Article pubs.acs.org/ac
Probing the Interactions between Boronic Acids and cis-DiolContaining Biomolecules by Affinity Capillary Electrophoresis Chenchen Lü, Hengye Li, Heye Wang, and Zhen Liu* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, People’s Republic of China S Supporting Information *
ABSTRACT: The affinity of boronic acids to cis-diolcontaining biomolecules has found wide applications in many fields, such as sensing, separation, drug delivery, and functional materials. A sound understanding of the binding interactions will greatly facilitate exquisite applications of this chemistry. Although a few analytical tools have been available for the characterization of the interactions, these techniques are associated with some apparent drawbacks, so they are only applicable to a limited range of boronic acids and cis-diolcontaining biomolecules. Therefore, a widely applicable method is still greatly needed. In this work, an affinity capillary electrophoresis (ACE) method was established and validated to probe the interactions between boronic acids and cisdiol-containing biomolecules. The method was proven to be applicable to almost all types of cis-diol-containing biomolecules and boronic acids. Based on this method, a quantitative, comparative study on the interactions between 14 boronic acids that have important potentials for application with 5 typical monosaccharides of biological importance was carried out. The findings provided new insights into boronate affinity interactions, particularly the relationship between the binding strength with the molecular structures of the binding species. Besides, effects of pH and temperature on the binding strength were also investigated. This method exhibited several significant advantages, including (1) possibility of simultaneous study of multiple interactions, (2) low requirement on the purity of the binding species, (3) wide applicability, and (4) high accuracy and precision.
B
important cis-diol-containing biomolecules such as sugars and glycoproteins is rarely known. Although the pKa values of boronic acids have been suggested as an important predictor for their affinity to cis-diol compounds,10 Wang and co-workers have warned that pKa is not a good indicator, because it does not correlate well with the affinity of boronic acids toward cisdiol compounds.28 At the same time, new boronic acids that are expected to show high affinity toward cis-diol-compounds, particularly sugars, at neutral or weak acidic pH environment had been widely synthesized out,3,4,29−31 but suitable methods for easy and precise measurement of their binding constants with cis-diol-containing biomolecules are limited. On the other hand, as one of the most important types of cis-diol-containing biomolecules, glycoproteins have multiple cis-diol groups and their interactions with boronic acids may involve in multiple binding. An open question is whether multiple-site binding (cooperativity) can dramatically enhance the binding strength. Although there were many studies on interactions between boronic acids and monosaccharides, few works were focused on the interactions between boronic acids and glycoproteins.32,33
oronic acids have been important ligands for the sensing and isolation of cis-diol-containing biomolecules, such as saccharides,1−3 nucleosides,4 and glycoproteins,5−7 and they are widely used in the construction of functional materials, such as self-assembled materials,8−10 nanoparticles 11 and nanotubes,12,13 polymer brushes,14 and macroporous monoliths.4,15 In the past decade, the applications of boronic acids have been extended to targeted drug delivery,16−19 enantiopurity analysis,20 living cell labeling and imaging,21 proteasome inhibitors,22 and HIV entry inhibitors.23−25 Recently, bortezomib, which is a boronic acid-based drug, had been approved for the treatment of multiple myeloma. 22,26,27 Most of these applications rely on the characteristics of boronic acids to covalently bind with cis-diol moieties to form five- or sixmembered cyclic esters in an alkaline aqueous solution while the esters dissociate when surrounding pH is changed to acidic or a cis-diol-containing compound with higher affinity is added to the surrounding solution. Clearly, a sound understanding of binding strength of boronic acids, as well as its relationship with the structures of boronic acids and cis-diol-containing biomolecules, is essential for further applications of boronic acids. Such a demand becomes more urgent because of two aspects of fact. On one hand, there are more than 1500 commercially available boronic acids and esters, but their affinity toward © 2013 American Chemical Society
Received: November 22, 2012 Accepted: January 12, 2013 Published: January 28, 2013 2361
dx.doi.org/10.1021/ac3033917 | Anal. Chem. 2013, 85, 2361−2369
Analytical Chemistry
Article
glycoproteins and cell glycans were measured, which offered a sound understanding of the relationship between the affinity and the chemical structures of the binding species. For the first time, definite binding parameters for the interactions between boronic acids and glycoproteins in free solution were reported.
