Affinity enhancement by dendritic side chains in synthetic carbohydrate receptors

Article abstract of DOI:10.1002/anie.201409124

Dendritic side chains have been used to modify the binding environment in anthracene-based synthetic carbohydrate receptors. Control of length, charge, and branching enabled the positioning of side-chain carboxylate groups in such a way that they assisted

Full text of DOI:10.1002/anie.201409124

Angewandte
Chemie
DOI: 10.1002/anie.201409124
Molecular Recognition
Affinity Enhancement by Dendritic Side Chains in Synthetic
Carbohydrate Receptors**
Harry Destecroix, Charles M. Renney, Tiddo J. Mooibroek, Tom S. Carter, Patrick F. N. Stewart,
Matthew P. Crump, and Anthony P. Davis*
Abstract: Dendritic side chains have been used to modify the
binding environment in anthracene-based synthetic carbohy-
drate receptors. Control of length, charge, and branching
enabled the positioning of side-chain carboxylate groups in
such a way that they assisted in binding substrates rather than
blocking the cavity. Conformational degeneracy in the den-
drimers resulted in effective preorganization despite the
flexibility of the system. Strong binding was observed to
glucosammonium ions in water, with Ka values up to 7000mÀ1
.
Affinities for uncharged substrates (glucose and N-acetylglu-
cosamine) were also enhanced, despite competition from
solvent and the absence of electrostatic interactions.
drive and control carbohydrate recognition, in both natural
and synthetic systems.[5] The role of polar interactions is
especially interesting. Although the contribution from CH–p
and hydrophobic effects is readily understood,[6] it is less
evident how polar binding groups can be deployed to increase
affinities in water. Moreover, studies of this problem are
handicapped by design and synthetic issues. In both 1 and 2,
the polar interactions are provided by amide groups, which
are intrinsic to the framework, and altering these groups
could change the positioning of the hydrophobic surfaces (in
some cases with destruction of the cavity). The addition of
polar groups to the receptor cores might be feasible at some
points, but all such changes would require major synthetic
effort.
Faced with this problem, we realized that one position
where changes could readily be made, especially in mono-
cyclic 2, is in the solubilizing groups X. At first sight, such
modifications should make little difference to the binding
properties. However, if X is dendritic[7] and the size and bulk
of the dendrimer is adjusted upwards, terminal groups are
located close to the opening of 2, as required to make contact
with the polar groups in bound substrates (Figure 1). The
T
he binding of polar molecules in aqueous solution remains
a major problem for supramolecular chemistry. Whereas
apolar molecules interact poorly with water and are readily
bound through the hydrophobic effect, polar species and
binding sites are well-hydrated. Binding requires that water
be displaced from both partners, and the energetic conse-
quences may be unpredictable. The problem is especially
difficult if the targets and/or binding sites bear hydroxy
groups, which resemble water molecules yet must be distin-
guished from solvent. Thus, carbohydrate recognition, an
important biological process,[1] is especially challenging.[2,3]
In previous studies we have shown that certain carbohy-
drates can be bound by amphiphilic cavities, which comple-
ment both polar and apolar regions in their targets.[4] For
example, both tricycle 1[4a] and monocycle 2[4b] bind glucose 3
by combining aromatic surfaces (complementary to axial CH
groups) with annular amides (complementary to equatorial
OH groups). Binding constants are modest at Ka ꢀ 60mÀ1, but
selectivities are good (e.g. 20:1 glucose/galactose). Moreover,
owing to the high concentrations of glucose in biological
fluids, the low affinities do not preclude applications in
medical glucose sensing.[4a]
A major aim of this research is to show how particular
interactions and supramolecular principles might help to
[*] H. Destecroix, C. M. Renney, Dr. T. J. Mooibroek, T. S. Carter,
P. F. N. Stewart, Prof. M. P. Crump, Prof. A. P. Davis
School of Chemistry, University of Bristol
Cantock’s Close, Bristol, BS8 1TS (UK)
E-mail: Anthony.Davis@bristol.ac.uk
[**] This research was supported by the Engineering and Physical
Sciences Research Council through grant number EP/I028501/1,
and studentships funded by the Bristol Chemical Synthesis Doctoral
Training Centre (EP/G036764/1) to C.M.R. and T.S.C.
