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Modulating the catalytic performance of an immobilized catalyst with matrix effects – a critical evaluation Babloo Sharma, Susanne Striegler, and Madison Whaley ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b01910 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018
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ACS Catalysis
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 72701, Arkansas, United States ABSTRACT: Microgels with embedded binuclear copper(II) complex were prepared in the presence of galactose and mannose 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-βD-mannopyranoside. The experimental 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.
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 catalysts,8-11 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 crosslinkers in 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 non-polymerizable 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 non-polymerizable compounds and subsequent re-activation 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 counter ion during material preparation.27, 32 To challenge the molecular imprinting (template polymerization) approach, we hypothesize here that matrix effects around a hydrolytic metal complex controls its catalytic activity whereas shapeselectivity 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 only a minor contribution to its catalytic performance, if any at all. X
X OH X Cu O N N N H H Cu
N
R
R 1a R = 1b R = H
O
Chart 1. Composition of metal complexes Cu2VBbpdpo (1a) and Cu2bpdpo (1b) in aqueous solution at pH 10.50; X = H2O As a first step to verify or reject this hypothesis, we developed a protocol for free-radical polymerization of sugar-metal complex assemblies at ambient temperature and below.33 Under these con-
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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 coordination 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). (a)
(b)
time [min] 15 30 45 60 75
0
0.00
time [min] 30 45
60
0.00
-1.00
-1.00
-2.00
-2.00
0.00
0.00
-2.00 -4.00 0.0
15
0.5
1.0 1.5 Molar Ratio
2.0
-2.00 -4.00
0.00 -0.10 -0.20 0
1 2 Molar Ratio
0.0 0.5 1.0 1.5 2.0 2.5 Molar Ratio
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
a.u. 0.1
0.1
0.0 500
0
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 10.50.
0.2
0.2
(b)
kcal/mole of injectant
2.1 Sugar-metal complex association evaluated by UV/Vis spectroscopy
(a)
µcal/sec
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 pre-polymerization 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 previously confirmed for even higher pH values.34
To measure the binding strength of the formed carbohydrateCu2bpdpo assemblies, isothermal titration calorimetry (ITC) was employed. All titrations were performed at 10 °C due to experimental limitations for the use of the instrument at 3 °C. The carbohydrate solutions were titrated into the metal complex solutions, and the released heat was recorded (Figure 2).
kcal/mole of injectant
2. Results and Discussion
2.2 Sugar-metal complex association evaluated by isothermal titration calorimetry
µcal/sec
However, investigations by Gagné et al indicate that chiral information of a chosen non-polymerizable 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 respective 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 crosslinked block copolymers prepared by thermallyinitiated polymerization and may not apply to water-dispersed microgels envisaged here. In this context, we selected mannose (2) and galactose (3) as chiral model carbohydrates and evaluated microgels synthesized therefrom for their catalytic performance related to matrix and templating effects. Our observations and conclusions are summarized below.
amounts of ethylene glycol (4) in place of a carbohydrate did not disclose interactions between the diol and 1b (see 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 formation of assemblies with different binding sites under the employed conditions likely, and require further characterization.
kcal/mole of injectant
ditions, 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 According to numerous reports in the field, a templated microgel with high selectivity and activity controlled by templating effects should result.12, 21-24
a.u.
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600
700 λ [nm]
800
0.0 500
K1 600
700 λ [nm]
800
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. 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 carbohydrate to the metal complex (Figure 1b). Galactose amounts in more than 5fold molar concentration relative to the metal complex concentration do not shift the absorbance maximum further and reveal saturation of the binding sites. Control experiments with equimolar
∆H1 ∆S1 K2 ∆H2 ∆S2 K3
Mannose
Galactose
492 ± 192 -8913 ± 1810
452 -130.6 ±
-19.2
11.7
1130 ± 642 16230 ± 3850
53.8 ± 2.6 334.7 ± 7
71.3
8.18
∆H3
7370 ± 4400 -9581 ± 3600
∆S3
-16.1
∆G
-12.5
-5.4
K in M−1; ∆H in cal mol−1, ∆S in cal mol−1K−1; ∆G in kcal/mol; ∆G = ∆H-T∆S.
