Selective Recognition of d -Aldohexoses in Water by Boronic Acid-Functionalized, Molecularly Imprinted Cross-Linked Micelles

Article abstract of DOI:10.1021/jacs.6b04613

Molecular imprinting within cross-linked micelles using 4-vinylphenylboronate derivatives of carbohydrates provided water-soluble nanoparticle receptors selective for the carbohydrate templates. Complete differentiation of d-aldohexoses could be achieved

Full text of DOI:10.1021/jacs.6b04613

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Communication
Selective Recognition of D-Aldohexoses in Water by Boronic
Acid-Functionalized Molecularly Imprinted Cross-Linked Micelles
Joseph K. Awino, Roshan W. Gunasekara, and Yan Zhao
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b04613 • Publication Date (Web): 21 Jul 2016
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Selective Recognition of D-Aldohexoses in Water by Boronic Acid-
Functionalized Molecularly Imprinted Cross-Linked Micelles
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Joseph K. Awino, Roshan W. Gunasekara, and Yan Zhao*
Department of Chemistry, Iowa State University, Ames, Iowa 50011-3111
Supporting Information Placeholder
Strongest binding is obtained when the maximum number of
boronate ester bonds are formed with minimal strain.
ABSTRACT: Molecular imprinting within cross-linked mi-
celles using 4-vinylphenylboronate derivatives of carbohy-
drates provided water-soluble nanoparticle receptors selec-
tive for the carbohydrate templates. Complete differentiation
of D-aldohexoses could be achieved by these receptors if a
single inversion of hydroxyl occurred at C2 or C4 of the sugar
or if two or more inversions took place. Glycosides with a hy-
drophobic aglycan displayed stronger binding due to in-
creased hydrophobic interactions.
The above results suggest that a general method to recog-
nize carbohydrates might result if the number, distance, and
orientation of boronic acids on the receptor can be tuned pre-
cisely to match the hydroxyl groups on the guest. Such struc-
tural control, however, is difficult to imagine given the mi-
nute structural differences of the carbohydrates.
Herein, we report that this level of structural precision can
be readily obtained through covalent molecular imprinting
within cross-linked micelles. The molecularly imprinted na-
noparticles (MINPs) obtained practically could distinguish
all eight D-aldohexoses based on the configurations of the hy-
droxyls. This work lays the foundation for the construction of
receptors to bind more complex carbohydrates, since differ-
ent hydroxyls contributed quite differently to the binding.
Carbohydrates are one of the most important classes of bi-
omolecules and involved in numerous biological processes
including cell–cell interaction, immune response, and viral
and bacterial infection.1,2 Synthetic analogues of carbohy-
drate-binding lectins are powerful tools for the detection of
biologically important sugars and intervention of carbohy-
drate-mediated interactions.3 Although receptors based on
both covalent4-8 and noncovalent9-12 interactions have been
reported, a general method for selectively binding carbohy-
drates in water remains elusive.13,14
With a tripropargylammonium headgroup, surfactant 1
could be cross-linked in the micellar form on the surface by
click chemistry with diazide 2 (Scheme 1), to afford alkynyl-
functionalized surface-cross-linked micelles (i.e., alkynyl-
SCM).22-24 Template 3 was solubilized in water together with
DVB and a photoinitiator (DMPA) by 1 in the very beginning.
