A general method for selective recognition of monosaccharides and oligosaccharides in water

Article abstract of DOI:10.1021/jacs.6b10773

Molecular recognition of carbohydrates plays vital roles in biology but has been difficult to achieve with synthetic receptors. Through covalent imprinting of carbohydrates in boroxole-functionalized cross-linked micelles, we prepared nanoparticle receptors for a wide variety of mono- and oligosaccharides. The boroxole functional monomer bound the sugar templates through cis-1,2-diol, cis-3,4-diol, and trans-4,6-diol. The protein-sized nanoparticles showed excellent selectivity for daldohexoses in water with submillimolar binding affinities and completely distinguished the three biologically important hexoses (glucose, mannose, and galactose). Glycosides with nonpolar aglycon showed stronger binding due to enhanced hydrophobic interactions. Oligosaccharides were distinguished on the basis of their monosaccharide building blocks, glycosidic linkages, chain length, as well as additional functional groups that could interact with the nanoparticles.

Full text of DOI:10.1021/jacs.6b10773

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Article
A General Method for Selective Recognition of
Monosaccharides and Oligosaccharides in Water
Roshan W. Gunasekara, and Yan Zhao
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b10773 • Publication Date (Web): 16 Dec 2016
Downloaded from http://pubs.acs.org on December 18, 2016
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A General Method for Selective Recognition of Monosaccharides and
Oligosaccharides in Water
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Roshan W. Gunasekara and Yan Zhao*
Department of Chemistry, Iowa State University, Ames, Iowa 50011ꢀ3111
ABSTRACT: Molecular recognition of carbohydrates plays vital roles in biology but has been difficult to achieve with
synthetic receptors. Through covalent imprinting of carbohydrates in boroxoleꢀfunctionalized crossꢀlinked micelles, we
prepared nanoparticle receptors for a wide variety of monoꢀ and oligosaccharides. The boroxole functional monomer
bound the sugar templates through cisꢀ1,2ꢀdiol, cisꢀ3,4ꢀdiol, and transꢀ4,6ꢀdiol. The proteinꢀsized nanoparticles
showed excellent selectivity for Dꢀaldohexoses in water with submillimolar binding affinities and completely distinꢀ
guished the three biologically important hexoses (glucose, mannose, and galactose). Glycosides with nonpolar aglycon
showed stronger binding due to enhanced hydrophobic interactions. Oligosaccharides were distinguished based on
their monosaccharide building blocks, glycosidic linkages, chain length, as well as additional functional groups that
could interact with the nanoparticles.
pletely different physical, chemical, and biological propꢀ
erties.
INTRODUCTION
Carbohydrates occupy a unique place in biology. Unꢀ
Molecular recognition of carbohydrates has progressed
like peptides and nucleic acids, they comprise entirely of
steadily in the last decades. Over the years, synthetic
hydrophilic building blocks and are thus solvated strongꢀ
receptors moved from organic to aqueous solution; carꢀ
ly by water. This feature implies that carbohydrates tend
bohydrate guests being studied transitioned from simple
to cover the surface of a cell and represent the first line
monosaccharides to functionalized oligosaccharides.
of interaction when other entities approach the cell. For
Chemists nowadays are able to distinguish glucosides
this reason, it is not surprising that carbohydrates are
from their isomeric sugars by their all equatorial substiꢀ
involved in many important biological processes includꢀ
ing fertilization, cell–cell interactions, immune response,
and viral and bacterial infection.1ꢀ3 In addition, they are
important sources of energy for most organisms and
form parts of the backbone for DNAs and RNAs.
tutions.7,8 Binding affinities for monosaccharides by synꢀ
thetic receptors in water could approach those by natural
lectins (binding constant Ka = 103–104 Mꢀ1).1,2 Despite
these impressive accomplishments, however, a general
method for molecular recognition of carbohydrates in
water is still not available, due to the many challenges
mentioned above.
Lectins are protein receptors that perform molecular
recognition of carbohydrates in nature. During the last
several decades, chemists have devoted great efforts toꢀ
wards developing synthetic analogues of lectins that can
bind sugars or their derivatives selectively.1ꢀ6 On the apꢀ
plied level, the research potentially can lead to tools useꢀ
ful in the study and intervention of carbohydrateꢀrelated
biological processes. On the fundamental level, the reꢀ
search tackles one of the most difficult challenges in suꢀ
pramolecular chemistry.
