Modulating the Catalytic Performance of an Immobilized Catalyst with Matrix Effects - A Critical Evaluation

1. Modulating the Catalytic Performance of an Immobilized Catalyst
with Matrix Effects - A Critical Evaluation
Babloo Sharma, Susanne Striegler,* and Madison Whaley
Department of Chemistry and Biochemistry, 345 North Campus Drive, University of Arkansas, Fayetteville, Arkansas 72701, United States
*S Supporting Information
ABSTRACT: Microgels with embedded binuclear copper(II)
complex were prepared in the presence of galactose and man-
nose as biomimetic catalysts for the hydrolysis of glycosidic
bonds. The study was designed to elucidate matrix effects
responsible for the high catalytic proficiency (kcat/KM × knon)
of the microgels that reaches up to 1.7 × 106
upon hydrolysis
of 4-methylumbelliferyl-β-D-mannopyranoside. The experi-
mental results reveal differences of sugar coordination to the
binuclear copper(II) complex in coordination sites, binding strength, overall geometry, and binding energies that differ by
7.1 kcal/mol and are based on experiments using UV−Vis spectroscopy and isothermal titration calorimetry. Accompanying
computational analyses, based on density functional theory (DFT) at the B3LYP/m6-31G(d) level of theory, further support
the experimental results of sugar coordination by suggesting plausible binding sites of sugar coordination and providing
additional insight into the cause of substrate discrimination during microgel-catalyzed glycoside hydrolyses. Subsequent kinetic
analyses correlate the catalytic proficiency of the microgels with contributions of the metal complex core, the surrounding cross-
linked matrix, and strongly binding mannose; however, the data reveal minor contributions of a templating effect to the overall
catalytic performance of the water-dispersed microgel catalysts.
KEYWORDS: macromolecular catalyst, matrix effects, polyacrylate microgels, hydrolysis, glycosides
1. INTRODUCTION
Microgels are cross-linked or highly branched polymers that
may be dispersed in water.1−3
Since their discovery as an
intriguing class of polymers, modifying the gels with stimuli-
responsive and performance-oriented properties has attracted
considerable attention.1
The inner core of the networked
microgel structure has often diffusion-controlled accessibility.
Microgels are frequently used as drug delivery vessels,4,5
as
nanoreactors capable of modulating the catalytic activity of
metal nanoparticles,3,6,7
and for functions in sensors and
optics.1
The facile one-pot synthesis of microgels from oil-in-
water nanodroplets constitutes an appealing alternate approach
for the preparation of biomimetic catalysts8−11
and has been
employed for the synthesis of templated catalytically active
microgels.12−24
In a typical synthesis, metastable colloidal structures are
formed by ultrasheering of monomers and cross-linkers in the
presence of surfactants and hydrophobes in aqueous solution
and secured by subsequent polymerization yielding water-
dispersed microgels.25
For the immobilization of a binuclear
copper(II) complex, we extended the formulation and added a
pentadentate backbone ligand, metal ion salts, and masking
nonpolymerizable ligands so that a free-radical polymerization
is not obstructed by the paramagnetic character of the Cu(II)
complex.26
Along these lines, a binuclear copper(II) complex
Cu2VBbpdpo (1a) was formed in situ from the backbone ligand
and copper(II) acetate (Chart 1),27
masked with a carbohydrate,
and immobilized in the microgel matrix by thermally initiated free
radical polymerization. The composition of the sugar-metal
complex assembly was deduced for the employed conditions
using speciation data of the nonpolymerizable low molecular
weight analog, Cu2bpdpo (1b).28−31
Extraction of all nonpoly-
merizable compounds and subsequent reactivation resulted
in microgel catalysts that hydrolyzed the glycosidic bond of
arylglycosides 160-fold faster than the low molecular weight
complex Cu2bpdpo but did not discriminate epimeric or anomeric
glycosides.27
The results indicate that the matrix was not templated
and that the sugar merely functions as a counterion during mate-
rial preparation.27,32
Received: May 17, 2018
Revised: July 5, 2018
Published: July 10, 2018
Chart 1. Composition of Metal Complexes Cu2VBbpdpo
(1a) and Cu2bpdpo (1b) in Aqueous Solution at pH 10.50;
X=H2O
Research Article
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2. To challenge the molecular imprinting (template polymer-
ization) approach, we hypothesize here that matrix effects around
a hydrolytic metal complex controls its catalytic activity, whereas
shape-selectivity of a “templated” matrix provides insignificant
contributions to the overall catalytic performance of the resulting
macromolecular catalysts. In other words, a cavity around a
catalytically active metal complex does not control the activity
and selectivity of the resulting microgel catalyst but may only
have a minor contribution to its catalytic performance, if any at all.