Furthermore, since these limited studies were on interaction analysis of surface-immobilized boronic acids, nonspecific adsorption and spatial hindrance could be involved. To date, the 11B NMR method34−37 and Alizarin Red S (ARS) assay28,38,39 are two widely used methods for the evaluation of the interactions between boronic acids and cisdiol-containing compounds. 11B NMR method relies on direct detection of the chemical shift of boronic acids and boronic esters. When the formed boronate esters can be completely separated from the boronic acids under investigation, this method offers direct and convenient determination of the binding constants. However, it suffers from apparent disadvantages, such as low sensitivity, poor peak resolution, and high concentration requirements.37 ARS assay is a method used especially for the measurement of the binding constants for boronic acids with cis-diol compounds, as proposed by Wang and co-workers.38,39 In this method, two competing equilibria between three species are involved. One is the equilibrium between a boronic acid under study and an optical reporter, ARS, which reports the binding through apparent changes in fluorescence intensity and color. The other is between the boronic acid and a cis-diol compound under investigation, which competes with the former equilibrium. This approach provides significant advantages of high sensitivity and general applicability to small molecules with good solubility. However, the requirement for high concentration of both boronic acid and cis-diol-containing compounds limits its application. Besides, surface plasmon resonance (SPR),32,40,41 ultraviolet−visible light (UV/vis) absorption spectroscopy,42 1H NMR spectroscopy,43 and circular-dichroism spectroscopy44 have also been used to examine the interactions between boronic acids and cis-diol-containing compounds. However, the requirement for complicated immobilization process or necessities for special spectral characteristics of boronic acids or cis-diol-containing molecules limits them to being only applicable for a small range of boronic acids and cis-diol-containing molecules. Therefore, a convenient method that is applicable to all types of boronic acids and cisdiol-containing compounds is of great importance, especially for cross comparison of different binding species. As a high-performance separation tool, capillary electrophoresis (CE) provides multiple advantages, including high resolution, speed, easy to automation, minute sample amount requirements, and the possibility to work under nearphysiological conditions. These benefits make CE a powerful tool for the characterization of biomolecular interactions.45−56 CE has been widely used in investigation of the interactions between small molecules,47,48 drugs and proteins,49−51 DNA and proteins,52−55 and proteins and proteins,46 as well as between protein and nanoparticle.56 Although boronic acids have already been frequently used in CE for the separation and detection of sugar or sugar alcohols57,58 and separation of nucleosides59 and glycoproteins,8 to the best of our knowledge, there is no particular work using CE to probe the interactions between boronic acids and cis-diol-containing biomolecules. In this work, an affinity capillary electrophoresis (ACE) approach was established and validated for probing the interactions between boronic acids and cis-diol-containing biomolecules. The validity of the method for a variety of important cis-diol-containing biomolecules and a variety of representative boronic acids was confirmed. The association constants for the interactions between 14 typical and important boronic acids and 5 monosaccharides that commonly occur in
■