Synthetic carbohydrate receptors can be classified in
two groups, depending on whether noncovalent or covaꢀ
lent bonds are used for binding. The first group often
utilizes strategically positioned hydrogen bonds in a relaꢀ
tively hydrophobic microenvironment to bind the
guest.6ꢀ10 The second group largely relies on the fast and
reversible boronate bonds formed between organic boꢀ
ronic acids and the diol functionalities on a sugar for the
molecular recognition.5,11ꢀ15
Selective binding of carbohydrates in water is difficult
for multiple reasons. Due to strong interactions between
water and the hydroxyls of a carbohydrate, a supramoꢀ
lecular host in aqueous solution has to pay a tremendous
amount of desolvation energy to bind its sugar guest.
Unlike proteins and DNAs, carbohydrates do not adopt
wellꢀdefined threeꢀdimensional conformations, making
the design of their complementary hosts difficult. Monoꢀ
saccharides, the building blocks of more complex carboꢀ
hydrates, differ minutely in structure, often by the stereꢀ
ochemistry of a single hydroxyl. Even with the same
building block, slightly different connections between the
monomers lead to oligoꢀ and polysaccharides with comꢀ
We recently reported a method to construct molecularꢀ
ly imprinted nanoparticles (MINPs) with precisely posiꢀ
tioned boronic acids to recognize monosaccharides in
water.16 The MINP receptors could distinguish
Dꢀ
aldohexoses with remarkable selectivity. For example,
MINP(glucose), i.e., MINP prepared with glucose as the
template, bound glucose with Ka = 1.18 × 103 Mꢀ1. Any
change in the C2, C4, or C6 hydroxyl essentially turned
off the binding and inversion of the C3 hydroxyl weakꢀ
ened the binding by over twoꢀfold.
Unfortunately,
functionalized MINPs showed impressive binding for
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monosaccharides, the synthetic method could not be situ imprinting are the highlights of this approach. The
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easily applied to oligosaccharides. Herein, we report
that, by modifying the key ingredients in the MINP prepꢀ
aration (i.e., the crossꢀlinkable surfactant, the crossꢀ
linker, and the sugarꢀbinding functional monomer) and
the imprinting procedure, we now can create nanopartiꢀ
cle receptors for oligosaccharides (and monosaccharides)
directly in water. The generality and simplicity of the in
preparation and purification took about 2 days and reꢀ
quired no special techniques, and thus could be potenꢀ
tially adopted by researchers without substantial training
in chemistry. These receptors are soluble in water, reꢀ
semble proteins in size, and displayed selectivity for
monosaccharides and oligosaccharides that has not been
achieved
by
previous
synthetic
materials.
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Scheme 1. Preparation of boroxoleꢀfunctionalized MINP(glucose).
fully soluble in water due to their hydrophoꢀ
bic/hydrophilic core–shell structure.29,30
RESULTS AND DISCUSSION
Design and Synthesis. Molecular imprinting is a
tremendously useful technique for creating guestꢀ
complementary binding sites in polymers or on
surface.17ꢀ28 However, conventional imprinting often
produces intractable highly crossꢀlinked polymers, hinꢀ
dering their usage in biology. To make the imprinted
materials soluble in water, we recently reported a proꢀ
cess to imprint within crossꢀlinked micelles. Because the
polymerization and crossꢀlinking took place within the
micelle boundaries, the resulting nanoparticles become
MINPs are generally prepared by first solubilizing a
hydrophobic template molecule with the micelles of a
crossꢀlinkable surfactant such as 1 (see Scheme 1 for
structure). The surfactant contains a propargylated
headgroup and a methacrylateꢀcontaining hydrophobic
tail that undergo orthogonal crossꢀlinking chemistries.
Crossꢀlinking by a diazide crossꢀlinker such as 2 yields
alkyneꢀfunctionalized
(SCMs), which can be functionalized by another round of
click reaction with an azideꢀcontaining ligand such as 3
surfaceꢀcrossꢀlinked
micelles
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(see Scheme 1 for structure). Afterwards, free radical conveniently done right afterwards. However, because
1
2
3
4
coreꢀcrossꢀlinking leads to the formation of a polymer
matrix around the template within the SCM, and thus
creates the binding site in the micellar core complemenꢀ
tary to the template in size, shape, and binding functionꢀ
ality.
the surface ligand (3) contains many hydroxyls and is
expected to compete with glucose for the boroxole bindꢀ
ing group, we reversed the order and performed the
coreꢀcrossꢀlinking in the second step, via UVꢀinitiated
radical polymerization of 1, 2′, 5, and DVB.