As a first step to verify or reject this hypothesis, we devel-
oped a protocol for free-radical polymerization of sugar-metal
complex assemblies at ambient temperature and below.33
Under
these conditions, the binding of a sugar-metal complex assembly
is strengthened to facilitate templating, while the possibility
for decomposition of a carbohydrate ligand during material
synthesis is minimized.34
In accordance with numerous reports
in the field, a templated microgel with high selectivity and
activity controlled by templating effects should result.12,21−24
However, investigations by Gagné et al. indicate that chiral
information on a chosen nonpolymerizable counter ligand may
not be satisfactorily transferred into a templated matrix.35−39
The synthesized catalytic polymers were nevertheless highly
active but did not show selectivity and acceleration of the respec-
tive reactions related to homogeneous, uniform binding sites.35,37
The noted catalytic performance is thus not determined by a
templating effect. These conclusions were reached using bulky,
insoluble and highly cross-linked block copolymers prepared
by thermally initiated polymerization and may not apply to
water-dispersed microgels envisaged here. In this context, we
selected mannose (2) and galactose (3) as chiral model carbo-
hydrates and evaluated microgels synthesized therefrom for
their catalytic performance related to matrix and templating
effects. Our observations and conclusions are summarized below.
2. RESULTS AND DISCUSSION
First, the coordination of mannose (2) and galactose (3) toward
Cu2bpdpo (1b) was established under polymerization conditions.
A combination of experiments using UV−Vis spectroscopy,
isothermal titration calorimetry, and computational analysis
was employed to disclose binding strengths and coordination
sites of the carbohydrates in the prepolymerization mixture.
The carbohydrate-metal complex interactions were evaluated
in 50 mM CAPS buffer at pH 10.50 and 10 °C or below.
Carbohydrate stability over the polymerization time was previ-
ously confirmed for even higher pH values.34
2.1. Sugar-Metal Complex Association Evaluated by
UV−Vis Spectroscopy. The UV−Vis spectra of Cu2bpdpo
(1b) show an absorbance maximum at 651 nm. The addition
of mannose in 5- and 10-fold molar excess at constant volume
and metal complex concentration reveals strong sugar coordi-
nation that is accompanied by a blue shift of the absorbance
maximum of 5 nm (λmax: 651 → 646 nm) and an isosbestic
point at 585 nm (Figure 1a).
By contrast, a blue shift of the absorbance maximum of less
than 1 nm is observed in the presence of galactose under
identical conditions, indicating weak coordination of this carbo-
hydrate to the metal complex (Figure 1b). Galactose amounts in
more than 5-fold molar concentration relative to the metal com-
plex concentration do not shift the absorbance maximum further
and reveal saturation of the binding sites. Control experiments
with equimolar amounts of ethylene glycol (4) in place of a
carbohydrate did not disclose interactions between the diol
and 1b (see the Supporting Information).
Previous studies for sugar coordination to Cu2bpdpo at
pH 12.40 revealed carbohydrate-discriminating behavior of the
metal complex upon binding of mannose over glucose that
was accompanied by a red-shift of the absorbance maximum.
Further analysis disclosed that various other pentoses and
hexoses coordinating over a cis, cis-diol patten involving the
hydroxyl groups at C-1, C-2, and C-3 show this effect.31
However, blue-shifts of the absorbance maximum of 1b upon
coordination of 2 or 3 are observed here, rendering the forma-
tion of assemblies with different binding sites under the employed
conditions likely, and require further characterization.
2.2. Sugar-Metal Complex Association Evaluated by
Isothermal Titration Calorimetry. To measure the binding
strength of the formed carbohydrate-Cu2bpdpo assemblies,
isothermal titration calorimetry (ITC) was employed. All titra-
tions were performed at 10 °C due to experimental limitations
for the use of the instrument at 3 °C. The carbohydrate solu-
tions were titrated into the metal complex solutions, and the
released heat was recorded (Figure 2).
To account for dilution effects during analysis, the sugar solu-
tions were also titrated into buffer solutions under otherwise
identical conditions. The corrected data were then analyzed by
nonlinear regression using a model for sequential binding with
two or three binding sites. The analysis suggests chelation of
both carbohydrates to 1b in a 1:1 molar ratio and exergonic
binding interreactions that are considerably stronger for man-
nose than for galactose. The difference in Gibb’s free energy of
binding is 7.1 kcal mol−1
(see Table 1). Control experiments
Figure 1. Coordination between Cu2bpdpo and (a) mannose (2) and
(b) galactose (3) in a 1:0, 1:5, and 1:10 molar ratio in aqueous
solution at pH 10.50 and 3.0 ± 0.1 °C.