EXPERIMENTAL SECTION Reagents and Materials. 3-Carboxyphenylboronic acid, 2furanboronic acid, and 3-furanboronic acid were purchased from Tokyo Chemical Industry (Tokyo, Japan). 4(Methylsulfonyl)benzene-boronic acid, 2-thiopheneboronic acid, 3-thiopheneboronic acid, 3-pyridinylboronic acid, 4pyridinylboronic acid, pyrimidine-5-boronic acid, galactose, glucose, xylose, and fucose were obtained from J&K Scientific (Beijing, PRC). Phenylboronic acid, benzoic acid, and dimethyl sulfoxide (DMSO) were obtained from Sinopharm Chemical Reagent (Shanghai, PRC). Uracil-5-boronic acid and Dmannose were obtained from Alfa Aesar China (Tianjin, PRC). 1-Pentenylboronic acid was obtained from Frontier Scientific (Logan, UT, USA). N-acetylneuraminic acid (Neu5Ac) was purchased from Maya Reagent (Zhejiang, PRC), D-fructose was obtained from Huixing Reagent (Shanghai, PRC). Nucleosides and proteins, as well as other chemicals, including 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), 2,4-difluoro-3-formylphenyl-boronic acid, and nbutylboronic acid were obtained from Sigma−Aldrich (St. Louis, MO, USA). All these reagents were used as-received. 3Carboxybenzoboroxole was synthesized in the laboratory, based on commercial boronic acid.4 Solutions were prepared in ultrapure water purified by a Milli-Q water purification system (Millipore, Billerica, MA, USA), and all solutions were filtered through hydrophilic membrane filters with pore sizes of 0.45 μm. Instruments. CE experiments were conducted on a P/ACE MDQ system (Beckman Coulter, Fullerton, CA, USA) equipped with a diode array detector (DAD) and an ultraviolet (UV) detector. A fused-silica capillary of 50 μm i.d. × 60 cm (50 cm to detector) from Yongnian Optical Fiber Factory (Hebei, PRC) was used in separation. Unless specified, CE experiments were performed at 30 °C under optimum voltage settings (typically 18 kV) and UV data were acquired using DAD. Prior to each run, the capillary was sequentially rinsed at 20 psi with 0.1 M NaOH for 2 min and running buffer for 2 min. Samples were injected under pressure at 0.5 psi for 5 s. Affinity Capillary Electrophoresis. For the investigation on the binding between boronic acids and monosaccharides, the running buffers were prepared with 50 mM phosphate buffer (pH 6.0 or 7.4) and monosaccharides of varied concentrations, and the samples contained 0.1% (v/v) DMSO (electro-osmotic flow (EOF) marker) and the boronic acids under investigation (10−5−10−4 M each). For the interactions between boronic acids and Neu5Ac, two stock solutions, including (1) 50 mM phosphate buffer containing 100 mM Neu5Ac, pH 7.4, and (2) 50 mM phosphate buffer containing 100 mM NaAc, pH 7.4, were mixed to prepare running buffers with different concentrations of Neu5Ac but with similar ion strength, while the samples contained 0.1% (v/ v) DMSO, boronic acids (10−3 M each), and Neu5Ac at different concentrations. The electropherograms were recorded under a wavelength of 214 nm. For the interactions between 3-carboxyphenylboronic acid and adenosine, the sample contained 20 μM adenosine while two stock solutions50 mM 3-carboxyphenylboronic acid and 2362
dx.doi.org/10.1021/ac3033917 | Anal. Chem. 2013, 85, 2361−2369
Analytical Chemistry
Article
50 mM benzoic acidwere mixed to prepare running buffers with different concentrations of 3-carboxyphenylboronic acid but with similar ion strength. The electropherograms were recorded at a wavelength of 254 nm. As for the interactions between butylboronic acid and nucleosides, butylboronic acid was added to a running buffer of 200 mM CHES at pH 10.0 and the concentration of butylboronic acid ranged from 2.0 mNM to 20.0 mM. The samples contained six nucleosides, 80 μM each, 0.1% DMSO (V/V), and butylboronic acid with the same concentration as that added in the running buffer. The migration of nucleosides was monitored at 214 nm. To assess the interactions between 3-carboxyphenylboronic acid and glycoproteins, two stock solutions50 mM phosphate buffer containing 50 mM 3-carboxyphenylboronic acid (pH 8.5) and 50 mM phosphate buffer containing 50 mM benzoic acid (pH 8.5)were mixed to prepare running buffers with different concentrations of 3-carboxyphenylboronic acid but with similar ion strength. The migration of glycoproteins was monitored at 214 nm. The viscosity correction factor was determined from separate viscosity measurements by measuring the migration time of a plug of 0.1% (v/v) DMSO, under a certain pressure, within the same capillary filled with the running buffer under investigation. Data Analysis. Apparent mobility was calculated by the 32Karat software provided by Beckman Coulter. Data analysis for nonlinear regression was performed using OriginPro8 software (OriginLab Corporation, Northampton, MA, USA). All of the association constants reported here were the averages of triplicate measurements.
