Platform Synthetic Lectins for Divalent Carbohydrate Recognition in Water

Article abstract of DOI:10.1002/anie.201603082

Biomimetic carbohydrate receptors (“synthetic lectins”) have potential as agents for biological research and medicine. However, although effective strategies are available for “all-equatorial” carbohydrates (glucose, etc.), the recognition of other types of saccharide under natural (aqueous) conditions is less well developed. Herein we report a new approach based on a pyrene platform with polar arches extending from aryl substituents. The receptors are compatible with axially substituted carbohydrates, and also feature two identical binding sites, thus mimicking the multivalency observed for natural lectins. A variant with negative charges forms 1:2 host/guest complexes with aminosugars, with K₁>3000 m^?1for axially substituted mannosamine, whereas a positively charged version binds the important α-sialyl unit with K₁≈1300 m^?1.

Full text of DOI:10.1002/anie.201603082

Angewandte
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International Edition: DOI: 10.1002/anie.201603082
German Edition: DOI: 10.1002/ange.201603082
Synthetic Receptors
Platform Synthetic Lectins for Divalent Carbohydrate Recognition in
Water
Tom S. Carter, Tiddo J. Mooibroek, Patrick F. N. Stewart, Matthew P. Crump, M. Carmen Galan,
and Anthony P. Davis*
Abstract: Biomimetic carbohydrate receptors (“synthetic
lectins”) have potential as agents for biological research and
medicine. However, although effective strategies are available
for “all-equatorial” carbohydrates (glucose, etc.), the recog-
nition of other types of saccharide under natural (aqueous)
conditions is less well developed. Herein we report a new
approach based on a pyrene platform with polar arches
extending from aryl substituents. The receptors are compatible
with axially substituted carbohydrates, and also feature two
identical binding sites, thus mimicking the multivalency
observed for natural lectins. A variant with negative charges
forms 1:2 host/guest complexes with aminosugars, with K1 >
3000mÀ1 for axially substituted mannosamine, whereas a pos-
itively charged version binds the important a-sialyl unit with K1
ꢀ 1300mÀ1
.
C
arbohydrate recognition is a central biological phenom-
enon that mediates a range of cellular processes.[1] Carbohy-
drate-binding molecules are important as research tools for
investigating these processes, and potentially as diagnostic
and therapeutic agents in medicine.[1–3] Studies in this area
most commonly use lectins, the major class of saccharide-
binding proteins, but lectins often lack the desired selectivities
and tend to show low affinities (generally 103–104 mÀ1 for
monosaccharides).[4] Moreover, as proteins, their therapeutic
potential is limited by issues such as immunogenicity.[3] There
is consequently much interest in small-molecule receptors,
which could complement lectins and perhaps be developed
for new types of application.[1,3,5] However, the design of such
molecules has proved difficult, especially for biomimetic
systems based on noncovalent bonding.[6] Although a variety
of structures have been shown to be active in organic
solvents,[7] there are few which can operate in the natural
but challenging environment of water.[8]
Figure 1. a) A receptor 1 for all-equatorial carbohydrates 2 (see
Ref. [9a]). The symmetrical cavity matches the polar (red) and apolar
(blue) groups in the substrate. b) A strategy for binding saccharides
with axial substituents, illustrated for the b-galactosyl group 3 as the
substrate. c) General design of the pyrene-based receptors described
herein.
We have approached this problem by constructing sym-
metrical cavities with an aromatic “roof” and “floor”
separated by polar spacers (e.g. 1, Figure 1a).[9] The designs
are complementary to saccharides with all-equatorial sub-
stitution patterns (e.g. 2), and have yielded encouraging
results. Selectivities are good, and some affinities are above
104 mÀ1, even for uncharged substrates.[9a,b] However, the
selectivity for all-equatorial carbohydrates is a constraint on
potential applications, as many substrates of interest do not
belong to this family. Herein we report an alternative design
strategy which rationally targets carbohydrates with axial
substituents and which, for the first time, mimics the multi-
valency exhibited by many lectins.[1]
[*] T. S. Carter, Dr. T. J. Mooibroek, P. F. N. Stewart, Prof. M. P. Crump,
Dr. M. C. Galan, Prof. A. P. Davis
School of Chemistry, University of Bristol
Cantock’s Close, Bristol, BS8 1TS (UK)
E-mail: Anthony.Davis@bristol.ac.uk
Supporting information, including all data underlying this study, and
the ORCID identification number(s) for the author(s) of this article
can be found under http://dx.doi.org/10.1002/anie.201603082.
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited.
