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Recent advances in monolithic column-based boronate-affinity chromatography
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Trends in Analytical Chemistry, Vol. 37, 2012
Recent advances in monolithic column-based boronate-affinity chromatography Hengye Li, Zhen Liu Cis-diol-containing biomolecules are an important class of compounds, including carbohydrates, glycoproteins, RNA, and nucleosides. Many are the main analytes at the frontiers of life science studies (e.g., proteomics, metabolomics and glycomics). As many cis-diol molecules of biological importance are present in very low abundance in samples while interfering substances are usually present in high abundance, specific capture and effective enrichment of target cis-diol biomolecules become key, challenging steps in the -omics analyses. Boronate-affinity chromatography (BAC) is a tool for specific isolation and enrichment of cis-diol compounds. In recent years, monolithic column-based BAC has attracted increasing attention. A variety of BAC monolithic columns have been developed and impressive applications in selective enrichment of glycopeptides and nucleosides have been demonstrated. We review recent advances in monolithic column-based BAC. We mainly focus on the common issues encountered during the development and application of monolithic columns for BAC. We suggest a set of strategies to guide how to select appropriate binding-buffer composition and how to design new BAC columns with the desired properties. We highlight progress and discuss trends in lowering the binding pH. ª 2012 Elsevier Ltd. All rights reserved. Keywords: Boronate-affinity chromatography (BAC); Boronic acid; Cis-diol; Glycopeptide; Glycoprotein; Metabolomics; Monolithic column; Nucleoside; Phenylboronic acid; Proteomics Abbreviations: AAPBA, 3-Acrylamidophenylboronic acid; ACN, acetonitrile; AFP, a-Fetoprotein; BAC, Boronate-affinity chromatography; BSPBA, 4(3-Butenylsulfonyl) phenylboronic acid; CEA, Carcinoembryonic antigen; Con A, Concanavalin A; DMAMAPB, 3-(Dimethylaminomethyl)-aniline-4pinacol boronate; DMF, N,N-dimethylformamide; DMSO, Dimethyl sulfoxide; EDMA, Ethylene glycol dimethacrylate; ESI, Electrospray ionization; GMA, Glycidyl methacrylate; HILIC, Hydrophilic interaction chromatagraphy; HMDA, 1,6-Hexamethylenediamine; HPBA, p-Hydroxyphenylboronic acid; HPLC, High-performance liquid chromatography; HRP, Horseradish peroxidase; MALDI-TOF-MS, Matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry; mAPBA, m-Aminophenylboronic acid; MBAA, N,N 0 -methylenebiacrylamide; MEKC, Micellar electrokinetic chromatography; MOG, Metal-organic gel; PEG, Polyethylene glycol; PEGDA, Poly(ethylene glycol) diacrylate; TEPIC, Tris(2,3epoxypropyl)isocyanurate; TMOS, Tetramethyloxysilane; VPBA, 4-Vinylphenylboronic acid; c-MAPS, 3-Methacryloxypropyltrimethoxysilane
1. Introduction Hengye Li, Zhen Liu*, State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
*
Corresponding author. Tel./Fax: +86 25 8368 5639; E-mail: [email protected]
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Since the beginning of the new century, a variety of ‘‘-omics’’, particularly proteomics, metabolomics and glycomics, have increasingly attracted attention. Cis-diolcontaining compounds comprise a class of biomolecules, which cover several major types of target molecules in -omics studies, including glycoproteins and glycopeptides in proteomics, nucleosides and modified nucleosides in metabolomics, and saccharides in glycomics. Cis-diol biomolecules play essential roles in many biological processes and are significant in disease diagnostics. Glycopro-
teins play important roles (e.g., molecular recognition, inter-cell and intra-cell signaling, immune response and sperm-egg interaction). Some glycoproteins [e.g., afetoprotein (AFP) and carcinoembryonic antigen (CEA)] have been employed as biomarkers for clinical cancer screening [1]. Nucleosides are building blocks of RNA. The turnover of tRNA generates modified nucleosides and their excretion concentrations in urines reflect the whole turnover level of tRNA [2]. Because neoplastic diseases are associated with increased tRNA turnover rate [3], urinary modified nucleosides have been suggested as potential diagnostic markers of cancer
0165-9936/$ - see front matter ª 2012 Elsevier Ltd. All rights reserved. doi:http://dx.doi.org/10.1016/j.trac.2012.03.010
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diseases [4–7]. A common feature of these cis-diol biomolecules is that they are usually present in very low abundance in the -omic samples, especially at the early stage of cancer development, while interfering substances are present in high abundance. Specific capture and effective enrichment of target cis-diol biomolecules are therefore key steps in -omics analyses. Boronate-affinity chromatography (BAC) is a powerful technique for the selective isolation and enrichment of cisdiol-containing compounds, such as glycoproteins [8,9], nucleosides [10,11] and saccharides [12]. The principle relies on the formation of cyclic esters between boronicacid ligands and the cis-diol moiety of analytes under alkaline conditions (usually pH of 8.5 or higher) and the dissociation of the cyclic esters when the environmental condition is changed to acidic. The general formula of the reaction between boronic acids and cis-diol-containing compounds is illustrated in Fig. 1. This pH-dependent chemistry makes boronic acids excellent ligands for molecular recognition, which had led to the generation of boronic acid-based chemical sensing for saccharides [13] and the invention of BAC in the 1970s [14]. BAC exhibits several features. First, it has broadspectrum affinity, so one boronic-acid ligand can capture most, if not all, cis-diol biomolecules, making it very useful for global -omics analyses. Second, BAC relies on covalent reactions, so adsorption induced by non-specific interactions can be suppressed or eliminated by choosing appropriate conditions, giving high specificity. Third, the capture/release process can be manipulated easily through switching pH. Fourth, the desorption kinetics is usually fast, so analyte carry-over is usually limited. Finally, as the elution is carried out at moderate acidic conditions, this approach is very compatible with mass spectrometry (MS), facilitating applications in -omics analyses. Due to these merits, boronic-acid-functionalized materials have increasingly attracted attention in recent years, particularly in proteomics [15–18] and metabolomics [19,20].