Figure 1. Schematic view of a bisanthracenyl carbohydrate receptor
(analogous to 2) binding glucose (Z=O) or glucosammonium
(Z=NH2+) with the aid of hydrogen bonding from a polycarboxylate
dendritic side chain (green).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201409124.
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

.
Angewandte
Communications
terminal groups will be connected to the core through
a flexible chain, so will not be preorganized for binding.
However, as one end group moves away, another can move
into range. A level of preorganization is therefore achieved
through the symmetry and degeneracy of the dendron
structure. Effects should be substantial if electrostatic forces
can be invoked, but might also be significant for neutral
substrates. Herein we report experiments which show that the
solubilizing side chains in synthetic carbohydrate receptors
can indeed be used to enhance binding. The effect has been
exploited to create some of the highest affinities yet observed
for biomimetic carbohydrate recognition in water.
The side chains in 2 are derived from the amine 10
(Scheme 1), available in one step from tert-butyl acrylate and
tris(hydroxymethyl)aminomethane.[8] As a first test of the
concept, we planned to use a second-generation dendrimer
constructed with this unit. Accordingly, we prepared the
receptor 7 (Scheme 1) by the general route shown in
Scheme 2[9] and investigated the binding of this macrocycle
Scheme 2. Synthetic route to the receptors in Scheme 1. TFA=tri-
fluoroacetic acid.
Although the effects were negative, the results with 7
confirmed that dendrimeric side chains could indeed influ-
ence binding properties. To exploit this principle construc-
tively, we synthesized a range of receptors with controlled
side-chain length, charge, and steric bulk (Scheme 1). The
side chains were constructed from 10, which gives units with
relatively long branches; the triester 11,[10] which gives
medium-length units; and di-tert-butyl iminodiacetate (12),
which gives short branches. In particular, by restricting the
overall length and terminating the dendrimers with 11 or 12,
we expected to prevent the threading which had disabled 7.
1
to glucose in water by H NMR spectroscopy. To our initial
surprise, the spectra yielded no evidence of complexation.
However, further investigation revealed NOE connections
between side-chain hydrogen atoms and inward-directed
receptor hydrogen atoms (NH, ArH), thus implying that
terminal strands from the side chain can thread through the
cavity (see Figure S37 in the Supporting Information).
Modeling confirmed that such threading was feasible (see
Figure S183).
Scheme 1. Carbohydrate receptors and side-chain components. Although side chains are shown as fully ionized for simplicity, some carboxylate
groups will be protonated at pH 7. For further discussion, see the Supporting Information.
2
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!

Angewandte
Chemie
Four of the series possessed second-generation (G2) dendri-
mer side chains (including 7), whereas two contained G3 side
chains (8 and 9). Side-chain lengths varied fairly smoothly
from 7 to 15 atoms, while the overall charge ranged from À6
to À54.
All receptors dissolved readily in D2O to give well-
resolved, concentration-independent 1H NMR spectra (up to
a concentration of about 4 mm). Their binding properties
towards carbohydrates were investigated primarily by
1H NMR titrations. The substrates included the aminosugars
glucosammmonium 13·H+ and galactosammonium 14·H+, for
Figure 2. Partial 1H NMR spectra, with assignments, from the titration
of 6 (0.23 mm) with d-glucose (0–218 mm) in D2O at 298 K. The
movement of the signal for hydrogen atom E (internally directed
isophthalimide CH) is highlighted by arrows.
dramatic effects were observed for glucosammonium 13·H+,
which is all-equatorial (and is therefore complementary to the
cavity) and which is charge-complementary to the side chains.
As the dendrimers expanded, affinities for 13·H+ increased
from 160mÀ1 (for 2) to 7000mÀ1 (for 8).[11] Modeling confirmed
that both side chains in 8 can contribute to binding through
strain-free salt bridges (Figure 3).[12] Support was provided by
the NOESY spectra of 8 and 8·glucosammonium. Both
showed NOE connections between anthracenyl hydrogen
atoms and the terminal side-chain CH2 groups, a result
consistent with the presence of side-chain carboxylate groups
near the entrance to the cavity (see Figures S38 and S39).
Interestingly, the next step to 9 was counterproductive,
despite the increase in negative charge. NOE measurements
on free 9 suggested that, as for 7, terminal side-chain groups
are able to penetrate the cavity (see Figure S40). The affinity
for glucosammonium was lowered by competing ions, as
expected for electrostatic interactions,[13] but Ka values
which electrostatic interactions could contribute to binding.