Figure 2. Isothermal titration of (a) mannose (2) and (b) galactose
(3) into a solution of 1b in 50 mM CAPS buffer at pH 10.50 and 10 °C.
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3. with diol 4 in place of a sugar did not reveal measurable
binding interactions (see Supporting Information). To define
the metal complex-carbohydrate assemblies further and evaluate
possible binding sites of the monosaccharides upon coordina-
tion, computational analyses were employed.
2.3. Sugar-Metal Complex Assemblies Evaluated by
Computational Methods. Density functional theory (DFT)
calculation were performed with the B3LYP exchange
correlation functional and the m6-31G(d) basis set implement-
ing improved functions for transition metals.40
Differences
in structures and Gibb’s free energies were calculated for
complexes derived from Cu2bpdpo and the selected mono-
saccharides in aqueous solution applying the COSMO model
under standard conditions. Results of the calculations in the
gas phase may be found in the Supporting Information.
The computational analysis involved coordination of manno-
and galactofuranoses and -pyranoses in α- and β-configuration
to reflect predominant structures of the carbohydrates in
aqueous solution and in coordination.41,42
Appropriate proton-
ation sites of the hydroxyl groups of the carbohydrates were
chosen to account for Lewis acidity of the metal complex upon
coordination and the overall experimental conditions in solu-
tion (pH = 10.50; pKa,man = 12.08; pKa,gal = 12.39 (in H2O at
25 °C).28,31,43
The sugar-Cu2bpdpo assemblies were computed
with three binding sites for mannose and two for galactose as
implied by the experimental results.
Notably, the computation suggests for both carbohydrates a
4
C1 pyranose conformation and deprotonation of the hydroxyl
group at the anomeric C atom as lowest energy assemblies
upon coordination to 1b. The association of Cu2bpdpo with
α-mannose (α-2) formed a conformer with the overall lowest
energy among 25 putative carbohydrate assemblies studied and
is therefore used as a standard during further discussion. The
coordination of α-2 involves chelation of the sugar to both
metal ions in Cu2bpdpo over the hydroxyl groups at C-1 and
C-6 as well as the ring oxygen atom (Figure 3a). The com-
puted distances of the Cu−O and Cu−OH bonds are well
within experimentally verified distances found by X-ray struc-
ture analysis of binuclear copper(II) complexes.44
By contrast,
the lowest energy conformer of a β-mannose-Cu2bpdpo com-
plex is more than 6.2 kcal mol−1
higher in energy than the
above-described assembly with α-mannose and thus not con-
sidered further.
The coordination between galactose and Cu2bpdpo was
evaluated likewise. The calculations propose association of
Cu2bpdpo with 3 in α-configuration as the lowest energy
conformer in this series, while the lowest energy complex
formed with β-galactose is 2.8 kcal mol−1
higher in energy and
thus not considered further. Although experimental data reveal
that α-galactopyranose is less favored in equilibrium in aqueous
solution (≤30%),41
the cis-diol configuration of the hydroxyl
groups at C-1 and C-2 constitutes a known binding site of
various carbohydrates upon interaction with metal ions and
complexes (Figure 3b).42
Overall, the α-galactose-Cu2bpdpo complex computed here
is 7.2 kcal mol−1
higher in Gibb’s free energy than the corre-
sponding assembly with α-mannose. The computational results
are thus in fairly good agreement with the difference in Gibb’s
free energies for the experimentally characterized sugar coor-
dination to Cu2bpdpo using ITC [Δ(ΔG) = 7.1 kcal mol−1
]
and reflect the noted weak binding of galactose to the metal
complex. Further details may be found in the Supporting
Information.
The different binding behavior of mannose (2) and galactose
(3) upon coordination to 1b renders these sugars ideal for an
in-depth investigation of matrix versus templating effects for
catalytic microgels prepared in their presence. The weak coor-
dination of 3 leaves microgels prepared in its presence purely
dependent on matrix effects upon catalytic hydrolysis of model
saccharides. By contrast, microgels prepared in the presence of
2 may show an additional templating effect that may contribute
to accelerate catalytic glycoside hydrolyses. Taking advantage
of a previously developed protocol for microgel synthesis at
subambient temperature,33
corresponding macromolecular
catalysts were synthesized and evaluated.