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The properties of the boronate-affinity-separation media are detemined by both the boronic-acid ligands and the support materials. Generally, the boronic-acid ligands can be any aromatic boronic acids or their derivatives. The pKa value of a boronic acid is a key parameter that influences its performance in BAC. Usually, a lower pKa value results in a stronger affinity and requires a lower environmental pH for binding. The nature of support materials is another key factor in BAC. An inappropriate support material may greatly degrade performance, particularly the specificity of the resulting BAC column. Monolithic columns, pioneered by Hjerten [21], Svec [22] and Tanaka [23], are defined as ‘‘continuous stationary phases that form as a homogeneous column in a single piece and prepared in various dimensions with agglomeration-type or fibrous microstructures’’ [24]. Due to the advantages of ease of preparation, low back pressure and convective mass transfer, versatile surface chemistry for ligand attachment and ease of miniaturization in channels and capillaries, great attention has been paid on monolithic columns in recent decades. Especially, monolithic capillaries based high-performance liquid chromatography (HPLC) have the advantages of improved chromatographic resolution, higher efficiency, lower sample consumption, convenient online coupling to MS and improved mass-detection sensitivity, compared with classical column-based HPLC [25–30]. Capillary monolithic columns have been widely used in the -omics studies [31–34]. Monolithic column-based BAC has developed rapidly since its first appearance in 2006 [35]. A variety of boronate-affinity monolithic columns have been developed and the analytes investigated included nucleosides [36–42], glycoproteins [37–39,41,43,44], glycopeptides [38], and catechol [36,37,42]. Also, some boronateaffinity monolithic materials in the format of disks integrated in microchip systems for the determination [45] and extraction [46] of catecholamines have been developed, but they are not the focus of this review. The
Figure 1. The general interaction between boronic acids and cis-diol-containing compounds.
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separation mechanism in monolithic column-based BAC has been systematically investigated [36], and this work provided a theoretical guideline for the selection of binding buffers and the design of new monolithic capillaries. Further, attempts have been made to improve the specificity especially to glycoproteins [37,43] and to lower the binding pH [40–42]. For better understanding of monolithic column-based BAC and even general BAC, it is necessary to survey the common issues and concerns encountered in this area. In this review, we examine the recent advances in the development of boronate-affinity monolithic columns, focusing on common issues and challenging tasks. We also discuss possible solutions to these challenges and further development of BAC. 2. Preparation and characterization Generally, boronate-functionalized monolithic columns can be prepared by direct or indirect methods. In direct methods, boronic-acid ligands with active groups (e.g., vinyl and amino group) participate in the reactions and directly generate the boronate-affinity monoliths [36– 41,43,44]. In indirect methods, a base monolithic column, which initially contains no boronic-acid ligands but certain active functional groups (e.g., epoxy group), is first prepared for subsequent surface modification, and then an appropriate boronic-acid ligand is immobilized onto the surface of the base monolith by appropriate chemical reactions [35,42]. Fig. 2 shows the boronic-acid ligands, co-monomers and cross-linkers that have been used in the literature. 2.1. Preparation According to the nature of monolithic materials, existing boronate-affinity monolithic columns can be classified into organic polymer and organic-inorganic hybrid monolithic columns. Table 1 summarizes the co-monomers, the boronic acids and the porogenic systems used in the literature. Two kinds of in-situ polymerization have been used to prepare polymer-based monolithic stationary phases. The first is in-situ polymerization initiated by free radicals through heating or UV exposure. Generally, a monomer, a cross-linker and an initiator are dissolved in porogenic solvents and the resulting mixture is injected into a column (e.g., a fused-silica capillary). With two ends sealed, the column is heated in a water bath or exposed to UV light to initiate polymerization. After the reaction, a wash step is employed to remove the unreacted monomer, cross-linker and porogenic solvents. Most boronate-affinity monolithic capillaries were prepared by this kind of polymerization [35–38,41–44]. Generally, the porogenic solvents used in this method are organic solvents. However, novel materials can also