5
6
7
8
9
At this point, the binding site was already formed inꢀ
side the surfaceꢀ and coreꢀ doublyꢀcrossꢀlinked micelles.
Surfaceꢀfunctionalization with 4 using the click reaction
afforded MINP(glucose) with the template still bound in
the binding site. The sugarꢀderived ligand 4 was inꢀ
stalled so that the final nanoparticles could be easily reꢀ
covered by precipitation into acetone.29 The template
molecules were removed by repeated washing using aceꢀ
tone/water, methanol/acetic acid, and acetone. The
power obtained was completely soluble in water.
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The templates used in the
Dꢀaldohexoseꢀbinding
MINPs were the boronate esters formed from the sugars
and 4ꢀvinylphenylboronic acid.16 They had to be syntheꢀ
sized in a separate step prior to the MINP preparation
through azeotropic removal of water in dioxane at 88
°C.31 Because oligosaccharides generally have extremely
low solubility in dioxane and many organic solvents, this
method is not suitable for imprinting oligosaccharides. If
we want to imprint more sensitive sugar derivatives such
as glycoproteins in a longer term, organic solvents and
high temperatures clearly have to be avoided.
1
The reaction progress was generally monitored by H
NMR spectroscopy.29,30 Dynamic light scattering (DLS)
afforded the size and molecular weight of the MINP. The
nanoparticles were typically 4–5 nm in diameter. In our
experience, the DLSꢀdetermined size showed good
agreement with the size obtained from transmission
electron microscopy (TEM) for similarly crossꢀlinked
micelles.44
In this work, we synthesized boroxoleꢀcontaining funcꢀ
tional monomer (FM) 432 and a new crossꢀlinker 2′ to
address the above challenges (Scheme 1). Benzoboroxole
is known to bind 1,2ꢀ and 1,3ꢀdiols with higher affinities
than phenylboronic acid33,34 and have been used to create
sugarꢀbinding polymers.35ꢀ43 We reasoned that the anionꢀ
ic boronate derivative formed (i.e., 5) might be especially
stable in the cationic micelles of 1. (As will be shown latꢀ
er, the structure of 5 was inferred from our binding studꢀ
ies, as well as the binding property of boroxole.)33,34 If
the complex can survive the surfaceꢀ and coreꢀcrossꢀ
linking of the micelles, we would be able to imprint a
sugar directly in the micellar solution. In situ imprinting
is highly desirable because it eliminates the separate
template preparation and may be more compatible with
templates sensitive to organic solvents and/or high temꢀ
peratures.
MINPs for Binding Monosaccharides. We examꢀ
ined the binding of the MINP by isothermal by isotherꢀ
mal titration calorimetry (ITC), a method of choice for
studying intermolecular interactions.47 In addition to its
accuracy, the method affords the number of binding sites
per particle (N), as well as other thermodynamic binding
parameters. We have demonstrated in several studies
that (for fluorescently labeled guests) ITC gave very simiꢀ
lar binding constants for MINPs as other spectroscopic
methods.29,30,48
As shown in Table 1, MINP(glucose) prepared with
template/FM = 1:2 bound glucose with Ka = 2.30 × 103
Mꢀ1 in 10 mM HEPES buffer at pH 7.4 (entry 1). Binding
was somewhat weaker at pH 8.5 or 6.5 (entries 6 and 7).
Reducing the template/FM ratio to 1:1 lowered the bindꢀ
ing constant (entry 2). Having an excess of FM (thee
equiv to the template) did not improve the binding (enꢀ
try 3). Binding was negligible by the nonimprinted mateꢀ
rials prepared without FM 3 and the glucose template
(entry 4) or with FM 3 but without glucose (entry 5).
These results demonstrated that molecular imprinting
was clearly in operation and the optimal binding stoichiꢀ
ometry was 1:2 between the template and the boroxole.49
There are two considerations behind the design of
crossꢀlinker 2′. First, since a noncovalently formed
FM•template complex (i.e., 5) is involved, we have to
avoid other diolꢀcontaining molecules such as 2 in the
MINP preparation, at least prior to the formation of the
binding site. Second, 2′ is amphiphilic and expected to
form mixed micelles with 1, enabling the alkyne and azꢀ
ide groups to be intimately mixed on the surface of the
micelles and in close proximity to one another. As a reꢀ
sult, the local concentrations of the reactive groups are
exceedingly high on the micelle surface, making the surꢀ
face crossꢀlinking particularly facile.44,45
MINP(glucose) displayed excellent selectivity: among
the seven isomeric sugars, only allose showed noticeable
binding with Ka = 0.37 × 103 Mꢀ1, while the rest were not
bound at all (Chart 1). Similar selectivity was found for
MINP(mannose), which only bound altrose among the
remaining seven Dꢀaldohexoses.