With the exception of 7 (see above), titrations of the receptors
with all-equatorial saccharides, such as glucose, caused signal
movements similar to those observed earlier for 3. In
particular, substantial downfield shifts were observed for
the inward-facing isophthaloyl hydrogen atoms (see, for
example, Figure 2). Plotting of the change in shift of these
hydrogen atoms against substrate concentration gave plots
that fitted well to a 1:1 binding model in most instances and
yielded the binding constants Ka listed in Table 1. In some
cases, supporting values were obtained from isothermal
titration calorimetry (ITC) and fluorescence titrations (see
Table 1).
The data reveal that the side chains in these bisanthra-
cenyl receptors can indeed be tuned to enhance binding
properties and adjust selectivity. As expected, the most
Table 1: Data from the measurement of binding constants to carbohydrates in aqueous solution.[a]
Receptor
2
4
5
6
8
9
Length of side chain[b] (overall charge)
7 (À6)
8 (À12)
10 (À12)
10 (À18)
13 (À36)
15 (À54)
Association constant (Ka [mÀ1]) with:
d-glucosamine (13)[c]
160[d]
1400 (1500[e])
330
2000 (1700[e])
2400 (2100[e])
7000 (9700[e])
610
151
53
[f]
d-glucosamine (13; 20 mm NaCl)[c]
d-glucosamine (13; 154 mm NaCl)[c]
d-galactosamine (14; 154 mm NaCl)[c]
d-glucose (3)
–
–
–
420
135
27
690
1660
340
[f]
97 (76[g])
222< (226[g])
[f]
[h]
–
33
98
4
56 (55,[g] 58[e])
70 (65,[g] 75[e])
89 (91[e])
124
25
90 (81,[h] 87[e])
69 (41[e])
4 (6[e])
[f]
methyl b-d-glucoside
N-acetyl-d-glucosamine (15)
d-galactose
96 (101,[g] 121[e])
87 (87[g])
115 (120[g])
92
33
3
–
[f]
9
19
6
31
7
–
4[d]
0[i]
6
–
[f]
[f]
d-mannose
0[i]
0[i]
0[i]
0[i]
–
Limiting fluorescence change (F/F0)[j] with:
d-glucose
2.5
3.7
3.4
3.7
2.0
2.2
[a] Association constants Ka were measured by 1H NMR titration in D2O at 298 K unless otherwise noted. Calculated errors from curve fitting were
typically ꢁ5%. Data for 2 are from Ref. [4b] unless otherwise noted. See the Supporting Information for experimental details, spectra, and binding
curves, including results with additional substrates. [b] Number of atoms from C1 outwards (see Scheme 1). [c] Measurements were made at pH 7.
For details of conditions and procedures, see the Supporting Information. [d] The Ka value was measured/remeasured as part of the present study.
[e] The Ka value was measured by fluorescence titration in H2O. [f] The Ka value was not determined. At these salt concentrations, receptor 2 gives
broadened 1H NMR spectra, presumably as a result of aggregation. [g] The Ka value was measured by ITC in H2O. [h] Poor fit to a 1:1 binding model,
thus suggesting multiple stoichiometries. [i] Approximate value. The relationship between the signal position and the concentration was almost linear.
[j] Emission at 423 nm, excitation at 395 nm.
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
reported[3g] and among the largest observed for any mono-
saccharide substrate. Effects were also observed for neutral
substrates, despite the absence of electrostatic attraction. The
study provides a rare example of the deliberate positioning of
polar groups to enhance carbohydrate binding in water.
Dendritic side chains will be useful components of future
designs, as they provide an element of control that is
independent of macrocycle structure while ensuring that
water solubility is maintained even for hydrophobic core
structures.
Received: September 15, 2014
Revised: December 1, 2014
Published online: && &&, &&&&
Figure 3. Model of 8 binding glucosammonium 13·H+, featuring salt-
bridge formation by side chains on both sides of the receptor.
Anthracene units are shown in the space-filling mode. Substrate atoms
are pink, side-chain atoms are pale green, and hydrogen bonds to
terminal carboxylate groups are cyan. The structure was minimized
without constraints by the use of MacroModel 10.3 (MMFFs force
field, aqueous GB/SA solvation).