2.4. Microgel Synthesis and Characterization. Micro-
gels with immobilized catalyst were synthesized in the presence
of a sugar or diol by free-radical polymerization of butyl
acrylate (BA) and ethylene glycol dimethacrylate (EGDMA)
following a recently disclosed protocol (Scheme 1).33
As a representative example, the synthesis of microgels in the
presence of mannose is described. In short, the cross-linking
content in the microgels was varied using 5, 25, 40, 60, and
80 mol % of EGDMA and corresponding amounts of butyl
acrylate to a total of 1.75 mmol of monomer in 9.6 g of
aqueous SDS/CAPS buffer solution at pH 10.50. Relative to
the overall monomer amount, 0.5 mol % of polymerizable
ligand VBbpdpo (5) and corresponding molar amounts of
copper(II) acetate were added to the prepolymerization mix-
ture. To mask the paramagnetic character of the in situ formed
binuclear complex Cu2VBbpdpo (1a) and saturate all binding
sites, a 5-fold molar amount of 2 was added as indicated by the
UV−Vis titrations and ITC experiments described above. The
polymerization was initiated after sonication in an ice−water
bath in the presence of UV light by addition of photoinitiator
Table 1. Thermodynamic Parameters of the Association of
Cu2bpdpo with Mannose (2) and Galactose (3) at 10 °C in
50 mM CAPS Buffer at pH 10.50a
mannose galactose
K1 492 ± 192 452
ΔH1 −8913 ± 1810 −130.6 ±
ΔS1 −19.2 11.7
K2 1130 ± 642 53.8 ± 2.6
ΔH2 16230 ± 3850 334.7 ± 7
ΔS2 71.3 8.18
K3 7370 ± 4400
ΔH3 −9581 ± 3600
ΔS3 −16.1
ΔG −12.5 −5.4
a
K in M−1
, ΔH in cal mol−1
, ΔS in cal mol−1
K−1
, ΔG in kcal/mol,
and ΔG = ΔH − TΔS.
Figure 3. Schematic display of coordination sites and distances
to Cu(II) ions in 1b upon coordination to (a) α-mannose and
(b) α-galactose in water; distances in [Å].
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4. and maintained over 60 min. The polymerization protocol was
optimized by gravimetric analysis using styrene in place of 5
following previously described methods (see the Supporting
Information).33
Microgels prepared in the presence of galac-
tose (3) and ethylene glycol (4) were synthesized for control
reactions as described.33
All dispersed microgel solutions were subsequently purified
by repetitive dialysis cycles against aqueous EDTA/SDS solu-
tion and nanopure water as elaborated.33
Combustion data of
freeze-dried microgel aliquots confirmed near quantitative
incorporation of ligand 5. Dynamic light scattering experiments
revealed hydrodynamic diameters Dh of the microgels between
204 and 280 nm that depend on their cross-linking content as
discussed previously.33
However, significant changes in hydro-
dynamic diameters are not apparent for microgels prepared
in the presence of differently coordinating carbohydrates and
ethylene glycol at a given cross-linking content (see the Supporting
Information). An analysis of the microgels prepared in the
presence of galactose by TEM imaging was already reported.33
Given the similarity of the prepolymerization mixtures, the
matching proceedings of the polymerizations, the comparable
hydrodynamic diameters, and the use of the same synthesis
protocol, mannose-templated microgels are unlikely to display
a different morphology. Further TEM images were thus not
obtained.
2.5. Microgel Evaluation As Catalysts for the
Hydrolysis of Glycosides. The macromolecular catalysts
were purified prior to catalytic evaluation by repetitive dialysis
cycles using SDS/EDTA, SDS, and CAPS/SDS solutions as
described.33
Subsequently, defined amounts of Cu(II) acetate
solutions were added as indicated by elemental analysis
accounting for the ligand (5) content of the respective micro-
gels. The metal ion reloading was previously shown to be near
quantitative when following this protocol and results in catalyst
activation. The microgel-catalyzed hydrolysis of 4-methylum-
belliferyl mannopyranosides (6) leading to formation of
4-methylumbelliferone (7) was monitored in 96-well plate assays
using fluorescence spectroscopy (Scheme 2).
The recorded data were plotted over time to deduce the
initial rate of each reaction. The rates were transformed into
concentrations using the apparent extinction coefficient deter-
mined for each microgel dispersion. The data were then corrected
for contributions of the uncatalyzed background reaction and
the catalyst concentration, plotted over the substrate concen-
tration, and analyzed by nonlinear regression. The application
of the Michaelis−Menten model yielded the kinetic rate
constant (kcat) and the binding affinity (KM). For control reac-
tions, the uncatalyzed substrate hydrolysis was treated like-
wise, yielding the kinetic rate constant in absence of a catalyst
(knon). The catalytic efficiency (kcat/KM) and proficiency (kcat/
KM × knon) were deduced from the kinetic parameters as
described.27,32
Microgels prepared with different cross-linking content in
the presence of mannose (2) display a higher catalytic rate and
efficiency toward the hydrolysis of the α-mannopyranoside (α-6)
than for its β-analog (Figure 4). However, the uncatalyzed
reaction of α-6 is about 3 orders of magnitude faster than the
hydrolysis of its β-analog and has to be considered for a fair
comparison of catalyst performances (see Table 2). Con-
sequently, only the catalytic proficiency (kcat/KM × knon) of the
microgel catalysts is discussed in further evaluations of different
substrates herein.