While all-equatorial saccharides possess two roughly
similar hydrophobic patches, other carbohydrates tend to be
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facially amphiphilic. The inversion of a stereocenter, as in b-
galactosyl 3, adds to the polarity of one face while decreasing
the polarity of the other. A complementary binding site
should therefore contain just one extended apolar surface, the
remainder being mainly polar.[10] An approach to such
structures might involve an aromatic platform with polar
substituents that can arch over a bound carbohydrate (e.g.
Figure 1b). When considering options for realizing this
architecture, we noted the potential of 1,3,6,8-tetraarylpyr-
enes (Figure 1c). These compounds are readily prepared from
tetrabromopyrene (8) by Suzuki–Miyaura methodology, and
are forced to adopt nonplanar conformations owing to
interactions between aryl and peri-H groups.[11] The meta
positions on the aryl groups could provide anchor points for
the polar “arches”. Stereoisomers would be possible in the
general case, but could be avoided by using symmetrically
substituted aryl groups, as shown in Figure 1c. Interestingly,
this arrangement would generate two equivalent binding sites,
thus mimicking the multivalency common in lectins.[1] Groups
Z could be used to confer water-solubility as well as to
provide polar interactions. For example, polyionic dendrim-
ers, which are highly water solubilizing and capable of
hydrogen bonding to polar carbohydrate substituents, could
be readily installed.[9c]
As a prototype for this design, we chose the tetracosa-
carboxylate 9. Modeling[12] showed that the dendrimers in 9,
though relatively small, possessed sufficient reach to interact
with axial groups on a substrate. Protonated mannosamine
10·H+, with an axial NH3 group, was found to be an
+
especially promising substrate (Figure 2). Receptor 9 was
prepared in 23% yield over four steps from diacid 4, amine
5,[13] and 1,3,6,8-tetrabromopyrene (8;[14] Scheme 1).[12] The
anionic receptor 9 dissolved freely in water to give well-
1
resolved H NMR spectra, which were concentration-inde-
Figure 2. Model of receptor 9 bound to the b anomer of protonated
mannosamine 10·H+. Aromatic portions of the receptor are shown in
space-filling mode, side chains in pale green, mannosamine carbon
atoms cyan. a) View from the side, with mannosamine in space-filling
mode. b) View from above, with mannosamine in stick mode. Hydro-
gen bonds are shown in yellow.
pendent below 1.2 mm, thus implying a monomeric species.
Fluorescence spectra showed an emission maximum at
425 nm (excitation wavelength: 380 nm), detectable down to
nanomolar concentrations and with intensity directly propor-
tional to concentration.
Carbohydrate recognition was studied by fluorescence
and 1H NMR titrations of 9 with a range of sugars.[12]
Solutions were adjusted to pH 7, and the pH value was
confirmed to be unchanged after each experiment. Titrations
with aminosugars 10–12 (protonated at pH 7) yielded clear
Scheme 1. Synthesis of receptor 9. a) SOCl2, reflux; then 5, EtNiPr2,
THF, 53%; b) [Pd(dppf)Cl2], bis(pinacolato)diboron, KOAc, dioxane,
808C, 80%; c) 8, [Pd(dppf)Cl2], Cs2CO3, dioxane, H2O, 908C, 53%;
d) CF3CO2H, SiHEt3, CH2Cl2; then aqueous NaOH, quantitative.
dppf=1,1’-bis(diphenylphosphino)ferrocene.
2
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Figure 3. Partial 1H NMR titration spectra of receptor 9 (0.20 mm) with d-mannosamine (10) in D2O at pH 7 and 298 K. a) Signals from receptor
aromatic hydrogen atoms. For numbering, see Figure 1c. b) Mannosamine signals, with intensities normalized to compensate for the increase in
concentration. The mannosamine H1(b) signal is affected by the water-suppression sequence and is artificially depressed in the early stages of
the titration.
Table 1: Cumulative association constants (K1, K2) for 1:1 and 1:2
evidence of complex formation. For example, in NMR
titrations, the addition of mannosamine (10) was accompa-
nied by downfield changes in the positions of the receptor
binding of receptor 9 with aminosugars in aqueous solution, as
1
determined by H NMR and fluorescence titrations.[a]
K1 [mÀ1
]
K2 [mÀ1
]
aromatic signals (see Figure 3a). A Job plot[15] based on the
receptor 4-H signal displacements gave a maximum at 9/10 =
1:2, thus confirming divalency (see Figure S14 in the Support-
ing Information). Analysis of the receptor signals, assuming
the 1:2 binding model, gave stepwise binding constants K1 =
3120mÀ1 and K2 = 540mÀ1 (see Figure S9).[16] As K1 = 4K2 for
noncooperative two-site binding,[17] it seems the two associ-
ations are almost independent. The results were supported by
fluorescence titrations of 9 with 10, which showed large
increases in receptor emission intensity and could be analysed
to give almost identical binding constants (Figure 4 and
Table 1).
