, 93%; (ix) MeOH/H2O, NaOH, 99%. DCM = dichloromethane, Boc = tert-butoxycarbonyl,
DIPEA = diisopropylethylamine, DMSO = dimethylsulfoxide, TFA = trifluoroacetic acid, dppf = 1,1′-bis(diphenylphosphanyl)ferrocene.
This journal is © The Royal Society of Chemistry 2012
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Fig. 2 Carbohydrates tested for their affinities with 6.
macrolactamisation product. Hydrogenolysis of the four azido
groups in this macrocycle (H2, Pd/C) yielded tetra-amine 11. Fol-
lowing the lead of Roelens et al.,10 the BAMP spacer units were
introduced through reductive amination. Condensation of 11
with dialdehyde 1213 in CHCl3–CH3OH 9 : 1 gave an apparently
1
clean transformation (by H NMR) to the corresponding macro-
tricyclic tetra-imine. Reduction with NaBH4 followed by protec-
tion with di-tert-butyl dicarbonate (to aid isolation and
characterisation) gave macrotricycle 13 in 34% yield over 3
steps. Finally the Boc protecting groups and t-butyl esters were
cleaved using DCM/TFA, and the resulting decacarboxylic acid
was neutralised with NaOH to pH = 7, followed by lyophilisa-
tion. Unfortunately the resulting solid, presumably tetra-N-proto-
nated 6, was insoluble in water. Thus the effect of bis-protonated
BAMP spacers, as in Fig. 1d, could not be studied. However the
receptor was readily soluble in D2O at pD = 13, giving clean and
well-resolved NMR spectra corresponding to 6. It was therefore
possible to test this structure, with native BAMP spacers
(Fig. 1c), as a carbohydrate receptor.
The binding of macrotricycle 6 to carbohydrates 3, 4 and
14–18 (Fig. 2) was studied by H NMR titration at pD = 13 in
Fig. 3 1H NMR titration of receptor 6 with D-glucose 4 in D2O at pD
1
= 13. (a) Partial spectra, with assignments. (b) Observed and calculated
D2O. Typically the addition of carbohydrate substrates caused
movements of most of the receptor aromatic signals, consistent
with complex formation in fast-medium exchange on the 1H
NMR chemical shift timescale. Fig. 3a shows portions of the
NMR spectra resulting from the titration of 6 with D-glucose 4,
in which the movements of protons B, C, E and F are especially
pronounced (see Fig. 3a for key). The movements were analysed
by non-linear curve-fitting according to a 1 : 1 binding model,
using an Excel spreadsheet. The fit between observed and calcu-
lated data was generally good, as illustrated for 6 + 4 in Fig. 3b,
supporting the assumption of 1 : 1 stoichiometry.
The binding constants resulting from these analyses are listed
in Table 1, along with the corresponding values for prototype
receptor 1. The first point to note is that 6 is a fairly effective
receptor, binding glucose 4 with Ka = 18 M−1 (roughly twice as
strongly as 1). The increase is achieved despite a lowering of
symmetry from D2h to C2v. This reduces the number of equival-
ent binding modes from 4 to 2, with a consequent loss of
binding entropy.14 At the same time, the change from isophthala-
mide to BAMP spacers has significant consequences for selec-
tivity. While the affinity for glucose has increased, binding to
GlcNAc 3 has been considerably weakened. Also much lower is
the affinity for methyl β-D-glucoside 14 which is bound >4 times
less strongly than glucose itself. The change in structure thus
seems to favour glucose itself over other all-equatorial substrates.
The weak binding to β-glucoside 14 is especially interesting as
binding curves for proton B; Ka = 18 M−1
.
Table 1 Binding constants Ka for 1 : 1 complexes of receptor 6 with
carbohydrate substrates in D2O as determined by 1H NMR titrations.
Data for 1 is given for comparisona
Ka [M−1
]
Carbohydrate
1b
6
D-Glucose 4
9
18
7
N-Acetyl-D-glucosamine (GlcNAc) 3
Methyl β-D-glucoside 14
D-Mannose 15
56
28
∼0c
2
4
∼0c
3
D-Galactose 16
D-Xylose 17
D-Lactose 18
5
4
∼0c
∼0c
a pD = 13, T = 298 K. b Values for 1 from ref. 9c. c Too small for reliable
analysis.
this has previously been a good substrate for biphenyl-based
receptors9a,c–e (not unexpectedly, given its all-equatorial
configuration and hydrophobic nature). The result may imply a
particular mode of binding for glucose which is not available to
14 for steric reasons, or may involve specific polar interactions
with the reducing anomeric centre. The geometry of the cavity
5762 | Org. Biomol. Chem., 2012, 10, 5760–5763
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may play a role, although molecular modelling15 on 6 predicted
an open conformation with similar dimensions to that of 1.
Whatever the detailed explanation, the change in selectivity is
presumably due to the recasting of H-bonding valencies at one
end of the cavity, from the D–A or D–D of isophthalamide to
the A–D–A of BAMP (Fig. 1a–c).
In conclusion, we report a new type of biphenyl-based syn-
thetic lectin in which, for the first time, the standard isophthala-
mide spacers have been replaced by units with alternative
arrangements of polar binding groups. The recognition properties
of this system confirm that such changes are permissible, and
can be used to tune selectivities. In this case the result is un-
precedented selectivity for glucose against all other tested carbo-
hydrates. The introduction of the BAMP spacer is clean and
high-yielding, encouraging its adoption in future designs. In par-
ticular it will be interesting to prepare analogues with improved
solubility at pH 7, to test the D–D–D H-bonding arrangement in
Fig. 1d.
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This work was supported by the EPSRC (EP/D060192/1)
and the Commonwealth Commission (Postgraduate Scholarship
to G.J.).
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This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 5760–5763 | 5763