Microgel Performance during Hydrolysis of Anomeric
Substrates. A microgel Cu
2
L
Pman prepared with 60 mol % of
EGDMA shows a catalytic proficiency of up to 1.7 × 106
upon
the hydrolysis of β-6 and 2.8 × 105
for the hydrolysis of its
α-analog (Table 2, entries 20 and 7). The catalyst thus shows
a more than 6-fold higher proficiency when hydrolyzing a
β- over an α-glycosidic bond in substrates 6. We relate this
observation to different interactions of the binuclear metal
complex with the substrates during the catalytic turnover.
A computational analysis of transition state structures sug-
gests different geometries and energies for assemblies derived
from β-6 and α-6 with Cu2bpdpo (Figure 5).
Scheme 1. Immobilizing Binuclear Cu(II) Complex 1a in the Presence of (2) into Water-Dispersed Microgels
Scheme 2. Model Reaction for Catalyst Screening
Figure 4. Catalytic efficiency (kcat/KM) of Cu
2
L
Pman toward hydrolysis
of α-6 (red) and β-6 (olive) in 5 mM CAPS buffer at pH 10.50 and
37 °C.
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5. The computed structures show a B2,5 boat conformation for
the sugar moiety in α-6 and a 2,5
B boat conformation for the
sugar moiety in β-6. The computed geometries correspond
very well with structures of the transition states during the
hydrolysis of manno-configured glycosides proposed by
others.45,46
In aqueous solution, the Gibb’s free energy of the
transition state assembly between 1b and α-6 is 3.5 kcal mol−1
lower than for a corresponding assembly with β-6 and accom-
panied by more stabilizing H-bond interactions. For α-6,
H bonds are found between the O atoms of the hydroxyl groups
at C-2 and C-3 of α-6 and the hydroxyl ion nucleophile in 1b.
The same binding sites in the sugar show further H bonds with
the coordinated water molecule in 1b. By contrast, the com-
puted structure for the transition state of an assembly with β-6
indicates H bonds between the hydroxyl group at C-2 of β-6
and the O atom of the 4-methylumbelliferone anion. Addi-
tional H bonds are noted between one of the NH groups of the
backbone ligand of 1b and the hydroxyl group at C-6 of β-6
(for more detail and animated structures, see the Supporting
Information). Overall, the computed transition state structures
support the noted differences in the experimental results well
and suggest reasonable stabilizing H-bond interactions between
1b and the substrates 6 that account for the different stability
of the studied assemblies.
The kinetic results for hydrolysis of anomeric glycosides
constitute a significant improvement in catalyst proficiency and
selectivity compared to previously prepared microgels that
were synthesized at elevated temperature using a thermally
initiated free radical polymerization protocol.32
Our second
generation microgels are thus among the most potent man-
made glycosidase mimics known today. For comparison, cata-
lytic antibodies hydrolyze phenyl glycosides with similar catalytic
proficiency but without selectivity among epimers, anomers, or
other substrates.47
Microgel Performance Relative to Cu2bpdpo. The catalytic
proficiency of the small molecular weight complex Cu2bpdpo
(1b) is considerably lower than that of microgels prepared in
the presence of mannose (2), noncoordinating ethylene glycol
(4), or the nonactivated metal-free microgel control (Figure 6).
Similar differences in catalytic performances of macromole-
cular versus low molecular weight catalysts were noted previ-
ously.27,32
However, the recently introduced protocol for
microgel preparation at subambient temperature increases the
relative differences in catalytic performances. The resulting
proficiency of the microgels prepared in the presence of man-
nose is remarkably 41-fold higher than that of 1b and decreases
for Cu
2
L
PEG and L
Pman to 25 and 18 upon hydrolysis of α-6.