As usual, we solubilized DVB (a free radical crossꢀ
linker) and DMPA (a photoinitiator) in the (mixed) miꢀ
celles prior to any crossꢀlinking. The presence of DVB
increases the crossꢀlinking density of the core and was
confirmed previously to be important to the molecular
recognition of the final MINP.29 The 3:2 ratio of 1 and 2′
left the SCM with alkynyl groups on the surface.44ꢀ46
Normally, we perform surfaceꢀfunctionalization before
coreꢀcrossꢀlinking because it uses the same Cu(I) cataꢀ
lysts as the surfaceꢀcrossꢀlinking step and thus can be
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and C4 hydroxyls were critical to the molecular recogniꢀ
1
2
3
4
5
6
7
8
tion and any inversion at these positions shuts off the
binding. The C6 hydroxyl was also essential, as xylose,
lacking this hydroxyl, showed no binding. The C3 hyꢀ
droxyl played a secondary role in the binding, with its
inversion lowering Ka by 74–86% from the template sugꢀ
ar.
MINP(galactose), on the other hand, behaved distincꢀ
tively differently. Among the eight
Dꢀaldohexoses, it
bound only its template and achieved stronger binding
(Ka = 3.37 × 103 Mꢀ1) than either MINP(glucose) or
MINP(mannose) for its template (Table 1).
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Hall and coꢀworkers reported that benzoboroxole
binds glucose in a 1:1 ratio in water, with Ka = 17 Mꢀ1.33,34
It is possible that the 2nd binding observed in our MINPs
was weaker than the first one in bulk aqueous solution
and simply not observed in Hall’s study. The hydrophoꢀ
bic and positive environment of the cationic micelle conꢀ
ceivably could stabilize the negatively charged boronate
and enable the second, less stable adduct to form under
Chart 1. Structures of selected Dꢀaldohexoses and glycoꢀ
sides.
The boroxoleꢀfunctionalized MINP(glucose) and
MINP(mannose) showed higher binding selectivity than
the boronic acidꢀfunctionalized MINPs, but the trend
remained the same.16 The selectivity suggests that the C2
our
imprinting
and
binding
Table 1. ITC binding data for monosaccharide guests.a
Entry
Host
Guest
Ka (× 103 Mꢀ1)
ꢀꢁG (kcal/mol)
N
1
2
MINP(glucose)
MINP(glucose)b
MINP(glucose)c
NINPd
glucose
glucose
glucose
glucose
glucose
glucoseg
glucoseh
allosei
mannose
altrosej
galactosek
6
2.30 ± 0.11
0.95 ± 0.01
2.33 ± 0.38
<0.05e
4.58
4.06
4.59
ꢀ
1.1 ± 0.1
1.2 ± 0.1
1.0 ± 0.1
ꢀ
3
4
5
NINPf
<0.05e
ꢀ
ꢀ
6
MINP(glucose)
MINP(glucose)
MINP(glucose)
MINP(mannose)
MINP(mannose)
MINP(galactose)
MINP(6)
1.30 ± 0.16
0.52 ± 0.09
0.37 ± 0.09
1.90 ± 0.34
0.50 ± 0.01
3.37 ± 0.30
65.3 ± 8.8
11.0 ± 1.2
4.24
3.70
3.51
4.47
3.68
4.81
6.56
5.51
5.00
1.0 ± 0.1
1.1 ± 0.1
0.8 ± 0.1
1.0 ± 0.3
1.0 ± 0.1
1.0 ± 0.1
1.1 ± 0.1
1.0 ± 0.1
1.1 ± 0.1
7
8
9
10
11
12
13
14
MINP(6)
7
MINP(6)
8
4.66 ± 0.39
a The FM/template ratio in the MINP synthesis was 1:2 unless otherwise indicated. The titrations were performed in 10 mM
HEPES buffer at pH 7.4. The ITC titration curves are reported in the Supporting Information, including the binding enthalpy
b
c
d
and entropy. The template/FM ratio was 1:1. The template/FM ratio was 1:3. Prepared without FM 3 and the glucose temꢀ
plate. e Binding was extremely weak. Because the binding constant was estimated from ITC, ꢀꢁG and N are not listed in the
f
g
table (Figure 62S in Supporting Information). Prepared with FM 4 but without the glucose template. The binding was in 10
i
mM HEPES buffer at pH 8.5. h The binding was in 10 mM HEPES buffer at pH 6.5. The binding for other
Dꢀaldohexoses inꢀ
cluding mannose, galactose, altrose, gulose, talose, idose, and xylose was extremely weak, with estimated Ka <0.02 × 103 Mꢀ1
j
(Figure 66S and 67S). The binding for other
D
ꢀaldohexoses including glucose, allose, galactose, gulose, talose, and idose was
k
extremely weak, with estimated Ka <0.02 × 103 Mꢀ1 (Figure S68). The binding for other
Dꢀaldohexoses including glucose,
mannose, allose, altrose, gulose, talose, and idose was extremely weak, with estimated Ka <0.05 × 103 Mꢀ1 (Figure 69S and
70S).