Keywords: carbohydrates · dendrimers · molecular recognition ·
.
receptors · supramolecular chemistry
[1] B. Wang, G.-J. Boons, Carbohydrate Recognition: Biological
Problems, Methods and Applications, Wiley, Hoboken, 2011; D.
Solꢀs, N. V. Bovin, A. P. Davis, J. Jimꢁnez-Barbero, A. Romero,
R. Roy, K. Smetana, Jr., H. J. Gabius, Biochim. Biophys. Acta
Gen. Subj. 2015, 1850, 186.
[2] For reviews, see: a) A. P. Davis, R. S. Wareham, Angew. Chem.
Int. Ed. 1999, 38, 2978; Angew. Chem. 1999, 111, 3160; b) A. P.
Davis, Org. Biomol. Chem. 2009, 7, 3629; c) S. Jin, Y. F. Cheng, S.
Reid, M. Y. Li, B. H. Wang, Med. Res. Rev. 2010, 30, 171; d) M.
Mazik, Chem. Soc. Rev. 2009, 38, 935; e) Y. Nakagawa, Y. Ito,
Trends Glycosci. Glycotechnol. 2012, 24, 1.
remained quite high even at physiological salt levels (154 mm
NaCl). Galactosammonium 14·H+ was bound 3–13 times less
strongly than glucosammmonium, thus showing that the all-
equatorial preference of the cavity is maintained. The simple
nonsaccharidic amine tris(hydroxymethyl)aminomethane
(Tris) was also tested as a control substrate for receptor 6.
A 1H NMR titration at pH 7 produced just minor changes to
the receptor signals, which could not be analyzed to give
a binding constant (see Figure S132).
The studies on uncharged carbohydrates show that effects
are not solely dependent on electrostatic interactions. Binding
to glucose showed a modest but definite increase from 56mÀ1
(for 2) to approximately 90mÀ1 (for 5 and 6). Moreover, the
affinity for N-acetylglucosamine (15) increased by a factor of
about 4 on moving from 2 to 8. Modeling indicates that
intracomplex OÀ···HO and OÀ···HN hydrogen bonds are
geometrically feasible in most of these systems, but are likely
to be more effective for the longer side chains owing to lower
strain.[14] The experiments suggest that both interactions, but
especially OÀ···HN, can be deployed to enhance binding in
water despite competing solvation.
Finally, an unexpected outcome of this study was the
discovery that the fluorescence response to glucose binding
was also affected by the side chains. For prototype 2, we had
previously observed that emission increased by a limiting
value of 2.5 upon titration with glucose (based on the 1:1
binding model). The G2 dendrons in 4, 5, and 6 raised this
value as high as 3.7 (Table 1). Combined with the higher
affinities for glucose, the result indicates an approximately
twofold increase in sensitivity to changes in the glucose
concentration in the range 0–10 mm (see Figure S182). As this
is the concentration range of greatest relevance to diabetes,
the new systems may have practical significance.
In conclusion, we have shown that dendritic side chains
can be used to moderate the binding properties of synthetic
carbohydrate receptors as well as serving as solubilizing
groups. In particular, the strategy was used to increase
affinities for glucosamine to values similar to the highest
[3] For recent examples, see: a) O. Francesconi, C. Nativi, G.
Gabrielli, M. Gentili, M. Palchetti, B. Bonora, S. Roelens, Chem.
Eur. J. 2013, 19, 11742; b) G. Fukuhara, Y. Inoue, J. Am. Chem.
Soc. 2011, 133, 768; c) A. Pal, M. Berube, D. G. Hall, Angew.
Chem. Int. Ed. 2010, 49, 1492; Angew. Chem. 2010, 122, 1534;
d) M. Rauschenberg, S. Bomke, U. Karst, B. J. Ravoo, Angew.
Chem. Int. Ed. 2010, 49, 7340; Angew. Chem. 2010, 122, 7498;
e) C. Geffert, M. Kuschel, M. Mazik, J. Org. Chem. 2013, 78, 292;
f) C. Givelet, B. Bibal, Org. Biomol. Chem. 2011, 9, 7457; g) Y.
Jang, R. Natarajan, Y. H. Ko, K. Kim, Angew. Chem. Int. Ed.
2014, 53, 1003; Angew. Chem. 2014, 126, 1021.