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be used as porogenic templates to generate macroporous materials. Yang et al. [44] first employed metal-organic gels (MOGs) as porogenic templates to fabricate a macroporous monolith. The resulting monolith showed lmsized pore distribution. This large pore-size distribution was essential for the analysis of macrobiomolecules (e.g., glycoproteins). The second kind of polymerization is based on condensation of multifunctional epoxy with di-amino or triamino compounds [23,47]. Our group [40] developed a novel approach, called ring-opening polymerization with synergistic co-monomers, to prepare boronate-functionalized monolithic columns. As shown in Fig. 3, this approach takes advantage of boron-nitrogen coordination between a regular boronic-acid ligand, m-aminophenylboronic acid (mAPBA), and a diamine, 1,6-hexamethylenediamine (HMDA). On being mixed together, mAPBA and HMDA can self-assemble into a relatively stable complex due to the boron–nitrogen coordination and the resultant complex can function as a bifunctional reagent to react with a multifunctional compound, tris(2,3-epoxypropyl)isocyanurate (TEPIC), to form a poly(TEPIC-co-(mAPBA-HMDA)) monolithic capillary. Relative to conventional silica monolithic columns, organic-inorganic hybrid monolithic columns are more hydrolytically stable [48]. Organic-inorganic hybrid monolithic columns were developed quickly in recent years. Initially, they were prepared by sol-gel technology based on the co-condensation of tetra-alkoxysilanes and organosiloxane precursors [49]. In this approach, the organosiloxane precursor is directly incorporated into the reaction mixture, so the desired functional monomer is directly introduced into the monolith obtained without additional surface modification [50–52]. However, the preparation procedure is time consuming. Recently, a novel ‘‘one-pot’’ approach was proposed by Zou et al. [53–55] for quick preparation of the organic-inorganic hybrid monolithic column. In this approach, vinylalkoxysilanes (as precursor) and an organic monomer with [53,55] or without [54] tetraalkoxysilane were mixed in one pot, along with an appropriate initiator. Condensation and polymerization occurred at the same time and the monolithic column could be obtained in about 24 h. By using this ‘‘one-pot’’ approach, Lin et al. [39] prepared an organic-inorganic boronateaffinity monolithic column using 4-vinylphenylboronic acid (VPBA) as a functional monomer. 2.2. Characterization Boronate-functionalized monolithic columns are usually characterized in terms of morphology, binding capacity and pore properties (e.g., pore-size distribution, specific surface area, average pore size and pore volume). The morphology of boronate-functionalized monolithic capillaries is usually characterized by scanning electron microscopy (SEM). The morphology of the pre-
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Figure 2. The structures of the boronic-acid ligands, monomers and cross-linkers used to prepare monolithic columns for boronate affinity chromatography (BAC).
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Table 1. Summary of monolithic columns for boronate-affinity chromatography (BAC) Comonomer system
Boronate ligands
Porogenic systems
Analytes Nucleosides Catechol Nucleosides Glycoproteins Catechol Glycoproteins Catechol Nucleosides Glycoproteins Nucleosides Glycoproteins Glycopeptides Catechol Nucleoside Nucleosides Glycoproteins Catechol Nucleoside Nucleosides Glycoproteins
GMA/EDMA VPBA/EDMA
HPBA VPBA
Cyclohexanol, 1-Dodecanol Diethylene glycol, Ethylene glycol
VPBA/EDMA VPBA/MBAA
VPBA VPBA
Diethylene glycol, Ethylene glycol DMSO, DMF, H2O, Na2HPO4
AAPBA/EDMA
AAPBA
MOGs
AAPBA/EDMA
AAPBA
Methanol, PEG 20000
TEPIC/HMDA + mAPBA
HMDA + mAPBA
PEG 200
BSPBA/MBAA
BSPBA
DMSO, 1-Dodecanol
GMA/PEGDA
DMAMABA
DMSO, 1,4-butanediol
c-MAPS/TMOS/VPBA
VPBA
Diethylene glycol, PEG 10000
Binding pH
Ref.
9.0 8.5
[35] [36]
8.5 8.5
[43] [37]
8.5
[44]
9.2
[38]
P 7.0
[40]
P 7.0
[41]
P 5.5
[42]
8.5
[39]
Figure 3. The preparation of a monolithic column through the approach ring-opening polymerization with synergistic co-monomers. (I) B-N coordination and (II) ring-opening polymerization with synergistic co-monomers (Reprinted from [40] with permission).
pared monolithic columns is determined by not only the nature of the monomers and cross-linkers but also the properties and the amounts of porogenic solvents used in the polymerization system. For the control of the morphology of monolithic columns, readers can refer to an excellent article [56]. The pore properties of boronate-functionalized monolithic columns are usually measured by the Brunauer-Emmett-Teller (BET) method in terms of poresize distribution, specific surface area, average pore size and pore volume. The pore properties can be regulated by systematically changing the recipe of the pre-polymerization solutions and the temperature at which the polymerization is carried out. Binding capacity is an important performance indicator of boronate-functionalized monolithic columns. It is usually measured by frontal chromatography. The binding capacity of a boronate-functionalized monolithic column varies with the pH value of the binding buffer used.
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The pKa values of boronic-acid ligands are usually measured by the absorbance change at 268 nm that occurs upon conversion from the trigonal form (at low pH) to the tetrahedral form (at high pH) during titration of the boronic-acid ligands in free solution [57]. Gillespie et al. [58] reported a non-invasive approach for the determination of the pKa values of boronic-acid ligands on the monolith surface. Although the value measured by this method showed some deviation from that obtained by free-solution titration, the information offered by this method may reflect, to some extent, the basic property of a boronic-acid-functionalized monolithic column as a whole.