conditions.
benzoboroxole has a strong preference for transꢀ4,6ꢀdiol
over transꢀ3,4ꢀdiol in glucosides, suggesting the C3 hyꢀ
droxyl would not be involved in binding in glucose and
mannose.50 Hall’s work also demonstrated that, for galaꢀ
topyranosides, cisꢀ3,4ꢀdiol is preferred by boroxole over
cisꢀ4,6ꢀdiol. This preference was also maintained by
MINP(galactose), because gulose, which differs from
galactose only by the C3 hydroxyl and contains the cisꢀ
4,6ꢀdiol, was not bound.51
Benzoboroxole binds the methyl pyranosides of gluꢀ
cose, mannose, and galactose with Ka = 10–30 Mꢀ1,33,34
thus lacking intrinsic selectivity for these sugars. The
much higher selectivity and binding affinity displayed by
our MINPs must come from the microenvironment of
the crossꢀlinked micelle and the twoꢀpoint binding as
revealed in the binding studies. It is known that that
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For MINP(6) prepared with 4ꢀnitrophenyl αꢀ
Dꢀ
Maltose was the first oligosaccharide template used in
our study and expected to form FM•template complex 9
based on the binding motifs identified in the monosacꢀ
charideꢀbinding MINPs. Because numerous hydrogenꢀ
bonding groups exist in the complex, we hypothesized
that the micelle/MINP should contain hydrogenꢀ
bonding groups that interact with 9 through hydrogen
bonds, in addition to hydrophobic and electrostatic inꢀ
teractions present in the normal micelle/MINP. Amideꢀ
functionalized crossꢀlinkable surfactant 10
1
mannopyranoside 6 as the template, the aromatic aglyꢀ
con was expected to create a complementary hydrophoꢀ
bic binding pocket in the MINP, as we have demonstratꢀ
ed in several recent studies.29,30,48 Indeed, a much
stronger binding of Ka = 65.3 × 103 Mꢀ1 was obtained.
Gratifyingly, excellent binding selectivity was maintained
for this MINP. The Ka values for the corresponding gluꢀ
coside 7 and galactoside 8 were ~1/6 and 1/14, respecꢀ
tively. Thus, inversion of one or two hydroxyl groups was
easily distinguished in the glycosides as well.
2
3
4
5
6
7
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By confining the polymerization/crossꢀlinking largely
within micelles, we not only made our materials waterꢀ
soluble but also were able to control the number of bindꢀ
ing sites on the nanosized MINP. This feature distinꢀ
guishes our MINP from other molecularly imprinted
nanoparticles in the literature.52ꢀ60 Our previous studies
indicate that the SCM of 1 has roughly 50 crossꢀlinked
surfactants. With surfactant/template = 50/1 in the synꢀ
thesis, the MINPs on average contained one binding site
per nanoparticle (Table 1).61 As demonstrated recently,
this number can be tuned easily through changing the
surface/template ratio.29
was recently found to enhance the binding of guest
through
hydrogen
bonds.62
To
our
delight,
MINP(maltose) prepared with 10 as the crossꢀlinkable
surfactant bound maltose with Ka = 20.5 × 103 Mꢀ1, subꢀ
stantially higher than the value obtained (Ka = 3.50 × 103
Mꢀ1) for MINP prepared with surfactant 1 (Table 2, enꢀ
tries 1 and 2). When the template/FM ratio was varied
(1:1, 1:2, and 1:3), 1:2 gave
MINPs for Binding Oligosaccharides. FM 4 not
only afforded MINPs with higher binding affinity and
selectivity than 4ꢀvinylphenylboronuc acid but also enaꢀ
bled us to imprint oligosaccharides.