Carbohydrate
d-mannosamine (10)
d-galactosamine (11)
d-glucosamine (12)
3120,[b] 3120[c]
1800,[b] 2000[c]
1040,[b] 1100[c]
540,[b] 600[c]
200,[b] 180[c]
410,[b] 360[c]
[a] pH 7, T=298 K. [b] 1H NMR titration. [c] Fluorescence titration.
a and b anomers, present in the ratio 1:1.9.[19] Signals for
hydrogen atoms on the a face of the b anomer [for example,
H1(b), H5(b)] showed especially large movements, consistent
with the modeled structure (Figure 2). From the a anomer,
the proton H5(a) signal also moved substantially. NOE data
(see Figure S15) confirmed that 9 and the carbohydrate are
closely associated. Binding could not be quantified reliably
because of the complexity of the system (two substrates, both
forming 1:1 and 1:2 complexes). However, the signal move-
ments were consistent with K1 ꢀ 3000mÀ1 for both anomers.
Titrations with galactosamine (11) and glucosamine (12)
gave similar changes in both 1H NMR and fluorescence
spectra. Analysis of the data gave the binding constants
shown in Table 1. Affinities were somewhat lower, suggesting
Changes in the positions of carbohydrate signals during
1
the H NMR titration were also informative (Figure 3b). All
the signals from 10 moved downfield during the titration
implying that, as expected, these protons are shielded in the
complex.[18] The spectra provide separate information for
+
that the binding sites of 9 favor axial NH3 substitution.
Uncharged monosaccharides did not appear to bind, but
titrations with the disaccharides cellobiose, lactose, and
maltose, and also methylamine, gave evidence of weak-
complex formation (K1 ꢁ 16mÀ1).[12] It thus seems that the
high affinities for aminosugars result from combining carbo-
hydrate-specific interactions (hydrophobic, CH–p, hydrogen
bonding) with electrostatic attraction.
Whereas cationic sugars are relatively uncommon in
nature, anionic carbohydrates are widespread and play
important roles. Especially significant is the a-linked N-
acetylneuraminic acid (a-sialyl) unit 13. This moiety com-
monly appears as a terminus of oligosaccharides, accessible
for binding and therefore an important potential target.[20]
Figure 4. Fluorescence emission spectra and binding curve of receptor
9 (0.33 mm) titrated with d-mannosamine (10) in H2O at pH 7 and
298 K. Excitation wavelength: 380 nm.
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The substitution pattern in 13 is all-equatorial with an
additional axial negatively charged substituent. This com-
pound should be nicely complementary to our platform
design (Figure 1b), provided that the polar arches are
positively charged.
To test this concept, we prepared the cationic receptor 16,
possessing 24 guanidinium units (see the Supporting Infor-
mation). Guanidinium substituents were chosen to ensure
Figure 5. Fluorescence emission titration spectra and binding model
fit of receptor 16 (0.50 mm) with methyl sialoside (14) in H2O at pH 7
and 298 K. Excitation wavelength: 380 nm.
the changes assuming a 1:2 binding model (see Figure 5) gave
stepwise binding constants K1, K2 = 1300, 790mÀ1, consistent
with the NMR data. The decrease in emission was found to
depend on the chloride counterions; when these were
replaced with trifluoroacetate, fluorescence increased on
binding. We presume that the addition of 14 to 16 causes
a rearrangement of the counterions, thus promoting fluores-
cence quenching.
Fluorescence titrations were also performed for receptor
16 with several uncharged monosaccharides.[12] Efficient
quenching was again observed, although analysis suggested
that binding was much weaker than for 14. Moderate affinities
were estimated for methyl b-d-glucoside (K1 = 43mÀ1),
methyl b-d-galactoside (K1 = 46mÀ1), and galactose (K1 =
17mÀ1). In these cases, NMR shifts were too small for analysis,
but association between the receptor and the carbohydrate
was confirmed by NOE enhancements.[22] Sodium acetate was
also bound weakly (K1 = 17mÀ1). The affinity for glucose was
too small to be quantified implying that, as intended, the
platform design can reverse the selectivity shown by our
earlier synthetic lectins.
that the receptor would be fully protonated at pH 7.[21]
Spectroscopic studies implied that receptor 16 is monomeric
in water below a concentration of 1.2 mm. As expected, the
1H NMR spectrum of 16 was unaffected by the pH value in
the range 6–8, confirming full protonation.