Ka =
[TLn] [Lf ]n = [T0] (1/K a) + [Lf ]n
(3)
where [T0] is the initial concentration of the target. In ACE, varying concentrations of ligand are added into the running buffer, to form a kinetic equilibrium between the free target and the bound target. The kinetic equilibrium will give rise to an electrophoretic mobility shift of the target. Assuming the mobility shift of the target is proportional to the number of ligands bound with each target molecule, the electrophoretic mobility of the target (μi) with the presence of ligand in the running buffer can be expressed as a combination of the electrophoretic mobility of free target (μf) and of bound target (μb), as shown in eq 4: νμi =
[TLn] [T ] μ b + f μf [T0] [T0]
(ν = η /η0)
(4)
where v is the viscosity correction factor to enhance the measurement accuracy, and η and η0 are the viscosity of the running buffer with or without ligand, respectively. The EOF can be experimentally measured using a neutral molecule, such as DMSO, and thus μf can be experimentally measured with the absence of ligand in the running buffer. When combining eqs 3 and 4, the relationship between the mobility shift of the target (νμi − μf) and the free concentration of the ligand can be expressed as
RESULTS AND DISCUSSION Basic Considerations. The selection of an appropriate mode is essential for CE-based assessment of intermolecular interactions. The experimental approach to determine binding constants of a given system was selected based on three factors: (1) kinetics of the association/dissociation equilibrium, (2) association constants, and (3) mobility differences between free and complex species.46 In this study, ACE was chosen for the boronic acid−cis-diol biomolecule binding systems because of the following reasons. First, ACE, as a dynamic equilibrium CE, is applicable for binding systems with fast kinetics while the boronic acid−cis-diol biomolecule interactions have been demonstrated to be of fast dissociation.60 Second, the range of association constants that ACE can determine accurately and conveniently without any labeling procedure or additional chemicals is 10−105 M−1,45 while weaker interactions could be estimated by introducing a correction factor.61,62 Such an applicable range well matches the binding strength between boronic acids and cis-diol compounds (usually smaller than 104 M−1).28,39 Third, ACE is a mobility shift-based method. In boronic acid−cis-diol systems, the changes in size, charge, and pKa value due to binding will result in significant mobility shifts for both boronic acid and cis-diol biomolecules.28,63 Thus, ACE should be highly feasible for probing the interactions between boronic acids and cis-diol-containing biomolecules. Affinity Calculation. For binding between target (T) and ligand (L) with a 1:n stoichiometry, the equilibrium and apparent association constant (Ka) can be expressed as Ka
(2)
where [TLn], [Tf], and [Lf] are the concentrations of the complex, the free target, and the free ligand, respectively. When the ligand is in large excess, the bound target fraction ([TLn]/ [T0]) can be expressed as
■
T + n L → TLn
[TLn] [Tf ][Lf ]n
(vμi − μf ) = (μ b − μf )
[Lf ]n (1/K a) + [Lf ]n
(5)
where [Lf] can be approximated as the initial concentration of the ligand added to the running buffer, provided that the ligand is in large excess. The mobility shift of target is experimentally measured, while the mobility shift of the bound target (μb − μf) is a constant. By varying the concentration of ligand, a series of mobility shift data can be obtained. Thus, Ka can be obtained through nonlinear regression of (μb − μf) against [Lf], according to eq 5. Equation 5 is also called the Hill equation. Although 1:n binding systems have not been frequently investigated, the validity of eq 5 has been confirmed by Zhong and co-workers.56 As a special case, for a 1:1 binding system, n = 1; thus, eq 5 becomes (νμi − μf ) =
(μ b − μf )[Lf ] (1/K a) + [Lf ]
(6)
Equation 6 has been widely used in ACE analysis of 1:1 binding systems.64 Method Validation. To establish an ACE method, two more aspects should also be taken into account: (1) the detectability of the two binding species, and (2) the concentration available for the experiment. They are both key factors to deciding which species acts as the target and which acts as the ligand that is added to running buffer. Here, boronic acid−monosaccharide systems were used for validation. Since most monosaccharides are UV-transparent, electrically neutral,
(1)
where 2363
dx.doi.org/10.1021/ac3033917 | Anal. Chem. 2013, 85, 2361−2369
Analytical Chemistry
Article