Table 2. ITC binding data for oligosaccharide guests.a
Ka
Entry
Host
Guest
Krel
ꢀꢁG (kcal/mol)
N
(103 Mꢀ1)
20.5 ± 3.2
3.50 ± 0.23
maltose
maltose
1
2
MINP(maltose)
MINP(maltose)b
MINP(maltose)c
MINP(maltose)d
MINP(maltose)
MINP(maltose)
MINP(maltose)
MINP(maltose)
MINP(maltose)
MINP(maltose)
MINP(maltose)
MINP(maltose)
MINP(cellobiose)
MINP(cellobiose)
MINP(cellobiose)
MINP(cellobiose)
MINP(cellobiose)
MINP(lactose)
1
ꢀ
5.88
4.83
5.12
5.85
5.32
4.96
ꢀ
1.0 ± 0.1
1.2 ± 0.1
1.2 ± 0.1
1.0 ± 0.1
1.2 ± 0.1
1.2 ± 0.1
ꢀ
maltose
3
5.72 ± 0.61
19.7 ± 2.5
7.99 ± 0.12
4.37 ± 0.53
<0.05
ꢀ
maltose
4
ꢀ
cellobiose
gentiobiose
maltulose
lactose
5
0.39
0.21
<0.002
0.04
<0.002
0.09
ꢀ
6
7
8
0.79 ± 0.16
<0.05
3.95
ꢀ
0.8 ± 0.1
ꢀ
maltotriose
9
glucose
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
1.81 ± 0.22
15.2 ± 2.0
18.8 ± 2.7
9.45 ± 0.14
32.9 ± 5.9
4.77 ± 0.67
<0.05
4.44
5.70
5.83
5.42
6.16
5.01
ꢀ
0.9 ± 0.1
0.8 ± 0.1
0.9 ± 0.1
maltosee
maltosef
maltose
ꢀ
0.29
1
1.1 ± 0.1
1.1 ± 0.1
cellobiose
gentiobiose
maltulose
lactose
ꢀꢀ
1.1 ± 0.1
0.14
<0.002
0.04
0.06
0.13
0.22
0.01
1
ꢀ
1.29 ± 0.09
3.24 ± 0.42
6.83 ± 0.92
11.6 ± 1.7
0.50 ± 0.13
52.2± 9.5
52.8 ± 8.6
14.1 ± 2.0
4.24
4.79
5.23
5.54
3.67
6.43
6.44
5.66
0.8 ± 0.1
1.0 ± 0.1
0.8 ± 0.1
0.9 ± 0.1
1.0 ± 0.1
1.3 ± 0.1
1.1 ± 0.1
1.0 ± 0.1
maltose
cellobiose
gentiobiose
maltulose
lactose
MINP(lactose)
MINP(lactose)
MINP(lactose)
MINP(lactose)
maltotriose
maltose
MINP(maltotriose)
MINP(maltotriose)
1
0.27
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glucose
The template/FM ratio in the MINP synthesis was 1:2 unless otherwise indicated. The crossꢀlinkable surfactants
25
MINP(maltotriose)
0.56 ± 0.02
0.01
3.75
1.0 ± 0.1
1
2
3
4
5
6
7
8
a
were a 3:2 mixture of 10 and 2′ unless otherwise indicated. The titrations were performed in 10 mM HEPES buffer at
pH 7.4. Krel is the binding constant of a guest relative to that of the template sugar for a particular MINP. The ITC titraꢀ
b
tion curves are reported in the Supporting Information, including the binding enthalpy and entropy. The crossꢀ
c
d
linkable surfactants were a 3:2 mixture of 1 and 2′. The template/FM ratio was 1:1. The template/FM ratio was 1:3.
eThe titration was performed in the presence of cellobiose in 10 mM HEPES buffer at pH 7.4. [MINP] = 15 ꢂM. [cellobiꢀ
ose] = 75 ꢂM. The titration was performed in the presence of lactose in 10 mM HEPES buffer at pH 7.4. [MINP] = 15
f
ꢂM. [lactose] = 75 ꢂM.