It was prepared from 4-vinylphenylboroxine and glucose ac-
cording a literature procedure.25 Because the diboronate
product contains different isomers (e.g., 3a, 3b, etc.) that un-
dergo transesterification during repeated crystallization, the
material obtained from a single crystallization was used with-
out further purification.26 After surface-cross-linking, azide
3 was added to decorate the resulting alkynyl-SCMs with a
layer of hydrophilic ligands. Free radical polymerization was
subsequently initiated by UV irradiation in the core to poly-
merize/cross-link the methacrylate of 1, DVB, and the poly-
merizible styrenyl groups of 3. The doubly cross-linked mi-
celles were recovered by precipitation from acetone. Glucose
was removed by repeated washing with acetone/water, meth-
anol/acetic acid, and acetone.27 Template 3 has a free hy-
droxyl. As demonstrated previously, hydrophilic groups on
the template anchor the template near the surface of the mi-
celle/MINP and is helpful to its removal/rebinding.28,29
As building blocks of oligo- and polysaccharides, the eight
D-aldohexoses differ only in the stereochemistry of 1–3 hy-
droxyls. Unfortunately, the hydroxyl group is not a good
functional group handle from the supramolecular viewpoint,
because its strong solvation by water makes it difficult to use
hydrogen bonds to bind a sugar. Having the same number of
carbon and hydroxyls, the aldohexoses have only minute dif-
ferences in hydrophobicity, mainly in the axial/equatorial
distribution of the hydroxyls. Although the difference has
been used successfully to distinguish glucose/glucoside from
other monosaccharides,11,15 it is not clear how the other D-al-
dohexoses can be differentiated in this regard.
Despite these tremendous challenges, literature suggests
boronic acid-based covalent receptors could potentially over-
come the difficulty. Boronic acid-functionalized molecularly
imprinted polymers (MIPs) were reported in the 1970s by
Wulff as the stationary phase to separate sugar derivatives by
chromatography.16 Monoboronic acids generally bind fruc-
tose more strongly than glucose due to its higher percentage
of furanose that contains the preferred synperiplanar 1,2-diol
for boronate formation.17 This selectivity, interestingly, could
be reversed with diboronic acids preorganized to form boro-
nate with the particular hydroxyls of glucose, whether the
furanose or pyranose form.18-21 The preorganization comes
from the organic scaffold whose structure and conformation
dictate how the diboronic acids interact with its guests.
The MINP preparation was adapted from our published
procedures.28-31 Cross-linking of the micelles and formation
of the MINPs likewise were monitored by 1H NMR spectros-
copy and dynamic light scattering (DLS), as shown by Figures
S1 and S2 in the Supporting Information. DLS also allowed
us to estimate the molecular weight of the nanoparticles.
SCMs were characterized previously also by transmission
electron microscopy (TEM) and mass spectrometry (after
cleaving the surface-cross-linkages).22
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× 103 M-1). Binding with selected non-aldohexoses was very
O
Br
N
weak, with Ka = 0.06 and 0.003 × 103 M-1 for fructose and
xylose, respectively. Thus, covalent imprinting within the
cross-linked micelles must have positioned the boronic acids
quite accurately to match the hydroxyls on the templating
sugar. Our MINPs were prepared with a 1:1 surfactant/DVB
ratio. This amount of DVB was the maximum that could be
solubilized by the micelle and corresponds to ~50 molecules
of DVB per micelle. The high cross-linking density was found
previously to be critical to the MINP binding selectivity.28
O
DVB
1
2
3
4
5
6
7
8
Cu(I),
DMPA
1
OH
+
OH
OH
OH
N3
N3
3
2
alkynyl-SCM
O
O
B
O
OH
O
B
4
3a
3b
(a) Cu(I),
(b)
O
+
hv
Remove
OH
(c)
glucose
=
B
3
O
OH
O
O
OH
OH
OH
HO
HO
O
B
O
OH
HO
HO
HN
HO
OH
OH
OH
9
HO
O
etc.