The combined observations indicate strong dependence of
the substrate hydrolysis on the cross-linking of the polyacrylate
matrix. Additional minor contributions that increase the catalytic
Table 2. Kinetic Parameter for Microgel-Catalyzed Hydrolysis of α-6 and β-6 in 50 mM CAPS Buffer at pH 10.50 and 37 °Ca
entry S catalyst EGDMA (%) kcat (min−1
) × 10−3
KM (mM) kcat/KM (min−1
M−1
) kcat/KM × knon
1 α-6 Cu2bpdpo − 0.030 ± 0.004 3.9 ± 0.7 0.0077 6800
2 L
Pman 60 − − 0.14 120000
3 Cu
2
L
PEG 60 2.7 ± 1.16 14 ± 8.0 0.20 170000
4 Cu
2
L
Pman 5 1.0 ± 0.07 5.5 ± 0.6 0.18 160000
5 25 1.8 ± 0.19 7.7 ± 1.1 0.23 200000
6 40 1.4 ± 0.12 5.3 ± 1.1 0.26 230000
7 60 1.5 ± 0.19 4.6 ± 1.1 0.32 280000
8 80 1.1 ± 0.12 5.2 ± 1.1 0.20 180000
9 Cu
2
L
Pgal 5 1.7 ± 0.24 8.9 ± 2.6 0.10 160000
10 25 2.9 ± 0.28 12 ± 2.4 0.24 200000
11 40 3.2 ± 0.33 12 ± 3.0 0.26 210000
12 60 1.6 ± 0.08 8.4 ± 1.0 0.20 160000
13 80 1.5 ± 0.33 13 ± 5.6 0.13 110000
14 β-6 Cu2bpdpo − 0.0017 ± 0.0008 7.9 ± 0.5 0.00021 59000
15 L
Pman 60 0.14 ± 0.006 99 ± 53. 0.0014 390000
16 Cu
2
L
PEG 60 − − 0.0036 1000000
17 Cu
2
L
Pman 5 0.020 ± 0.003 9.2 ± 1.5 0.0022 600000
18 25 0.070 ± 0.004 23 ± 1.7 0.0030 840000
19 40 0.020 ± 0.006 5.3 ± 3.3 0.0038 1000000
20 60 0.040 ± 0.007 6.4 ± 2.1 0.0063 1700000
21 80 0.080 ± 0.006 19 ± 1.6 0.0043 1200000
22 Cu
2
L
Pgal 5 0.070 ± 0.001 35 ± 7.4 0.0020 560000
23 25 0.010 ± 0.001 3.4 ± 0.5 0.0029 810000
24 40 0.010 ± 0.005 2.6 ± 0.2 0.0039 1100000
25 60 0.070 ± 0.005 19 ± 1.3 0.0036 1000000
26 80 0.060 ± 0.001 18 ± 3.4 0.0033 930000
a
knon (α-6) = 1.1 × 10−6
min−1
M−1
, knon (β-6) = 3.6 × 10−9
min−1
M−1
, and 50 mM CAPS buffer pH 10.50, 37 °C.
Figure 5. Computed structures for the transition states of the
Cu2bpdpo-catalyzed hydrolysis of (a) α-6 and (b) β-6.
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6. performance of the microgels may be ascribed to the strength
of coordinating sugar ligand used during microgel preparation
(Table 2, entries 3, 7, and 12). For microgels prepared at a
constant cross-linking content of the matrix, a comparable
catalytic performance upon hydrolysis of α-6 is observed when
galactose and ethylene glycol are used as counter ligands
during material synthesis. The catalytic performance increases
about 2-fold in the presence of strongly coordinating mannose.
The microgels behave likewise upon hydrolysis of β-6
(Table 2, entries 16, 20, and 25) but show a lower normalized
catalytic proficiency (Figure 6) that relates to the somewhat
higher activation energy required for the hydrolysis of the
β-glycosidic bond in the β-6 substrate. A corresponding com-
putational analysis in the gas phase reveals a free energy of
activation of 15.5 kcal/mol for the hydrolysis of β-6 compared
to 9.5 kcal/mol for its α-analog.