or electrically neutral. Under these conditions, ACE is also applicable with slight modification. The boronic acid−Neu5Ac system can be considered as an example. Unlike most monosaccharides, Neu5Ac has relatively high UV absorption and is negatively charged at physiological pH. When high concentration of Neu5Ac is added into the running buffer, it will result in high background signal and the negative peak of Neu5Ac may interfere with the peaks of the boronic acid ligands. At the same time, ionic strength of the running buffer will change and apparent change in the current of electrophoresis will occur, which may influence the mobility of the boronic acids. To reduce these negative influences, Neu5Ac at the same concentrations within the running buffers was added to the boronic acid samples to avoid the formation of a negative peak and, meanwhile, sodium acetate was added to the running buffer as a co-ion to minimize current change (see Figures S1 and S2 in Supporting Information for representative raw electropherograms). R2 values from 0.991 to 0.999 were obtained for the 14 boronic acids investigated. This case showed high flexibility of the ACE method. The ACE method was further validated by comparison of the measured association constants for the interactions between phenylboronic acid and a variety of monosaccharides with those by ARS assay and 11B NMR. The Ka values for fructose, galactose, glucose, mannose, xylose, and Neu5Ac were measured by the ACE method to be 140 ± 3, 12.6 ± 0.1, 3.3 ± 0.2, 7.0 ± 0.1, 8.3 ± 0.4, and 16.0 ± 0.7 M−1, respectively. In comparison, the association constants for fructose, glucose, and Neu5Ac were measured to be 79, 0, and 12 M−1, respectively, by 11B NMR,29 while the association constants for the abovementioned six monosaccharide were 160, 15, 4.6, 13, 14, and 21 M−1, respectively, by ARS assay.39 Clearly, the data measured by the ACE method are in good agreement with those from 11B NMR and ARS assay. Besides, error level for the binding parameters was also reported in this work, which indicates high precision and reproducibility of the method. Insights into Boronic Acid−Monosaccharide Interactions. The association constants between 14 representative boronic acids with 5 typical monosaccharides at physiological pH were determined by the ACE method, and the results are listed in Table 1. Most boronic acids analyzed here, including arylboronic acids with electron-withdrawing substituents (3carboxyphenylboronic acid, 4-(methylsulfonyl)benzene-boronic acid and 2,4-difluoro-3-formylphenyl-boronic acid), heterocyclic boronic acids (2-furanboronic acid, 3-furanboronic acid, 2-thiopheneboronic acid, 3-thiopheneboronic acid, 3-pyridinylboronic acid, 4-pyridinylboronic acid, pyrimidine-5-boronic acid, and uracil-5-boronic acid) and intramolecularly B−Ocoordinated (3-carboxybenzoboroxole), were expected to possess high affinity to cis-diol-containing biomolecules at neutral or physiological pH conditions, according to our previous studies.4,10,30,31 The affinities of some of them, such as benzoboroxoles, had already been experimentally confirmed,4 while affinities of some others have never been investigated. The 5 monosaccharides chosen here, including fructose, mannose, N-acetylneuraminic acid, xylose, and fucose, had been all found as terminal monosaccharides of glycans in glycoproteins and cell walls.65 Based on the association constants determined by ACE, the following conclusions could be drawn for different boronic acids. First, compared to phenylboronic acid, the presence of electron-withdrawing groups on the phenyl ring surly increased the affinity to cis-diols at physiological pH, as 3-carboxyphe-
and highly water-soluble, they can be conveniently added to running buffer as ligands. On the other hand, most boronic acids, particularly arylboronic acids, heterocyclic boronic acids, and benzoboroxoles, exhibit relatively strong UV absorbance and therefore can be used as targets. The interactions between three boronic acids (phenylboronic acid, 3-carboxyphenylboronic acid, and 3-carboxybenzoboroxole) and fructose were simultaneous studied by adding fructose to the running buffer and using a mixture of the three boronic acids (0.10 mM each) as the sample. Representative electropherograms are shown in Figure 1A. Association with fructose prolonged the migration
Figure 1. (A) Representative electropherograms for the interactions between boronic acids and fructose and (B) model fitting curves ((1) phenylboronic acid, (2) 3-carboxyphenylboronic acid, and (3) 3carboxybenzoboroxole). [From bottom to top, the concentrations of fructose added to running buffer were 0, 1.5, 3.0, 6.0, and 15.0 mM.]