Studies of 16 as a receptor for the a-sialyl unit 13 required
a model substrate. The parent saccharide N-acetylneuraminic
acid is readily available, but exists mainly (> 90%) as the
b anomer 15, and is thus unrepresentative of 13. Simple a-
sialosides are not commercially available, and we therefore
synthesized the methyl derivative 14 through a variation of
a literature procedure.[12] The binding of 14 to 16 was studied
1
by H NMR and fluorescence spectroscopy. NOESY cross-
peaks between substrate and receptor aromatic proton signals
supported complex formation (see Figure S55). On the other
hand, NMR titrations yielded relatively small changes in the
signal positions, implying a looser geometry than for 9 + 10. A
Job plot based on a receptor aromatic signal confirmed the
expected 16/14 = 1:2 binding stoichiometry (see Figure S54).
For quantitative analysis, we employed a titration in which
receptor 16 was added to substrate 14. The carbohydrate
signals moved upfield as expected, and several could be
followed throughout (see Figure S57). Simultaneous analysis
of four of these signals was consistent with three successive
In conclusion, we have reported a rational design for
synthetic lectins which mimic the multivalency shown by
natural lectins and, unlike earlier systems, can accommodate
substrates with axial substituents. There is scope for varying
the system, for example, by altering/extending the side chains
or changing one aryl substituent. The potential for binding the
b-sialyl group 13 is especially significant, and will be a focus of
future efforts.
binding events, with K1, K2, and K3 = 1310, 570, and 30mÀ1
,
Acknowledgements
respectively. Given the high density of positive charge on 16,
it is reasonable to suppose that a third (and possibly a fourth)
molecule of 14 might bind to the receptor.
Fluorescence titration of 16 with 14 yielded a surprisingly
strong effect (Figure 5); receptor emission was reduced
almost to zero by addition of the carbohydrate. Analysis of
This research was supported by the Engineering and Physical
Sciences Research Council through grant number EP/
I028501/1 and by a studentship funded by the Bristol
Chemical Synthesis Doctoral Training Centre (EP/G036764/
1) to T.S.C.
4
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[9] For recent examples, see: a) P. Rios, T. S. Carter, T. J. Mooi-
Keywords: carbohydrates · dendrimers · molecular recognition ·
broek, M. P. Crump, M. Lisbjerg, M. Pittelkow, N. T. Supekar,
G.-J. Boons, A. P. Davis, Angew. Chem. Int. Ed. 2016, 55, 3387;
Angew. Chem. 2016, 128, 3448; b) T. J. Mooibroek, J. M. Casas-
Solvas, R. L. Harniman, C. M. Renney, T. S. Carter, M. P.
Crump, A. P. Davis, Nat. Chem. 2016, 8, 69; c) H. Destecroix,
C. M. Renney, T. J. Mooibroek, T. S. Carter, P. F. N. Stewart,
M. P. Crump, A. P. Davis, Angew. Chem. Int. Ed. 2015, 54, 2057;
Angew. Chem. 2015, 127, 2085; d) C. Ke, H. Destecroix, M. P.
Crump, A. P. Davis, Nat. Chem. 2012, 4, 718.
receptors · supramolecular chemistry
[1] a) D. Solꢀs, N. V. Bovin, A. P. Davis, J. Jimꢁnez-Barbero, A.
Romero, R. Roy, K. Smetana, H. J. Gabius, Biochim. Biophys.
Acta Gen. Subj. 2015, 1850, 186; b) B. Wang, G.-J. Boons,
Carbohydrate Recognition: Biological Problems, Methods and
Applications, Wiley, Hoboken, 2011; c) The Sugar Code—
Fundamentals of Glycoscience (Ed.: H.-J. Gabius), Wiley-Black-
well, Weinheim, 2009.
[2] J. Balzarini, Nat. Rev. Microbiol. 2007, 5, 583.
[3] Y. Nakagawa, T. Doi, T. Taketani, K. Takegoshi, Y. Igarashi, Y.
Ito, Chem. Eur. J. 2013, 19, 10516.
[4] E. J. Toone, Curr. Opin. Struct. Biol. 1994, 4, 719.
[5] C. E. Miron, A. Petitjean, ChemBioChem 2015, 16, 365; Y.
Nakagawa, Y. Ito, Trends Glycosci. Glycotechnol. 2012, 24, 1; M.
Mazik, RSC Adv. 2012, 2, 2630; A. P. Davis, Org. Biomol. Chem.
2009, 7, 3629; A. P. Davis, R. S. Wareham, Angew. Chem. Int. Ed.