9
We then created MINPs for all the other oligosacchaꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
rides and studied their binding. Good selectivity was
generally obtained and each MINP always bound its own
the highest Ka, supporting the 1:2 binding model shown
in 9.
template sugar better than other sugars (Table 2 and
Binding of the oligosaccharides (Chart 2) worked fully
Table 3S). As far as the absolute binding strength is conꢀ
as expected (Table 2). The selectivity of a particular
cerned, gentiobiose, lactose, and maltotriose gave someꢀ
MINP is indicated by Krel, which is the binding constant
what higher Ka values than the other sugars. The strongꢀ
of a sugar guest relative to that of the template. Cellobiꢀ
er binding for maltotriose could result from the addiꢀ
ose and gentiobiose had a Krel value of 0.39 and 0.21 toꢀ
tional hydroxyls on the template that interacted with the
ward MINP(maltose), indicating that changing the α 1,4ꢀ
amideꢀfunctionalized MINP by hydrogen bonds. For
glycosidic linkage to the β 1,4 or α 1,6 weakened the
MINP(maltotriose), as the guest became smaller (i.e.,
binding significantly. Replacing one of the two glucoses
from maltotriose to maltose to glucose), binding expectꢀ
in maltose with fructose and galactose was even less tolꢀ
edly weakened monotonously (Table 2, entries 21–23).
erated, yielding Krel of <0.002 and 0.04 for maltulose
To test whether these boroxoleꢀfunctionalized recepꢀ
and lactose, respectively. To probe the sensitivity, we
tors could distinguish more challenging targets, we preꢀ
also measured the binding of maltose by MINP(maltose)
pared MINPs for the three sugars that determine the
in the presence of 5 equiv of competing sugars (cellobiꢀ
human blood type: type O has sugar H on the surface of
ose and lactose). As shown by entries 11 and 12, the bindꢀ
its blood cells, type A has A, type B has B, and type AB
ing constant obtained was about 74% and 92%, respecꢀ
tively, of the original value (entry 1). These numbers
were in line with the selectivity indicated by Krel.
has both A and B.
As shown in Table 3, MINP(H), generated from sugar
H, bound its template with Ka = 35.6 × 103 Mꢀ1 and
showed no binding for the other two sugars. The differꢀ
ence between sugar A and B was extremely subtle:
among the numerous functional groups, the only differꢀ
ence is a single acetoamido group in sugar A versus a
hydroxyl in B (Chart 2). Impressively, MINP(A) was
found to bind sugar A twice as strongly as sugar B, and
MINP(B) displayed even higher selectivity. Meantime,
sugar H showed weak binding to MINP(A) and MINP(B),
with Krel = 0.13 in both cases.
Table 3. ITC binding data for blood sugars.a
ꢀꢁG
(kcal/mol
Ka
(103 Mꢀ1)
35.6 ±
5.2
Entry Host
Guest
Krel
N
MINP(H
1.0 ±
1
2
3
4
5
6
7
8
sugar H
1
6.2
ꢀ
)
0.1
MINP(H
<0.00
sugar A <0.02
sugar B <0.02
ꢀ
Chart 2. Structures of oligosaccharides used in this
study. The arrows indicate the hydroxyls potentially inꢀ
volved in the boronate formation with FM 4.
)
1
MINP(H
<0.00
ꢀ
ꢀ
)
1
MINP(A
1.0 ±
sugar H 9.9 ± 0.1 0.13
5.45
6.66
6.26
5.29
5.91
6.48
)
0.1
Interestingly, shortening the chain length was better
tolerated than lengthening the chain length, as glucose
was bound with Krel = 0.09 but maltotriose with Krel
<0.002. The result is reasonable because maltotriose
should not fit into the binding pocket generated from the
smaller maltose but glucose should be able to fit it, altꢀ
hough only expected to bind one of the two boroxoles.
MINP(A
76.7 ±
1.0 ±
sugar A
sugar B
1
)
1.2
0.1
MINP(A
39.0 ±
1.1 ±
0.51
)
4.8
0.1
MINP(B
1.1 ±
sugar H 7.6 ± 0.8 0.13
)
0.1
MINP(B
21.8 ±
1.0 ±
sugar A
sugar B
0.38
1
)
4.3
0.1
MINP(B
57.1 ±
1.1 ±
9
)
7.5
0.1
a
Note that Ka (= 1.81
×
103 Mꢀ1) for glucose by
The template/FM ratio in the MINP synthesis was 1:2
MINP(maltose) was close to that (= 2.30 × 103 Mꢀ1) by
MINP(glucose) in Table 1. It seems that the hydrogenꢀ
bonding interactions between the bound glucose and the
amideꢀfunctionalized MINP nearly compensated for the
loss of one boronate binding interaction.
for MINP(H) and 1:3 for MINP(A) and MINP(B). The crossꢀ
linkable surfactants were a 3:2 mixture of 10 and 2′. The
titrations were performed in 10 mM HEPES buffer at pH
7.4. The ITC titration curves are reported in the Supporting
Information, including the binding enthalpy and entropy.