HO
HO
O
O
HN
=
OH
OH
HO
HN
DVB
HO
HO
OH
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11
12
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O
N
OH
OH
Table 1. ITC binding data for boronic acid-func-
tionalized MINPs.a
N
N
N
N
HO
N
HN
HO
N
N
N
N
HN
O
N
N
OH
OH
HO
O
O
HO
N
N
N
=
N
N
DMPA
=
N
N
HO
H
B
MeO
HO OH
OMe
Ka
-ΔG
OH
B
Entry
Host
Guest
N
N
N
N
O
OHOH
HO
OH
(103 M-1) (kcal/mol)
OH
HO
O
4
N3
OH
H
O
OH
N
N
HO
NH
N
N
H
N
OHOH
OH
HO
HO
N
glucose
mannose
allose
1
2
MINP(glucose)
MINP(glucose)
MINP(glucose)
MINP(glucose)
MINP(glucose)
MINP(glucose)
MINP(glucose)
MINP(glucose)
MINP(glucose)
MINP(glucose)
MINP(glucose)
MINP(glucose)
MINP(mannose)
MINP(mannose)
MINP(mannose)
MINP(mannose)
MINP(mannose)
MINP(mannose)
MINP(mannose)
MINP(mannose)
MINP(5)
1.18 ± 0.20
0.003b
4.19
1.0 ± 0.1
N
OH
N
N
N
OH
N
N
N
N
N
N
O
N
N
NH
OH
OH
NH
HO
N
N
OH
HO
HO
O
HO
NH
NH
3
0.52 ± 0.02
0.002b
0.004b
0.009b
0.011b
3.7
--b
--b
--b
--b
0.9 ± 0.1
OH
O
NH
OH
OH
OH
HO
OH
OH
O
O
OH
HO
HO
--b
--b
--b
--b
--b
1.0 ± 0.1
galactose
altrose
gulose
talose
HO
4
HO
HO
OH
HO
HO
OH
HO
5
6
MINP(glucose)
7
Scheme 1. Preparation of boronic acid-functionalized
MINP(glucose).
0.004b
--b
idose
8
fructose
xylose
glucosec
glucosed
glucose
mannose
allose
9
0.06 ± 0.01
0.003b
2.4
--b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
--b
1.1 ± 0.1
0.6 ± 0.1
--b
To understand the potential of MINPs in carbohydrate
recognition, we prepared boronic acid-functionalized recep-
tors first for glucose, mannose, and galactose. Among the
eight D-aldohexoses, these three are most biologically rele-
vant, serving as building blocks for many naturally occurring
oligo- and polysaccharides. We also prepared MINP(5), us-
ing the diboronate derivative of 4-nitrophenyl α-D-mannopy-
ranoside 5 as the template. We are interested in the glycoside
because the added hydrophobic aglycan potentially could
contribute to the binding. In addition, the glycoside will be
bound in the pyranoside form, different from the free sugars.
0.54 ± 0.17
1.20 ± 0.30
0.012b
3.7
4.2
--b
0.94 ± 0.06
0.003b
0.003b
4.1
--b
--b
1.1 ± 0.1
--b
--b
galactose
altrose
gulose
talose
0.56 ± 0.02
0.001b
0.013b
3.8
--b
--b
0.9 ± 0.1
--b
--b
0.001b
--b
--b
idose
5
26.3 ± 3.0
8.1 ± 0.16
4.1 ± 0.15
0.80 ± 0.07
6.0
5.3
4.9
4.0
0.5 ± 0.1
1.1 ± 0.1
1.1 ± 0.1
0.6 ± 0.1
OH
OH
OH
OH
HO
HO
O
O
6
O
O
MINP(5)
HO
HO
HO
HO
HO
HO
7
OH
OH
OH
OH
OH
OH
MINP(5)
OH
OH
OH
OH
OH
mannose
MINP(5)
O
HO
a 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. b Binding was extremely
weak. Because the binding constant was estimated from ITC, -ΔG
and N are not listed (see SI for details). c The binding was in 10 mM
HEPES buffer at pH 6.5. d The binding was in 10 mM HEPES buffer
at pH 8.5.
O
HO
O
O
HO
HO
HO
HO
HO
HO
D
HO
OH
OHHO
OH
HO
OH
OH
HO
-Glucose
D
-Allose
D
-Mannose
D
-Altrose
OH
HO
OH
OH
OH
HO
O
O
O
HO
HO
HO
HO
OH
OH
O
NO2
NO2
NO2
O
O
To our delight, the binding selectivity was reproduced
when mannose and galactose were used as the templates.