1999, 38, 2978; Angew. Chem. 1999, 111, 3160.
[10] As commonly observed for proteins; see, for example: K. L.
Hudson, G. J. Bartlett, R. C. Diehl, J. Agirre, T. Gallagher, L. L.
Kiessling, D. N. Woolfson, J. Am. Chem. Soc. 2015, 137, 15152.
[11] X. Feng, H. Tomiyasu, J. Y. Hu, X. Wei, C. Redshaw, M. R. J.
Elsegood, L. Horsburgh, S. J. Teat, T. Yamato, J. Org. Chem.
2015, 80, 10973.
[12] For further details, see the Supporting Information.
[13] G. R. Newkome, C. D. Weis, Org. Prep. Proced. Int. 1996, 28,
495 – 498.
[14] K. Ogino, S. Iwashima, H. Inokuchi, Y. Harada, Bull. Chem. Soc.
Jpn. 1965, 38, 473 – 477.
[15] C. Y. Huang, Methods Enzymol. 1982, 87, 509.
[16] Conditions were chosen to minimize ionic strength and max-
imize electrostatic interactions (see the Supporting Informa-
tion). As expected, binding constants are lowered by the
addition of salts.
[17] P. Thordarson, Chem. Soc. Rev. 2011, 40, 1305.
[18] The proportion of carbohydrate bound is highest early in the
titration.
[19] Binding-constant measurements based on receptor properties
(fluorescence, receptor NMR signals) should give average values
for the two anomers; for a discussion, see: N. P. Barwell, M. P.
Crump, A. P. Davis, Angew. Chem. Int. Ed. 2009, 48, 7673;
Angew. Chem. 2009, 121, 7809.
À
[6] An alternative strategy is to employ covalent B O bonds; see,
for example, Ref. [1b] and T. D. James, M. D. Phillips, S. Shinkai,
Boronic Acids in Saccharide Recognition, RSC, Cambridge,
2006.
[7] For recent examples, see: Y. Ohishi, H. Abe, M. Inouye, Chem.
Eur. J. 2015, 21, 16504; J. Lippe, M. Mazik, J. Org. Chem. 2015,
80, 1427; N. Chandramouli, Y. Ferrand, G. Lautrette, B.
Kauffmann, C. D. Mackereth, M. Laguerre, D. Dubreuil, I.
Huc, Nat. Chem. 2015, 7, 334; A. Schmitt, O. Perraud, E. Payet,
B. Chatelet, B. Bousquet, M. Valls, D. Padula, L. Di Bari, J. P.
Dutasta, A. Martinez, Org. Biomol. Chem. 2014, 12, 4211; O.
Francesconi, M. Gentili, C. Nativi, A. Arda, F. J. Canada, J.
Jimꢁnez-Barbero, S. Roelens, Chem. Eur. J. 2014, 20, 6081; S.
Rieth, M. R. Miner, C. M. Chang, B. Hurlocker, A. B. Braunsch-
weig, Chem. Sci. 2013, 4, 357.
[8] See, for example: Y. Jang, R. Natarajan, Y. H. Ko, K. Kim,
Angew. Chem. Int. Ed. 2014, 53, 1003; Angew. Chem. 2014, 126,
1021; M. Rauschenberg, S. Bandaru, M. P. Waller, B. J. Ravoo,
Chem. Eur. J. 2014, 20, 2770; S. M. Bromfield, A. Barnard, P.
Posocco, M. Fermeglia, S. Pricl, D. K. Smith, J. Am. Chem. Soc.
2013, 135, 2911; C. Geffert, M. Kuschel, M. Mazik, J. Org. Chem.
2013, 78, 292.
[20] T. Angata, A. Varki, Chem. Rev. 2002, 102, 439; A. Varki, Trends
Mol. Med. 2008, 14, 351.
[21] The corresponding tetracosaamine precursor to 16 was also
studied. However, partial protonation of the amino groups
resulted in broadened spectra, which could not be interpreted.
[22] As the affinities for 16 + uncharged monosaccharides were
quantified by just one technique, they should be seen as
tentative.
Received: March 29, 2016
Published online: && &&, &&&&
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Communications
Synthetic Receptors
Be my guests: Carbohydrate receptors
featuring two equivalent binding sites
were designed to target facially amphi-
philic substrates. A version with anionic
side chains favors aminosugars, whereas
a cationic variant binds the biologically
important a-sialyl unit (as shown).
T. S. Carter, T. J. Mooibroek,
P. F. N. Stewart, M. P. Crump,
M. C. Galan, A. P. Davis*
&&&& — &&&&
Platform Synthetic Lectins for Divalent
Carbohydrate Recognition in Water
6
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