times of phenylboronic acid and 3-carboxyphenylboronic acid but shortened the migration time of 3-carboxybenzoboroxole, indicating different binding strength of different boronic acids with the same sugar. Since boronic acids used herein were all monoboronic acids and excess monosaccharide was used, the boronic acid−monosaccharide system was regarded as 1:1 binding and the association constants were calculated using eq 6. To minimize the error, only data measured from monosaccharide concentrations between 0.25Ka and 4Ka were used to calculate the binding parameters and a viscosity correction factor was used. Model fitting curves are shown in Figure 1B. Correlation coefficients (R2) from 0.994 to 0.999 were obtained. Such excellent correlation ensured the accuracy of the method. For some boronic acid−cis-diol-containing biomolecule systems, none of the two binding species are UV-transparent 2364
dx.doi.org/10.1021/ac3033917 | Anal. Chem. 2013, 85, 2361−2369
Analytical Chemistry
Article
Table 1. Experimental Association Constants Measured by ACE for the Interactions between Fourteen Representative Boronic Acids and Five Typical Monosaccharides at pH 7.4 Association Constant (M−1)
a
boronic acid
fructose
mannose
phenylboronic acid 3-carboxybenzoboroxole 3-carboxyphenylboronic acid 4-(methylsulfonyl)benzene-boronic acid 2,4-difluoro-3-formylphenyl-boronic acid 2-furanboronic acid 3-furanboronic acid 2-thiopheneboronic acid 3-thiopheneboronic acid 3-pyridinylboronic acid 4-pyridinylboronic acid pyrimidine-5-boronic acid uracil-5-boronic acid 1-pentenylboronic acid
140 ± 3 293 ± 21 261 ± 7 1450 ± 40 2790 ± 770 372 ± 3 139 ± 2 483 ± 6 169 ± 2 833 ± 34 794 ± 33 1020 ± 40 185 ± 8 N/Aa
7.0 ± 0.1 14.6 ± 0.9 14.3 ± 0.9 58.7 ± 5.3 80.4 ± 2.3 20.8 ± 0.2 5.8 ± 0.1 27.1 ± 0.4 7.8 ± 0.3 N/Aa N/Aa 59.0 ± 3.3 12.0 ± 1.5 0.3 ± 0.2
Neu5Ac
xylose
± ± ± ± ± ± ± ± ± ± ± ± ± ±
7.9 ± 0.4 42.7 ± 1.7 10.4 ± 1.0 98.5 ± 3.4 224 ± 18 35.0 ± 0.7 5.7 ± 0.5 33.6 ± 1.3 8.0 ± 0.5 52.3 ± 16.5 N/Aa 105 ± 5 22.2 ± 0.9 1.9 ± 0.9
16.0 14.5 35.7 104 42.5 23.7 9.2 24.0 8.8 39.4 35.7 22.6 21.7 0.8
0.7 1.1 4.7 14 4.7 0.6 0.8 1.0 0.8 1.8 1.1 3.1 0.4 0.5
fucose 3.8 17.7 5.1 23.8 47.9 8.6 1.9 10.4 3.4 8.7 8.8 27.9 3.0 0.7
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.3 1.3 0.4 1.9 3.3 0.5 0.5 0.4 0.4 1.9 2.1 0.6 0.7 0.3
N/A = not applicable.