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To account for dilution effects during analysis, the sugar solutions were also titrated into buffer solutions under otherwise identical conditions. The corrected data were then analyzed by non-linear 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 mannose than for galactose. The difference in Gibb’s free energy of binding is 7.1 kcal mol−1 (Table 1, previous page). Control experiments with diol 4 in place of a sugar did not reveal measurable binding interactions (see Supporting Information). To define the metal complexcarbohydrate assemblies further and evaluate possible binding sites of the monosaccharides upon coordination, computational analyses were employed.
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 corresponding 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 coordination to Cu2bpdpo using ITC, (∆(∆G) = 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.
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 implementing improved functions for transition metals.40 Differences in structures and Gibb’s free energies were calculated for complexes derived from Cu2bpdpo and the selected monosaccharides 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.
(a)
(b) OH 2.2439
HO HO HO HO
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 protonation 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 solution (pH = 10.50; pKa, man = 12.08; pKa, gal = 12.39 (in H2O at 25 °C).28, 31, 43 The sugarCu2bpdpo assemblies were computed with three binding sites for mannose and two for galactose as implied by the experimental results.
HO HO
VBbpdpo (5) Cu(OAc)2
OH O (2)
OH
CAPS/SDS buffer solution
HO
2.0205
O
2.0148
1.8985
1.8986
Cu
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 coordination of 3 leaves microgels prepared in its presence purely dependent on matrix effects upon catalytic hydrolysis of model saccharides. By contrast, microgels prepared in 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 sub-ambient temperature,33 corresponding macromolecular catalysts were synthesized and evaluated.
2.4 Microgel synthesis and characterization Microgels with immobilized catalyst were synthesized in 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
2+
HN (1a) O
HO OH O
O
Cu
Figure 3. Schematic display of coordination sites and distances to Cu(II) ions in 1b upon coordination to (a) α-mannose and (b) αgalactose in water; distances in [Å]
Cu
HO HO
O
HO Cu
R
OH
2+
Cu
O
Notably, the computation suggests for both carbohydrates a 4C1 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 computed distances of the Cu-O and Cu-OH bonds are well within experimentally verified distances found by X-ray structure analysis of binuclear copper(II) complexes.44 By contrast, the lowest energy conformer of a βmannose-Cu2bpdpo complex is more than 6.2 kcal mol−1 higher in energy than the above described assembly with α-mannose, and thus not considered further.
N
2+
HO
+ NH R
Cu N
R=
EGDMA BA decane
(i) sonication (ii) polymerization (iii) dialysis (iv) catalyst activation
O
O
Scheme 1. Immobilizing binuclear Cu(II) complex 1a in presence of (2) into water-dispersed microgels
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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 microgels. 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-methylumbelliferyl mannopyranosides (6) leading to formation of 4-methylumbelliferone (7) was monitored in 96-well plate assays using fluorescence spectroscopy (Scheme 2).
HO HO
OHOH O (α-6) (α
H2O, catalyst
OR
HO HO
CAPS buffer pH 10.50
O
OHOH O
0.3
0.2
0.1
0.0
0
20
40 60 EGDMA [%]
80
Figure 4. Catalytic efficiency (kcat/KM) of Cu2LPman toward hydrolysis of α-6 (red) and β-6 (olive) in 5 mM CAPS buffer at pH 10.50 and 37 °C. Microgel performance during hydrolysis of anomeric substrates. A microgel Cu2LP man prepared with 60 mol% of EGDMA shows a catalytic proficiency of up to 1.7 x 106 upon the hydrolysis of β-6 and 2.8 x 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 suggests different geometries and energies for assemblies derived from β-6 and α-6 with Cu2bpdpo (Figure 5). (a)
(b)
(2) OH
+ O
Microgels prepared with different crosslinking content in 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 three orders of magnitude faster than the hydrolysis of its βanalog and has to be considered for a fair comparison of catalyst performances (Table 2, next page). Consequently, only the catalytic proficiency (kcat/KM × knon) of the microgel catalysts is discussed in further evaluations of different substrates herein. −1
All dispersed microgel solutions were subsequently purified by repetitive dialysis cycles against aqueous EDTA/SDS solution 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 crosslinking content as discussed previously.33 However, significant changes in hydrodynamic diameters are not apparent for microgels prepared in presence of differently coordinating carbohydrates and ethylene glycol at a given crosslinking content (see Supporting Information). An analysis of the microgels prepared in 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.