3. Separation mechanism and selectivity manipulation Specificity is an essential concern in BAC and is often a challenging task for macromolecules. To reach a pure
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Table 2. Strategies for selectivity manipulation in boronate-affinity chromatography (BAC) Interaction type Boronate-affinity interaction
Hydrophobic interaction Ionic interaction Hydrogen bonding interaction
Changing stationary phase Use B-N coordinated molecular team; use Wulff-type boronic acid; use improved Wulfftype boronic acid; increase ligand density Use hydrophilic monomer/cross-linker; use hydrophilic support material Use B-N coordinated molecular team; use Wulff-type boronic acid None
boronate-affinity-governed separation, a sound understanding of the separation mechanism is indispensable. The boronate-analyte interactions involved in regular BAC have been well documented in an excellent review [59]. In addition to boronate-affinity interaction, four secondary interactions, including hydrophobic, ionic, hydrogen bonding and coordination interactions, can occur in BAC. Our group [36] systematically investigated the retention mechanism in monolithic capillary-based BAC on a single monolithic capillary. It was observed that multiple intermolecular interactions can occur between the analytes and the boronateaffinity monolith, including boronate-affinity, hydrophobic, ionic and hydrogen-bonding interactions. Under certain conditions, the secondary interactions could generate significant undesirable secondary retention. In addition to the previous conclusion that unprotonated amines and carboxyl groups can serve as electron donors and thus can coordinate with boronic acids [59], the coordination effect of Lewis bases (e.g., fluoride ion) was confirmed, and can enhance the complexation between cis-diol-containing compounds with boronic acids. Knowledge of the separation mechanism has been used as a theoretical guide for how not only to manipulate the separation selectivity through choosing appropriate binding buffer composition on a certain column [36,37,41] but also to design new boronate-affinity monolithic columns [37,40–42]. Based on recent publications and our experience, we propose herein a set of strategies for manipulating selectivity in monolithic column-based BAC, as listed in Table 2. These strategies can be classified into two categories: (1) choosing/designing appropriate boronate-affinity stationary phases; and, (2) choosing appropriate binding buffer composition. As in common LC, the nature of the BAC stationary phase is a major selectivity-determining factor, while the composition of the binding buffer is an auxiliary factor that can tune the selectivity to a certain extent. For a given boronate-affinity column, the composition of an appropriate binding buffer changes, depending on the structure and the property of the analytes,
Changing binding buffer composition Increase pH; add Lewis base
Add organic solvent; lower ionic strength Increase ionic strength Add urea
which may vary dramatically from small compounds to macromolecules. When adjusting the binding-buffer composition fails to provide satisfactory selectivity, changing to a more appropriate BAC column is the only solution. Boronate-affinity interaction can be generally enhanced by choosing boronic acids with low pKa values {e.g., the Wulff-type boronic acid [42] and electronwithdrawing group substituted boronic acid [41]} as the affinity ligands. However, for some specific analytes, such a general approach may be not true and other factors should be taken into account [57]. A boronnitrogen molecular team [40] (see later context for detailed explanation) is also an effective way to improve boronate affinity. Also, the density of the accessible affinity ligand on the surface of the stationary phase is an important factor that determines boronate-affinity retention. A higher density is favorable for boronateaffinity binding. However, it is difficult to predict the density of accessible affinity ligand under any selected route and procedure for monolith synthesis. A reasonable solution is to immobilize an appropriate boronicacid ligand onto a known base monolithic bed that exhibits high specific surface area and high density of accessible reactive functional. However, boronate-affinity retention can be enhanced by increasing the pH value of the binding buffer or adding a Lewis base (e.g., sodium fluoride) into the binding buffer. However, higher pH will make the affinity ligand more negatively charged and thereby cause increased ionic interaction if the analyte is charged. Moreover, too high concentration of Lewis base may result in increased hydrophobic interaction of proteins with the stationary phases. Hydrophobic interaction is one of the main secondary interactions that influence the selectivity of BAC. This interaction is due to the hydrophobicity of both the support materials and the boronate ligands. The boronate-affinity ligands are usually aromatic boronic acids, which can bring about hydrophobic interaction and aromatic p-p interaction. Hydrophobic interaction is undesirable because it can cause non-specific adsorption. Hydrophobic interaction can be reduced by choosing hydrophilic cross-linkers and/or hydrophilic-affinity li-
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Figure 4. Specific capture of glycoproteins on the poly(VPBA-co-MBAA) monolithic column and comparison with non-glycoproteins. Mobile phase: 250 mM ammonium acetate (pH 8.5), switched to 100 mM HOAc (pH 2.7) at 10 min; Sample: 0.5 mg/mL protein dissolved in 250 mM ammonium acetate (pH 8.5) (Reprinted from [37] with permission).
gands. For example, a poly(VPBA-co-EDMA) monolith [36], which exhibited significant hydrophobicity, failed to capture glycoproteins specifically when no organic solvent was added to the binding buffer (unpublished data). After replacing ethylene glycol dimethacrylate (EDMA) with a hydrophilic cross-linker, N,N 0 -methylenebiacrylamide (MBAA), reversed-phase retention on the resultant poly(VPBA-co-MBAA) monolith was reduced by 70% and the monolithic capillary exhibited specificity towards glycoproteins (see Fig. 4) [37]. Another example is that, by substituting the relatively hydrophobic boronic-acid VPBA with a relatively hydrophilic one, 3acrylamidophenylboronic acid (AAPBA) [38,44], the resultant poly(AAPBA-co-EDMA) monoliths exhibited specificity to glycoproteins. The poly(AAPBA-co-EDMA) monolith, especially, using MOGs as porogenic templates showed high specificity towards glycoproteins and a high analysis rate [44]. The combination of a hydrophilic cross-linker and a hydrophilic affinity ligand generated a more hydrophilic monolithic column {e.g., the poly(BSPBA-co-MBAA) monolithic column [41] exhibits a highly hydrophilic nature}. For monolithic columns prepared by post-modification approaches, the nature of the base monolith is an important factor that generates hydrophobic interaction. For example, poly(GMA-co-PEGDA) monolith was relatively hydrophobic, and a Wulff-type boronic-acidimmobilized poly(GMA-co-PEGDA) monolithic column [42] exhibited very poor specificity towards glycoproteins (unpublished data).