Table 2. Association Constants for the Binding between Boronic Acids with Fructose and Mannose at Various pHs Association Constant (M−1) monosaccharide fructose pH 6.0 pH 7.4 pH 8.5 mannose pH 6.0 pH 7.4 pH 8.5 a
phenylboronic acid
3-carboxyphenylboronic acid
3-carboxybenzoboroxole
8.3 ± 0.2 140 ± 3 862 ± 17
13.6 ± 0.6 261 ± 7 1080 ± 100
42.4 ± 5.0 293 ± 21 498 ± 27
N/Da 7.0 ± 0.1 33.0 ± 1.6
N/Da 14.3 ± 0.9 45.8 ± 3.8
2.6 ± 0.4 14.6 ± 0.9 22.4 ± 1.1
N/D = not determined.
affinity that is slightly higher than that of phenylboronic acid. Fourth, as an example of alkylboronic acid, 1-pentenylboronic acid showed very low affinity to monosaccharides. The strength of boronic acid−monosaccharide interactions was dependent on not only the structures of boronic acids but also the structures of the sugars. For all the boronic acids examined, fructose exhibited the highest association constants among all the monosaccharides investigated, which can be attributed to its predominant furanose form that facilitates the complexation with boronic acids.67 Mannose and Neu5Ac are both very common in glycan terminal of mammalian glycoproteins, and their affinity to boronic acids greatly contribute the affinity of glycoproteins to boronic acid ligands. It was reported that Neu5Ac had anomalously high complexing ability to phenylboronic acid at physiological pH,68 and this anomalous binding characteristic had already been used as the basis for a strategy to preferentially extract sialylated glycoproteins from nonsialylated glycoproteins by boronate affinity monolith through pH manipulation.69 However, according to the ACE results shown in Table 1, this strategy should be amended to limit to only a certain range of arylboronic acids, such as phenylboronic acid, 3-carboxyphenylboronic acid, and 4-(methylsulfonyl)benzene-boronic acid. For some other boronic acids, such as 2,4-difluoro-3formylphenyl-boronic acid and pyrimidine-5-boronic acid, their affinities toward Neu5Ac were much lower than those to neutral sugars. The Ka values of xylose with boronic acids that had moderate affinity to monosaccharides were almost the
nylboronic acid, 4-(methylsulfonyl)benzene-boronic acid, and 2,4-difluoro-3-formylphenyl-boronic acid behaved. 2,4-Difluoro-3-formylphenyl-boronic acid exhibited an affinity ∼20 times higher than that of phenylboronic acid. Second, benzoboroxoles were reported to exhibit high affinity;29,66 therefore, as a benzoboroxole derivative, 3-carboxybenzoboroxole was expected to exhibit strong affinity toward the monosaccharides. Beyond our expectation, however, the measured Ka values for 3-carboxybenzoboroxole were not apparently higher than that of 3-carboxyphenylboronic acid and even much lower than those of 4-(methylsulfonyl)benzeneboronic acid and 2,4-difluoro-3-formylphenyl-boronic acid. Third, the affinity of heterocyclic boronic acids investigated here, including thiophene boronic acids, furan boronic acids, pyridinylboronic acids, and pyrimidine boronic acids with the model monosaccharides, varied apparently. 3-furanboronic acid and 3-thiopheneboronic acid showed moderate affinity similar to phenylboronic acid, while 2-furanboronic acid and 2thiopheneboronic acid exhibited affinities to most sugars of nearly three times higher than those of their meta-isomer and phenylboronic acid at neutral pH. Distinctly, pyridinylboronic acids and pyrimidine-5-boronic acid, especially the latter, exhibited the highest binding constants among all the heterocyclic boronic acids investigated. However, the affinity difference between 3-pyridinylboronic acid and 4-pyridinylboronic acid was very little (