using the apparent extinction coefficient determined for each microgel dispersion. The data were then corrected for contributions of the uncatalyzed background reaction and the catalyst concentration, plotted over the substrate concentration, and analyzed by non-linear regression. The application of the Michaelis-Menten model yielded the kinetic rate constant (kcat) and the binding affinity (KM). For control reactions, the uncatalyzed substrate hydrolysis was treated likewise, 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
−1
As a representative example, the synthesis of microgels in the presence of mannose is described. In short, the crosslinking 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 mixture. 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 icewater bath in presence of UV light by addition of photoinitiator and maintained over 60 min. The polymerization protocol was optimized by gravimetric analysis using styrene in place of 5 following previously described methods (see Supporting Information).33 Microgels prepared in presence of galactose (3) and ethylene glycol (4) were synthesized for control reactions as described.33
catalytic efficiency [min M ]
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RO− (7)
R=
Scheme 2. Model reaction for catalyst screening The recorded data were plotted over time to deduce the initial rate of each reaction. The rates were transformed into concentrations
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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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°C Entry 1
S
Catalyst
EGDMA [%]
kcat [min−1] × 10-3
KM [mM]
kcat/KM [min−1M−1]
kcat/KM × knon
α-6
Cu2bpdpo
--
0.030 ± 0.004
3.9 ± 0.7
0.0077
6,800
L
60 60 5 25 40 60 80 5 25 40 60 80
-2.7 ± 1.16 1.0 ± 0.07 1.8 ± 0.19 1.4 ± 0.12 1.5 ± 0.19 1.1 ± 0.12 1.7 ± 0.24 2.9 ± 0.28 3.2 ± 0.33 1.6 ± 0.08 1.5 ± 0.33
-14 ± 8.0 5.5 ± 0.6 7.7 ± 1.1 5.3 ± 1.1 4.6 ± 1.1 5.2 ± 1.1 8.9 ± 2.6 12 ± 2.4 12 ± 3.0 8.4 ± 1.0 13 ± 5.6
0.14 0.20 0.18 0.23 0.26 0.32 0.20 0.10 0.24 0.26 0.20 0.13
120,000 170,000 160,000 200,000 230,000 280,000 180,000 160,000 200,000 210,000 160,000 110,000
Cu2bpdpo
--
0.0017 ± 0.0008
7.9 ± 0.5
0.00021
59,000
L
60 60 5 25 40 60 80 5 25 40 60 80
0.14 ± 0.006 -0.020 ± 0.003 0.070 ± 0.004 0.020 ± 0.006 0.040 ± 0.007 0.080 ± 0.006 0.070 ± 0.001 0.010 ± 0.001 0.010 ± 0.005 0.070 ± 0.005 0.060 ± 0.001
99 ± 53. -9.2 ± 1.5 23 ± 1.7 5.3 ± 3.3 6.4 ± 2.1 19 ± 1.6 35 ± 7.4 3.4 ± 0.5 2.6 ± 0.2 19 ± 1.3 18 ± 3.4
0.0014 0.0036 0.0022 0.0030 0.0038 0.0063 0.0043 0.0020 0.0029 0.0039 0.0036 0.0033
390,000 1,000,000 600,000 840,000 1,000,000 1,700,000 1,200,000 560,000 810,000 1,100,000 1,000,000 930,000
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Pman Cu L 2 PEG Cu L 2 P man
Cu L 2 Pgal
β-6
Pman Cu L 2 PEG Cu L 2 P man
Cu L 2 Pgal
knon (α-6) = 1.1 × 10−6 min−1 M−1; knon (β-6) = 3.6 × 10−9 min−1 M−1; 50 mM CAPS buffer pH 10.50, 37°C The computed structures show a B2,5 boat conformation for the sugar moiety in α-6, and a 2,5B 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 accompanied by more stabilizing Hbond interactions. For α-6, H-bonds are found between the Oatoms 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 computed 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. Additional 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 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 syn-
thesized 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, catalytic 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 presence of mannose (2), non-coordinating ethylene glycol (4) or the nonactivated metal-free microgel control (Figure 6). Similar differences in catalytic performances of macromolecular versus low molecular weight catalysts were noted previously.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 presence of mannose is remarkably 41-fold higher than that of 1b and decreases for Cu2LPEG and LPman to 25 and 18 upon hydrolysis of α-6. The combined observations indicate strong dependence of the substrate hydrolysis on the crosslinking of the polyacrylate matrix. Additional minor contributions that increase the catalytic 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
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crosslinking 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 two-fold in presence of strongly coordinating mannose. The microgels behave like-wise 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 computational 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.