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Silica-based monoliths should be excellent support materials for BAC, especially the organic-inorganic hybrid monolith, which combines the virtues of silica monolith and organic polymer monolith. Lin et al. [39] prepared an organic-inorganic hybrid monolithic column using VPBA as a functionalized organic monomer in a one-pot approach. The resultant hybrid boronate-affinity monolithic column showed high boronate affinity and basically eliminated the non-specific retention originating from hydrophobic interaction. However, hydrophobic interaction can also be suppressed by choosing an appropriate binding-buffer composition. For small cis-diol-containing compounds, adding adequate appropriate organic solvent [e.g., acetonitrile (ACN)] is effective. By adding 20% ACN to aqueous buffer to suppress the non-specific hydrophobic interactions, Lin et al. [43] improved the specificity of the hydrophobic poly (VPBA-co-EDMA) monolith towards glycoproteins. However, as the addition of too much organic solvent may increase the risk of protein precipitation, lowering the ionic strength is a possibility. Ionic interaction is another main secondary interaction that influences the selectivity of BAC. For regular boronic acids, the boron atom must have tetrahedral sp3 orbital hybridization status for strong binding, at which the boron is natively charged (see Fig. 1). Thereby, for charged analytes, ionic interaction always accompanies a boronate-affinity interaction. A solution to this issue seems to be the employment of Wulff-type boronic acid or a B-N coordinated molecular team because the B-N
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Figure 5. Separation of a mixture of myoglobin and HRP on the poly(BSPBA-co-MBAA) monolithic column. Sample, myoglobin and HRP, 1 mg/mL each, dissolved in 50 mM ammonium bicarbonate buffer (pH 7.4). Mobile phase: 50 mM ammonium bicarbonate (pH 7.4), switched to 100 mM HOAc (pH 2.7) containing 3 M urea at 15 min (Reprinted from [41] with permission).
coordination can reduce the charge of the boron. Although such a strategy has not been confirmed with boronate-affinity monolithic columns, it has been demonstrated that B-N coordinated molecular team-functionalized magnetic nanoparticles (MNPs) exhibit enhanced specificity toward adenosine monophosphate (negatively charged) as compared with MNPs functionalized with a regular boronic acid [60]. Ionic interaction in BAC can also be suppressed effectively by increasing the ionic strength of the binding buffer. The ionic strength becomes critical for the specificity towards significantly negatively charged glycoproteins (e.g., a1 acid glycoprotein). On a poly (BSPBA-co-MBAA) monolithic capillary, when the sodium-chloride concentration was less than 500 mM, a1 acid glycoprotein could not be captured, due to strong electrostatic repulsion [41]. Therefore, to suppress electrostatic attraction between non-glycoproteins with boronic-acid ligand, the presence of adequate salt in the binding buffer is a precondition. However too high ionic strength may increase the hydrophobic interaction between proteins and stationary phase. Although the presence of ionic interaction is an undesirable factor, this secondary interaction can be employed as a beneficial driving force for other purposes. For example, cationexchange separation of non-glycoproteins was realized by using boronate-affinity polymeric monolith as a cation-exchange column [61].
Hydrogen binding is an inevitable secondary interaction, since boronic acids always carry hydroxyl group(s). The effect of hydrogen bonding is usually negligible. However, in some special circumstances, hydrogen bonding can be strong enough to become the main mechanism for retention on a boronate-affinity column. An example is the apparent secondary separation capability of the poly(BSPBA-co-MBAA) monolithic column [41], on which glycoprotein horseradish peroxidase (HRP) was separated into two sharp peaks under acidic elution (see Fig. 5) and similar secondary separation of nucleosides was also observed. The mechanism for this secondary separation was attributed to hydrogen-bonding interaction. Such a finding opened a new opportunity for the separation of hydrophilic compounds, since effective columns are still lacking for high-resolution separation of hydrophilic compounds. This boronateaffinity column enables two-dimensional separation on a single column. However, as the separation capability was still poor, further development is necessary to make hydrogen-bonding-driven separation of practical use. Based on the above discussion, it is clear that appropriate boronate-affinity columns are the key to highperformance BAC, though appropriate binding-buffer composition is very important for the specificity. It should be noted that the operational parameters of binding-buffer composition do not work independently and compromises among several factors are often
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encountered. To obtain high specificity towards cis-diol compounds, especially glycoprotein and RNA, all the factors that govern the selectivity must be taken into account.