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(11) Kim, K. T.; Cornelissen, J. J. L. M.; Nolte, R. J. M.; van
CONCLUSIONS
1
2
3
4
5
6
7
8
Hest, J. C. M. J. Am. Chem. Soc. 2009, 131, 13908.
(12) Pal, A.; Bérubé, M.; Hall, D. G. Angew. Chem. Int. Ed.
2010, 49, 1492.
(13) Wu, X.; Li, Z.; Chen, X.ꢀX.; Fossey, J. S.; James, T. D.;
Jiang, Y.ꢀB. Chem. Soc. Rev. 2013, 42, 8032.
(14) Bull, S. D.; Davidson, M. G.; Van den Elsen, J. M. H.;
Fossey, J. S.; Jenkins, A. T. A.; Jiang, Y. B.; Kubo, Y.;
Marken, F.; Sakurai, K.; Zhao, J. Z.; James, T. D. Acc.
Chem. Res. 2013, 46, 312.
(15) Wulff, G.; Vesper, W. J. Chromatogr. 1978, 167, 171.
(16) Awino, J. K.; Gunasekara, R. W.; Zhao, Y. J. Am. Chem.
Soc. 2016, 138, 9759.
In summary, we have reported a facile and general
method to create proteinꢀsized waterꢀsoluble nanopartiꢀ
cle receptors for a wide range of monoꢀ and oligosacchaꢀ
rides. The in situ imprinting was enabled by the strong
interactions between FM 4 and the appropriate diol
functionalities on the sugar in the micellar environment.
The number of binding sites on these “synthetic lectins”
could be controlled easily. Importantly, the binding sites
on the sugar can be identified prior to imprinting (nameꢀ
ly, cisꢀ1,2ꢀdiol, cisꢀ3,4ꢀdiol, and transꢀ4,6ꢀdiol), making
the molecular recognition highly predictable. Among the
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(17) Wulff, G. Angew. Chem. Int. Ed. Engl. 1995, 34, 1812.
(18) Wulff, G. Chem. Rev. 2001, 102, 1.
eight Dꢀaldohexoses, glucose, mannose, and galactose are
the most biologically relevant and can be distinguished
completely. With the ability to differentiate oligosacchaꢀ
rides by their building blocks, chain length, and glycosidꢀ
ic linkages, we expect these “synthetic lectins” could beꢀ
come highly useful in biology and chemistry in the fuꢀ
ture.
(19) Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495.
(20)Ye, L.; Mosbach, K. Chem. Mater. 2008, 20, 859.
(21) Shea, K. J. Trends Polym. Sci. 1994, 2, 166.
(22)Sellergren, B. Molecularly imprinted polymers: man-
made mimics of antibodies and their applications in
analytical chemistry; Elsevier: Amsterdam, 2001.
(23) Komiyama,
M.
Molecular
imprinting:
from
ASSOCIATED CONTENT
Supporting Information
fundamentals to applications; WileyꢀVCH: Weinheim,
2003.
(24)Zimmerman, S. C.; Lemcoff, N. G. Chem. Commun.
2004, 5.
(25) Yan, M.; Ramström, O. Molecularly imprinted
materials: science and technology; Marcel Dekker:
New York, 2005.
(26)Alexander, C.; Andersson, H. S.; Andersson, L. I.;
Ansell, R. J.; Kirsch, N.; Nicholls, I. A.; O'Mahony, J.;
Whitcombe, M. J. J. Mol. Recognit. 2006, 19, 106.
(27) Sellergren, B.; Hall, A. J. In Supramol Chem; John
Wiley & Sons, Ltd: 2012.
(28)Haupt, K.; Ayela, C. Molecular Imprinting; Springer:
Heidelberg ; New York, 2012.
(29)Awino, J. K.; Zhao, Y. J. Am. Chem. Soc. 2013, 135,
12552.
Experimental details, ITC titration curves, and additional
data. This material is available free of charge via the Interꢀ
net at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
zhaoy@iastate.edu
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
(30)Awino, J. K.; Zhao, Y. Chem.-Eur. J. 2015, 21, 655.
(31) Wulff, G.; Schauhoff, S. J. Org. Chem. 1991, 56, 395.
(32) Kim, H.; Kang, Y. J.; Kang, S.; Kim, K. T. J. Am. Chem.
Soc. 2012, 134, 4030.
We thank the National Institute of General Medical Sciences
of the National Institutes of Health (R01GM113883) for
financial support of the research.
(33) Dowlut, M.; Hall, D. G. J. Am. Chem. Soc. 2006, 128,
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