200,000
100,000
0
0
20
40 60 EGDMA [%]
80
0
20
40 60 EGDMA [%]
80
30
(b)
20 6
2.0x10
10 1
0 L
bp u2
an
dp
P EG
an
Pm
Pm
L u2
C
C
L u2
catalyst
o
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. Microgel performance dependent on sugar coordination. Microgels prepared in the presence of strongly coordinating 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 presence of different amounts of EGDMA monomer to account for previously identified matrix rigidity caused by its crosslinking content.33 While all polymers Cu2LPgal 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 Cu2LP man with 60 and 80 mol % of crosslinking content. The microgels prepared in presence of mannose show an up to 1.7-fold higher proficiency than those prepared in 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 templating 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 crosslinking content. Therefore, designing a catalytically 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 non-related reactions.35-38
Conclusions The coordination of galactose and mannose to 1b was evaluated 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 complex assemblies. The study reveals a weak two-point binding for galactose and a strong three-point coordination of mannose. The resulting mannose-
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Figure 7. Catalytic proficiency of microgels Cu2LP man(blue) and Cu L 2 Pgal(cyan) with crosslinking 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. Cu2bpdpo association involves remarkably 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 microgels provides catalysts that are up to 41fold 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 catalysts.11, 16, 21, 23-24 For comparison, corresponding catalytic antibodies hydrolyze phenyl glycosides with similar catalytic proficiency, 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 insignificant 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 mannosides. Additionally, the composition of the matrix, e.g. its crosslinking 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 statestabilizing interactions. Further studies in this regard are very appealing and will be reported in near future.
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ACS Catalysis 3.3.3 Analysis of sugar coordination and transition states of glycoside hydrolysis by computation
3 Experimental 3.3 Experimental Methods Buffer solutions. All 50 mM CAPS buffer solutions were prepared for pH 10.50 at 3 or 10 °C using standard methods.
3.3.1 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 solutions of galactose (3) and ethylene glycol (4) were prepared likewise. All solutions were cooled and kept in ice until to use. Stock solution of Cu2bpdpo. In a typical experiment, 8.21 mg (0.0125 mmol) of Cu2bpdpo were 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.
3.3.2 Analysis of coordination by isothermal titration calorimetry General remarks. All stock solutions were prepared 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 solution 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 experiments, 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 ∆H, and the entropy ∆S. The Gibb’s free Energy (∆G) was deduced from these values as ∆G = ∆H-T∆S; T = 283.15 K.
All electronic 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 functional theory using the B3LYP exchange correlation functional and the m631G(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 calculations. 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 re-optimized 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
3.3.4 Microgel synthesis and characterization Microgels were prepared in 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 characterization data of the mannose-templated microgels may be found in the Supporting Information.
ASSOCIATED CONTENT Supporting Information. 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. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author *Email:
[email protected]; phone: +1-479-5755079; fax: +1-479-575-4049 ORCID Babloo Sharma: 0000-0002-0265-322X Susanne Striegler: 0000-0002-2233-3784 Madison Whaley: 0000-0001-9100-440X
Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Funding Sources Support of this research to S.S. from the National Science Foundation (CHE-1305543) and the Arkansas Biosciences Institute is gratefully acknowledged.
ACKNOWLEDGMENT The authors thank Peter Pulay for advice and access to PQSmol, and Feng Wang for critical reading of the manuscript.
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