4. Binding pH The binding pH of BAC is generally determined by the pKa value of the boronic-acid ligands, although some other factors can make optimal binding pH deviate from the pKa-determining rule {e.g., the structure of analytes [57] and the species and the concentrations of the ions in the binding buffers [62,63]}. Generally, boronic-acid ligands of lower pKa values generate stronger affinity towards the cis-diol-containing compounds. However, most of the commercially-available boronic acids are weak acids with a pKa 8–9, so a basic medium is required to form stable cyclic esters between boronic-acid ligands and cis-diol-containing compounds. However, the use of basic pH gives rise to not only the inconvenience of pH adjustment but also the risk of degradation of labile biomolecules [64,65]. In order to reduce the binding pH, novel boronic-acid ligands and innovative methods are required. Although this issue has not been satisfactorily solved in conventional BAC, the binding pH has been reduced by a great extent in monolithic column-based BAC in recent years.
So far, there are four main strategies to lower the pKa values of boronic-acid ligands: (1) introduction of an electron-withdrawing group (e.g., nitro and sulfonyl) into the phenyl ring [66,67]; (2) incorporation of an amino group adjacent to the boron atom of a boronic acid to form an intramolecular B-N coordination (Wulff-type boronic acids) [68,69]; (3) introduction of intramolecular B-O coordination into the boronic-acid molecules (improved Wulfftype boronic acids) [70,71]; and, (4) ‘‘molecular team’’ or ‘‘teamed boronate-affinity (TBA)’’ proposed by our group [40,60]. 4-(3-Butenylsulfonyl) phenylboronic acid (BSPBA), which carries a strong electron-withdrawing group in the phenyl ring, exhibited a pKa value of 7.0 [67]. Using this boronic acid as the affinity ligand, the poly(BSPBAco-MBAA) monolithic column exhibited strong affinity to both cis-diol small molecules and glycoproteins at neutral pH conditions [41]. An additional advantage of this ligand is that the strong electron-withdrawing sulfonyl group greatly enhanced the hydrophilicity of the ligand, so the monolithic column exhibited excellent specificity towards both cis-diol small molecules and glycoproteins. Among the four strategies mentioned above, Wulfftype boronic acids exhibit the lowest pKa, which is as
Figure 6. The chromatographic retention of deoxyadenosine (5) and adenosine (6) on Wulff-type boronate modified (a–g) and non-modified poly(GMA-co-PEGDA) monolithic column (h). Mobile phase: (a) 10 mM sodium phosphate buffer (pH 7.4); (b) 100 mM HOAc (pH 2.7); (c–h) 10 mM sodium phosphate buffer at pH 7.0, 6.0, 5.5, 5.0, 5.0 and 7.4, respectively, switched to 100 mM HOAc (pH 2.7) at 30 min. Sample: (a) 0.5 mg/mL deoxyadenosine dissolved in the loading buffer; (b–f, h) 0.1 mg/mL adenosine dissolved in corresponding loading buffers; (g) the loading buffer at pH 5.0 as a blank control (Reprinted from [42] with permission).
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Figure 7. Intermolecular synergistic action of a boronic-acid molecular team for the reversible capture/release of cis-diol-containing compounds at neutral conditions (Reprinted from [40] with permission).
Figure 8. Chromatographic retention of quinol (6) and catechol (7) on the poly(TEPIC-co-(mAPBA-HMDA)) monolithic column. Mobile phase: (a) 100 mM HOAc (pH 2.7); (b–e) 10 mM sodium phosphate buffer at pH 6.0, 6.5, 7.0, and 7.5, respectively; the mobile phase was changed to 100 mM HOAc (pH 2.7) at 30 min. Sample: Quinol and catechol were dissolved in the mobile phase at a concentration of 1 mg/mL each (Reprinted from [40] with permission).
low as 5.2 [68]. Thus, Wulff-type boronic-acid ligands may greatly facilitate the application of boronate-affinity techniques to biological samples of acidic pH (e.g., urine, tears and saliva). Although Wulff-type boronic acids have been widely employed in biological sensing [72– 74], the available Wulff-type boronic acids do not usually carry a reactive moiety for immobilization onto support materials, and this limited the use of Wulff-type boronic acid in BAC.
Our group [42] synthesized a novel Wulff-type boronate, 3-(dimethylaminomethyl) aniline-4-pinacol boronate (DMAMAPB), which possesses an amino group in the phenyl ring for its attachment to various support materials. The synthesized boronate was attached to a poly(GMAco-PEGDA) monolith, and the resulting boronate-affinity monolithic column could specifically capture cis-diol-containing compounds at or above pH 5.5 (See Fig. 6). This is the lowest binding pH in BAC reported in the literature so
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far. Such a Wulff-type boronate can greatly expand the application range of BAC-based approaches to samples of medium acidic pH (e.g., urine and saliva). In strategies (1)–(3) mentioned above, multi-step reaction routes are required to synthesize appropriate boronic-acid ligands (e.g., a four-step reaction route is needed to synthesize the DMAMAPB). To overcome this problem, our group [40] presented a new approach, called ring-opening polymerization with synergistic comonomers, for the preparation of a boronate-affinity monolith that can bind cis-diol compounds under neutral conditions. The principle of this approach is based on a core concept – ‘‘molecular team’’ or ‘‘molecular teamwork’’. A boronic-acid molecular team comprises a regular boronic acid (e.g., mAPBA) and a neighboring diamine (e.g., HMDA) (see Fig. 7). By virtue of boronnitrogen coordination between the boronic acid and the diamine, the two compounds can form a complex or a molecular team. Through ring-opening polymerization, the molecular team was fixed in the polymer, and the resulting monolith can function as a single Wulff-type boronic-acid ligand to enable the specific capture of cisdiol-containing compounds under neutral conditions. When the medium is made more acidic, the amine group is protonated, and B–N coordination is broken, which results in the release of the cis-diol compound from the monolith. As shown in Fig. 8, the binding pH was reduced to 7.0. Recently, our group [60] proposed the concept of molecular team and coined a term ‘‘teamed boronate-
affinity (TBA)’’, which means the boronate-affinity originated from a boronic-acid molecular team, instead of a single boronic acid in the conventional way. The concept of TBA opened new possibilities for the preparation of boronate-affinity functionalized materials of lower binding pH. In addition to the ring-open polymerization approach, other chemical-processing approaches, which can fix the team members to proper positions, can also be used to build a molecular team. For example, molecular self-assembly on a gold surface was employed as the driving-force to build TBA-MNPs and TBA-functionalized matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) target-plate analysis. Both exhibited enhanced affinity towards cis-diol-containing biomolecules [60].