[4] See, for example: a) N. P. Barwell, M. P. Crump, A. P. Davis,
Angew. Chem. Int. Ed. 2009, 48, 7673; Angew. Chem. 2009, 121,
7809; b) C. Ke, H. Destecroix, M. P. Crump, A. P. Davis, Nat.
Chem. 2012, 4, 718; c) B. Sookcharoenpinyo, E. Klein, Y.
Ferrand, D. B. Walker, P. R. Brotherhood, C. F. Ke, M. P.
Crump, A. P. Davis, Angew. Chem. Int. Ed. 2012, 51, 4586;
Angew. Chem. 2012, 124, 4664; d) F. Corzana, A. Fernandez-
Tejada, J. H. Busto, G. Joshi, A. P. Davis, J. Jimꢁnez-Barbero, A.
Avenoza, J. M. Peregrina, ChemBioChem 2011, 12, 110; e) Y.
Ferrand, E. Klein, N. P. Barwell, M. P. Crump, J. Jimꢁnez-
Barbero, C. Vicent, G.-J. Boons, S. Ingale, A. P. Davis, Angew.
Chem. Int. Ed. 2009, 48, 1775; Angew. Chem. 2009, 121, 1807;
f) Y. Ferrand, M. P. Crump, A. P. Davis, Science 2007, 318, 619.
[5] For a study which demonstrates the role of hydrophobic forces in
carbohydrate recognition, see: E. Klein, Y. Ferrand, N. P.
Barwell, A. P. Davis, Angew. Chem. Int. Ed. 2008, 47, 2693;
Angew. Chem. 2008, 120, 2733.
[6] M. Nishio, Phys. Chem. Chem. Phys. 2011, 13, 13873; K.
Ramꢀrez-Gualito, R. Alonso-Rꢀos, B. Quiroz-Garcꢀa, A. Rojas-
Aguilar, D. Dꢀaz, J. Jimꢁnez-Barbero, G. Cuevas, J. Am. Chem.
Soc. 2009, 131, 18129; M. C. Fernꢂndez-Alonso, F. J. CaÇada, J.
Jimꢁnez-Barbero, G. Cuevas, J. Am. Chem. Soc. 2005, 127, 7379;
Z. R. Laughrey, S. E. Kiehna, A. J. Rierrien, M. L. Waters, J.
Am. Chem. Soc. 2008, 130, 14625.
4
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!

Angewandte
Chemie
[7] Macrocyclic receptors with dendrimeric side chains have been
termed “dendrophanes” and have been studied extensively by
Diedreich and co-workers; see, for example: F. Diederich, B.
Felber, Proc. Natl. Acad. Sci. USA 2002, 99, 4778.
[8] C. M. Cardona, R. E. Gawley, J. Org. Chem. 2002, 67, 1411.
[9] For details of syntheses and binding experiments, see the
Supporting Information.
[10] G. R. Newkome, C. D. Weis, Org. Prep. Proced. Int. 1996, 28, 495.
[11] As the experiments are performed at pH 7.0, the glucosamine
(pKa = 7.58) is only partially protonated (ca. 80% according to
the Henderson–Hasselbalch equation). Assuming that glucos-
amine itself is only weakly bound, the affinities for glucosam-
monium are somewhat higher than those reported in Table 1.
[12] Similar connections can be found for the shorter side chains as in
4, and even 2, but in these cases there are clear signs of strain
(see Figure S184).
[13] S. M. Bromfield, A. Barnard, P. Posocco, M. Fermeglia, S. Pricl,
D. K. Smith, J. Am. Chem. Soc. 2013, 135, 2911.
[14] See, for example, Figure S185 in the Supporting Information.
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

.
Angewandte
Communications
Communications
Molecular Recognition
Sticky branches: The carboxylate-termi-
nated side chains in a series of carbohy-
drate receptors contributed to binding if
their length was just right. If they were too
long, they blocked the cavity, but shorter
variants could reach round to interact
with polar groups on the substrate.
Binding is favored by conformational
degeneracy: If one carboxylate group
moves away, another can take its place.
H. Destecroix, C. M. Renney,
T. J. Mooibroek, T. S. Carter,
P. F. N. Stewart, M. P. Crump,
A. P. Davis*
&&& — &&&
Affinity Enhancement by Dendritic Side
Chains in Synthetic Carbohydrate
Receptors
6
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!