MINP(mannose), for example, bound its sugar template with
Ka of 0.94 × 103 M-1 and no other D-aldohexoses except al-
trose (Ka = 0.56 × 103 M-1) (Table 1, entries 13–20; see Chart
1 for structures). MINP(galactose), likewise, only bound its
template (Ka = 1.41 × 103 M-1) and one other sugar (gulose, Ka
= 0.80 × 103 M-1) among the D-aldohexoses (Table 1S).
5
6
7
4-nitrophenyl-
-mannopyranoside
4-nitrophenyl-
4-nitrophenyl-
D
-galactopyranoside
-
-
-
α
α
α
D
D
-glucopyranoside
Chart 1. Selected template/guest molecules in this study.
All the bindings were studied by isothermal titration calo-
rimetry (ITC). The method previously was found to yield sim-
ilar binding constants (Ka) as fluorescence titration for fluo-
rescently labelled templates.28-31 As shown in Table 1, Ka be-
tween MINP(glucose) and glucose was 1.18 × 103 M-1 in 10
mM HEPES buffer at pH 7.4. This binding affinity compares
favorably with those between lectins and their monosaccha-
ride ligands (typically 103–104 M-1).1,2 Importantly, the MINP
receptor displayed excellent selectivity, showing negligible
binding toward other D-aldohexoses except allose (Ka = 0.52
Molecular imprinting has become a very powerful tech-
nique to create guest-complimentary materials.32-43 Conven-
tional typical MIPs, however, are intractable highly cross-
linked polymers. In our case, because polymerization and
cross-linking largely occurred within the boundary of the mi-
celles, the MINPs were completely soluble in water due to
their nanosize and hydrophobic/hydrophilic core–shell
structure.28-31 Note that most MINPs in Table 1 had a single
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4-Nitrophenyl α-D-mannopyranoside 5 should interact
binding site per nanoparticle on average.44 The number of
1
2
3
4
5
6
7
8
binding site per nanoparticle is controlled by the surfac-
tant/template ratio used in the MINP preparation and, as
demonstrated earlier, is fully tunable if desired.28 This num-
ber was determined by the ITC titration from the guest/host
ratio.45 Previously, the ITC-determined binding stoichiome-
try was found to agree well with those determined by the Job
plots for fluorescently labeled substrates.46
with the MINP receptor through hydrophobic interactions, in
addition to two boronate esters through the C2,3 and C5,6
hydroxyls.25 After template removal, a binding site is ex-
pected to form in the hydrophobic core of the cross-linked
micelle, complementary to the 4-nitrophenyl group in size
and shape. The hydrophobic imprinting worked very well for
many substrates in our previous work.28-31 Consistent with
the added hydrophobic interactions, binding between 5 and
MINP(5) was over twenty times stronger than those between
the sugars and their MINPs (Table 1, entry 21). Encourag-
ingly, significant selectivity was found for the binding of sim-
ilar glycosides. Glucopyranoside 6 was bound by MINP(5)
with <1/3 of the binding affinity and mannopyranoside 7
with ~1/6. Apparently, inversion of one or two hydroxyl
groups could still be easily distinguished, even for the pyra-
nosides. Lastly, MINP(5) was also able to bind mannose with
Ka = 0.5 × 103 M-1 (entry 24). The binding constant was sim-
ilar to that between mannose and its own MINP (entry 14).
Thus, the binding site imprinted from the mannopyranoside
was able to bind mannose, most likely in the pyranose form.
It is very interesting that a consistent binding selectivity
was displayed by all three MINPs. Strong binding for the
template sugar was fully expected. Why did the MINP bind
only one of the remaining D-aldohexoses? The answer be-
comes clear when the following trends are considered.