5. Applications So far, published works on monolithic column-based BAC have mainly focused on fundamental aspects, especially how to achieve high specificity and how to reduce the binding pH. A few impressive applications have been demonstrated. Feng et al. [38] successfully employed a poly(AAPBA-co-EDMA) monolith as a boronate-affinity sorbent for selective enrichment of glycopeptides. MALDI-TOF-MS analysis indicated that glycopeptides from the tryptic digests of HRP were specifically en-
Figure 9. MEKC analysis of (a) urine sample and (b) extracted components by the Wulff-type monolithic column for boronate-affinity chromatography (BAC). Peak identity: 5, cytidine; 12, guanosine; other, unknown (Reprinted from [42] with permission).
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riched. Without enrichment, the glycopeptides from HRP digests were hardly detected. With enrichment, the glycopeptides were all readily detected because the target molecules were enriched while the interfering non-glycopeptides were removed. We demonstrated [42] the application potential of a Wulff-type boronate-functionalized monolithic column for specific enrichment of modified nucleosides directly from a human-urine sample. Urinary-modified nucleosides have been suggested as biomarkers for early cancer diagnostics [4–7]. Urine samples without and with extraction using the boronate-affinity column were analyzed by micellar electrokinetic chromatography (MEKC). The results revealed that nucleosides (e.g., cytidine and guanosine) were significantly enriched (see Fig. 9). Compared with conventional boronate-affinity absorbents, the Wulff-type boronate-functionalized monolithic column has an apparent advantage that sample-pH adjustment prior to the enrichment was avoided. In addition to these applications, approaches to the preparation of boronate-affinity monolithic columns can be used to prepare open tubular boronate-affinity capillaries for on-line extraction through slight modification of the procedures. For example, our group [75] reported on-line coupling of open tubular capillary-based boronate-affinity microextraction with HPLC-electrospray ionization (ESI)-tandem MS for the specific, sensitive determination of dopamine in urine samples.
6. Conclusion and outlook Boronate-affinity monolithic columns have developed quickly in recent years, due to their potential as affinity platforms for the specific enrichment of cis-diol-containing target molecules. A variety of monolithic columns have been successfully developed. To lower the binding pH, most existing strategies have been carried out and new strategies have been proposed. The appearance of Wulff-type boronate-functionalized monolithic columns widely expanded the application range of BAC, while TBA provided new access to boronate-affinity functionalized materials for applications to physiological samples. Previous understanding of the separation mechanism has guided the selection of appropriate experimental conditions and the design of monolithic columns. Based on recent practices and experience in this area, more comprehensive understanding has been established. A set of strategies has been suggested for further development and application of boronate-affinity monolithic columns. All these advances have paved a solid way for further in-depth development and wider applications. Although boronic acids exhibited attractive features, they also showed some weaknesses. A major weakness comes from the relatively weak binding strength of
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boronic acids with cis-diol-containing compounds. The binding constants of regular boronic acids are in the range 101–104/M, depending on the structure of the cisdiol-containing compounds [57], so boronic-acid functionalized materials will fail the specific capture of target molecules of very low abundance. Another disadvantage is that specificity and affinity greatly depend on the structure and the properties of the cis-diol biomolecules under investigation. This will cause different enrichment efficiency for different target molecules under the binding conditions used. Apparently, boronate-affinity monolithic columns inherit these disadvantages. As to future development trends, we anticipate that several aspects will be the focus in this area. One is the development of high-performance boronate-affinity monolithic columns. Here, high performance means high affinity, high specificity and high capacity towards small and big cis-diol biomolecules. Wulff-type boronic acids have exhibited improved affinity at medium acidic pH. Another type of excellent boronic-acid ligand will probably be benzoboroxoles (improved Wulff-type boronic acids), which have shown excellent water solubility and improved sugar-binding capacity, even superior to Wulff-type boronic acids, in neutral water [70,71]. We predict that MBAA or similar cross-linkers and silicaorganic hybrid monolithic columns will get more attention due to their hydrophilic nature. A second aspect is the development of novel boronateaffinity-based monolithic columns (e.g., molecularly imprinted monolithic columns), in which boronate affinity will play important roles. A third aspect is to develop boronate-affinity-based separation techniques. So far, high-resolution separation of hydrophilic compounds, particularly glycoprotein isoforms, is still challenging. Boronate affinity could be a useful driving force if it is used appropriately. Meanwhile, hydrogen-binding interaction could provide a new driving force, as its potential has been demonstrated. Finally, more important applications will appear in near future. In particular, boronate-affinity monolithic columns would be promising on-line and off-line platforms for coupling with MALDI-TOF-MS and ESI-MS for analysis and identification of cis-diol biomolecules in omics. The chemistry of boronic acids binding with cisdiol compounds has been recognized over 160 years, and BAC appeared more than 40 years ago. Can one teach old dogs new tricks? Our answer is YES!