Microgel Performance Dependent on Sugar Coordina-
tion. Microgels prepared in the presence of strongly coor-
dinating mannose and weakly coordinating galactose were
finally evaluated to assess contributions of a putative templating
effect to the overall catalytic performance (Figure 7). The study
encompasses microgels prepared in the presence of different
amounts of EGDMA monomer to account for previously
identified matrix rigidity caused by its cross-linking content.33
While all polymers Cu
2
L
Pgal can only show matrix effects due to
the weak coordination of the sugar during material preparation,
a very small templating effect is noted for microgels Cu
2
L
Pman
with 60 and 80 mol % of cross-linking content. The microgels
prepared in the presence of mannose show an up to 1.7-fold
higher proficiency than those prepared in the presence of
galactose during hydrolysis of substrates α-6 (Figure 7a) and
up to 1.4-fold higher proficiency for the hydrolysis of β-6
(Figure 7b). However, the overall small contribution of the tem-
plating effect to the overall catalytic performance of microgels at
higher material rigidity is negligible in comparison to the other
contributions identified and nonexistent for microgels prepared
at lower cross-linking content. Therefore, designing a catalyti-
cally active microgel by solely relying on templating of its matrix
is unlikely to result in a potent catalyst with significant activity or
regio- and stereoselectivity. Similar conclusions were reached by
others evaluating organometallic catalysts embedded in insoluble
bulk polymers using nonrelated reactions.35−38
■ CONCLUSIONS
The coordination of galactose and mannose to 1b was eval-
uated at pH 10.50 with UV−Vis spectroscopy, isothermal
titration calorimetry, and computational analyses. The results
suggest discrimination of the carbohydrates by the binuclear
complex due to different binding strength and altered number
and location of binding sites in the resulting sugar-metal com-
plex assemblies. The study reveals a weak two-point binding
for galactose and a strong three-point coordination of mannose.
The resulting mannose-Cu2bpdpo association involves remark-
ably the hydroxyl group at C-6 instead of C-3 of the sugar
moiety. The immobilization of the sugar-Cu2bpdpo assemblies
in the macromolecular environment of water-dispersed micro-
gels provides catalysts that are up to 41-fold more proficient in
the hydrolysis of model substrates than the metal complex alone.
The microgels show a catalytic proficiency of up to 1.7 × 106
when considering the uncatalyzed background hydrolysis
rendering them among the most potent biomimetic cata-
lysts.11,16,21,23,24
For comparison, corresponding catalytic anti-
bodies hydrolyze phenyl glycosides with similar catalytic pro-
ficiency but without selectivity among epimers, anomers, or
other substrates.47
A putative templating effect ascribed to the sugars used as
counter ligands during microgel synthesis was found insignifi-
cant and cannot be correlated to the apparent high catalytic
proficiency. Thus, the overall noteworthy catalytic performance
of the microgels is not controlled by shape selectivity effects
but rather dependent on interactions of the substrates with the
catalytic center (e.g., during discrimination of anomeric man-
nosides). Additionally, the composition of the matrix (e.g., its
cross-linking content and polarity) provide major contributions to
the proficiency of the synthesized microgels. Our results are very
remarkable as high potency and catalytic proficiency of water-
dispersed microgels was achieved without the incorporation of
transition state-stabilizing interactions. Further studies in this
regard are very appealing and will be reported in the near future.
Figure 6. Catalytic proficiency of selected microgels relative to
Cu2bpdpo (1b) toward hydrolysis of α-6 (wine) and β-6 (olive) in
50 mM CAPS buffer at pH 10.50 and 37 °C.
Figure 7. Catalytic proficiency of microgels Cu
2
L
Pman(blue) and
Cu
2
L
Pgal(cyan) with cross-linking content between 5 and 80 mol %
toward the hydrolysis of (a) α-6 and (b) β-6 in 50 mM CAPS buffer
at pH 10.50 and 37 °C.
ACS Catalysis Research Article
DOI: 10.1021/acscatal.8b01910
ACS Catal. 2018, 8, 7710−7718
7715

7. 3. EXPERIMENTAL SECTION
3.1. Experimental Methods. Buffer Solutions. All 50 mM
CAPS buffer solutions were prepared for pH 10.50 at 3 or
10 °C using standard methods.
3.1.1. Analysis of Coordination by UV−Vis Spectroscopy.
Aqueous Sugar Stock Solutions. Typically, 45 mg (0.25 mmol)
of mannose (2) were dissolved into 5 mL using the aqueous
buffer solution yielding a 50 mM stock solution. Stock solu-
tions of galactose (3) and ethylene glycol (4) were prepared
likewise. All solutions were cooled and kept in ice until use.
Stock Solution of Cu2bpdpo. In a typical experiment,
8.21 mg (0.0125 mmol) of Cu2bpdpo was dissolved into 10.0 mL
using the aqueous buffer solution to yield a 1.25 mM solution
of the metal complex. The resulting stock solution was kept in
ice until use.
Binding Assay. Three different solutions were prepared that
are each 1 mM in their respective metal complex concentration
and 5 and 10 mM in their respective sugar concentration.
Along these lines, an 800 μL aliquot of the aqueous stock
solution of Cu2bpdpo was mixed with 200 μL buffer solution, a
100 μL aliquot of the sugar stock solution, and a 100 μL aliquot
of buffer solution, or a 200 μL aliquot of sugar stock solution.
The absorbance of the resulting solutions were recorded at 3 °C
between 200 and 800 nm immediately after mixing with a
resolution of 0.5 nm.