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10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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32
33
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(a) Inversion of two (or more) hydroxyl groups of the tem-
plating sugar turns off the binding essentially completely.
This trend was seen from glucose to any of the diaxial sugars,
and from mannose to allose/galactose/idose. The anomeric
hydroxyl at C1 was not considered because it exchanges be-
tween the α and β form as the hemiacetal.
(b) The effect of a single hydroxyl inversion depends on the
position of the hydroxyl. Consistently for all three MINPs, in-
version at either C2 or C4 turned off the binding—e.g., from
glucose to mannose/galactose or from mannose to glu-
cose/talose. Inversion at C3, however, weakened the binding
by 40–60%, e.g., from glucose to allose, mannose to altrose,
or galactose to gulose. As shown by xylose, missing the C6
hydroxyl also caused a complete loss of binding.
The most significant discovery of this research is the gen-
eral applicability of the MINP receptors, which are com-
pletely water-soluble and similar to proteins in size. It is very
important that the MINP receptors displayed a very clear and
consistent trend in the binding, mainly controlled by the C2,
C4, and C6 hydroxyls of the D-aldohexoses. The milli- and
submillimolar binding affinities already approached those
found in natural lectins for monosaccharides. The observed
binding selectivity suggests that the cross-linked micelles can
be used as a platform to precisely position and orient binding
groups even to distinguish minute structural changes in car-
bohydrates. If the same holds true for oligo- and polysaccha-
rides, a general method for selective binding of carbohy-
drates will become available.
When monoboronic acids bind free monosaccharides, lit-
erature generally agrees that binding of the first boronic acid
occurs through the C1,2 hydroxyls of the sugar.4,47,48 Binding
of the second boronic acid, however, could differ depending
on the reaction conditions (e.g., aqueous or nonaqueous sol-
vent, solution pH, concentration) and the structure of the bo-
ronic acid. The situation is complicated further by pyranose–
furanose interconversion. Furanose, having the preferred
synperiplanar 1,2-diols for boronate formation,17 is generally
the minor component, sometimes representing <1% of the
mixture. Nonetheless, two or all three of the C3,5,6 hydroxyls
are frequently involved in the second boronate formation for
furanose.4 There is also evidence that, for certain sugars, ad-
ditional conformations (e.g., twist boat) may play roles in the
binding, at least in aqueous alkaline media.48
ASSOCIATED CONTENT
Supporting Information
Experimental details, ITC titration curves, and additional
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
In our case, the hydroxyls at C2, C4, and C6 were critical to
the binding and the C3 hydroxyl played a secondary role. The
results suggest that, under our conditions, the boronic acids
probably bound two pairs of hydroxyls, at C1,2 and C4,6, re-
spectively. Because the boronate template was synthesized in
organic solvent under azeotropic distillation, neutral triva-
lent boronate esters instead of negatively charged tetravalent
structures are expected.4
AUTHOR INFORMATION
Corresponding Author
zhaoy@iastate.edu
Notes
The authors declare no competing financial interests.
Solution pH has a large effect on the binding of small-mol-
ecule boronic acids. A change of pH from 6.5 to 8.5 increased
the binding constant between phenyl boronic acid and glu-
cose by over an order of magnitude.49 The pH effect came
from the acid–based equilibria involved in the binding and
the tetrahedral vs. trigonal forms of boronic acid/boronate.
In our case, the same pH change barely had any effect on the
MINP binding (compare entries 1, 11, and 12 of Table 1). Pre-
sumably, the overall hydrophobicity of the diboronate tem-
plate means that the boronic acids would reside in a relatively
hydrophobic region of the MINP. With poor solvent expo-
sure, neither the boronic acids nor the boronate esters
formed after binding would be very sensitive to the solution
pH.
ACKNOWLEDGMENT
We thank the National Institute of General Medical Sciences
of the National Institutes of Health (R01GM113883) for fi-
nancial support of this research.
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