Data Analysis. The absorbance for each metal complex-
sugar solution was corrected for buffer effects and plotted over
the wavelength. The absorbance maxima were determined
from the recorded data.
3.1.2. Analysis of Coordination by Isothermal Titration
Calorimetry. General Remarks. All stock solutions were pre-
pared in aqueous CAPS buffer solution at ambient temperature
and cooled and stored in an ice bath until use. The reference
cell of the calorimeter was filled with nanopure water. All
experiments were conducted at 10.0258 ± 0.0005 °C.
Aqueous Sugar Stock Solutions. Typically, 35.77 mg
(198.7 mmol) of galactose were dissolved in 10 mL of buffer
solution yielding a 20 mM aqueous galactose stock solution.
Likewise, stock solutions of mannose and ethylene glycol were
prepared.
Stock Solution of Cu2bpdpo. A 2 mM aqueous stock solu-
tion of the metal complex was prepared by dissolving 12.96 mg
(19.74 mmol) of Cu2bpdpo into 10.0 mL of buffer solution.
Binding Assay. A 200 μL aliquot of the metal complex stock
solution was titrated with the stock solution of the selected
carbohydrate. After an initial 0.4 μL aliquot, 18 2.0 μL aliquots
were titrated into the same solution with a spacing between
injections of 150 and 250 s. The heat of the coordination was
recorded over 60 or 90 min, respectively. For control experi-
ments, the sugar solution was titrated into the aqueous buffer
solution in a similar fashion.
Data Analysis. The recorded data were corrected for
dilution effects and then analyzed by applying a fitting model
implemented in the supplied Origin software of the instrument
for one, two, or three sequential binding sites to determine the
thermodynamic parameters for the apparent binding constant
K, the enthalpy ΔH, and the entropy ΔS. The Gibb’s free
energy (ΔG) was deduced from these values as ΔG = ΔH −
TΔS, where T = 283.15 K.
3.1.3. Analysis of Sugar Coordination and Transition
States of Glycoside Hydrolysis by Computation. All elec-
tronic structure calculations in the gas phase and in aqueous
solution were performed with PQSmol.48
Low energy
conformers of the complexes derived from carbohydrates 2
or 3 with Cu2bpdpo (1b) were calculated with density func-
tional theory using the B3LYP exchange correlation functional
and the m6-31G(d) basis set, which is the 6-31G(d) basis set
with improved functions for transition metals.40
All stationary
points were examined with vibrational analyses and confirmed
as minima with zero imaginary frequency for gas phase calcu-
lations. The Gibb’s free energies were calculated after geometry
optimization of the assemblies in water using the COSMO
model at 298.15 K and 1 atm.49,50
Transition state structures derived from substrates 6 and
Cu2bpdpo (1b) were initially computed in the gas phase using
the same level of theory. First, the distances of the O−C−OH
bonds at the reaction center were scanned to their highest
energy and the obtained structures then reoptimized at fixed
O−C−OH distances as low energy conformers. All transition
states were characterized by a single imaginary frequency
(νimag). The Gibb’s free energies of solvation were finally
computed as a single point calculation using the COSMO
model under standard conditions.49,50
3.1.4. Microgel Synthesis and Characterization. Microgels
were prepared in the presence of galactose and ethylene glycol
as described.33
Mannose-templated microgels were synthesized
likewise here. Protocols for dialysis, catalyst activation, and
spectroscopic evaluation were followed as described.33
Further
details on instrumentation, methods and material, and char-
acterization data of the mannose-templated microgels may be
found in the Supporting Information.
■ ASSOCIATED CONTENT
*S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acscatal.8b01910.
Experimental details and characterization of mannose-
templated polymers, UV−Vis and ITC data for EG
interactions with Cu2bpdpo, DLS data, and details of
computational analysis (PDF)
Animated structures (AVI)
Animated structures (AVI)
Animated structures (AVI)
Animated structures (AVI)
■ AUTHOR INFORMATION
Corresponding Author
*E-mail: Susanne.Striegler@uark.edu. Tel: +1-479-575-5079.
Fax: +1-479-575-4049.
ORCID
Babloo Sharma: 0000-0002-0265-322X
Susanne Striegler: 0000-0002-2233-3784
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
The authors thank Peter Pulay for advice and access to
PQSmol and Feng Wang for critical reading of the manuscript.
Support of this research to S.S. from the National Science
ACS Catalysis Research Article
DOI: 10.1021/acscatal.8b01910
ACS Catal. 2018, 8, 7710−7718
7716

8. Foundation (CHE-1305543) and the Arkansas Biosciences
Institute is gratefully acknowledged.
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