Acknowledgements This work was supported by the General Grant (No. 21075063) from the National Natural Science Foundation of China, the Fundamental Research Funds (No. 1093020506) and the New Century Excellent Talents Program (No. NCET-08-0270) from the Ministry of
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Recent advances in monolithic column-based boronate-affinity chromatography Hengye Li, Zhen Liu Cis-diol-containing biomolecules are an important class of compounds, including carbohydrates, glycoproteins, RNA, and nucleosides. Many are the main analytes at the frontiers of life science studies (e.g., proteomics, metabolomics and glycomics). As many cis-diol molecules of biological importance are present in very low abundance in samples while interfering substances are usually present in high abundance, specific capture and effective enrichment of target cis-diol biomolecules become key, challenging steps in the -omics analyses. Boronate-affinity chromatography (BAC) is a tool for specific isolation and enrichment of cis-diol compounds. In recent years, monolithic column-based BAC has attracted increasing attention. A variety of BAC monolithic columns have been developed and impressive applications in selective enrichment of glycopeptides and nucleosides have been demonstrated. We review recent advances in monolithic column-based BAC. We mainly focus on the common issues encountered during the development and application of monolithic columns for BAC. We suggest a set of strategies to guide how to select appropriate binding-buffer composition and how to design new BAC columns with the desired properties. We highlight progress and discuss trends in lowering the binding pH. ª 2012 Elsevier Ltd. All rights reserved. Keywords: Boronate-affinity chromatography (BAC); Boronic acid; Cis-diol; Glycopeptide; Glycoprotein; Metabolomics; Monolithic column; Nucleoside; Phenylboronic acid; Proteomics Abbreviations: AAPBA, 3-Acrylamidophenylboronic acid; ACN, acetonitrile; AFP, a-Fetoprotein; BAC, Boronate-affinity chromatography; BSPBA, 4(3-Butenylsulfonyl) phenylboronic acid; CEA, Carcinoembryonic antigen; Con A, Concanavalin A; DMAMAPB, 3-(Dimethylaminomethyl)-aniline-4pinacol boronate; DMF, N,N-dimethylformamide; DMSO, Dimethyl sulfoxide; EDMA, Ethylene glycol dimethacrylate; ESI, Electrospray ionization; GMA, Glycidyl methacrylate; HILIC, Hydrophilic interaction chromatagraphy; HMDA, 1,6-Hexamethylenediamine; HPBA, p-Hydroxyphenylboronic acid; HPLC, High-performance liquid chromatography; HRP, Horseradish peroxidase; MALDI-TOF-MS, Matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry; mAPBA, m-Aminophenylboronic acid; MBAA, N,N 0 -methylenebiacrylamide; MEKC, Micellar electrokinetic chromatography; MOG, Metal-organic gel; PEG, Polyethylene glycol; PEGDA, Poly(ethylene glycol) diacrylate; TEPIC, Tris(2,3epoxypropyl)isocyanurate; TMOS, Tetramethyloxysilane; VPBA, 4-Vinylphenylboronic acid; c-MAPS, 3-Methacryloxypropyltrimethoxysilane
1. Introduction Hengye Li, Zhen Liu*, State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
*
Corresponding author. Tel./Fax: +86 25 8368 5639; E-mail: [email protected]
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Since the beginning of the new century, a variety of ‘‘-omics’’, particularly proteomics, metabolomics and glycomics, have increasingly attracted attention. Cis-diolcontaining compounds comprise a class of biomolecules, which cover several major types of target molecules in -omics studies, including glycoproteins and glycopeptides in proteomics, nucleosides and modified nucleosides in metabolomics, and saccharides in glycomics. Cis-diol biomolecules play essential roles in many biological processes and are significant in disease diagnostics. Glycopro-
teins play important roles (e.g., molecular recognition, inter-cell and intra-cell signaling, immune response and sperm-egg interaction). Some glycoproteins [e.g., afetoprotein (AFP) and carcinoembryonic antigen (CEA)] have been employed as biomarkers for clinical cancer screening [1]. Nucleosides are building blocks of RNA. The turnover of tRNA generates modified nucleosides and their excretion concentrations in urines reflect the whole turnover level of tRNA [2]. Because neoplastic diseases are associated with increased tRNA turnover rate [3], urinary modified nucleosides have been suggested as potential diagnostic markers of cancer
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diseases [4–7]. A common feature of these cis-diol biomolecules is that they are usually present in very low abundance in the -omic samples, especially at the early stage of cancer development, while interfering substances are present in high abundance. Specific capture and effective enrichment of target cis-diol biomolecules are therefore key steps in -omics analyses. Boronate-affinity chromatography (BAC) is a powerful technique for the selective isolation and enrichment of cisdiol-containing compounds, such as glycoproteins [8,9], nucleosides [10,11] and saccharides [12]. The principle relies on the formation of cyclic esters between boronicacid ligands and the cis-diol moiety of analytes under alkaline conditions (usually pH of 8.5 or higher) and the dissociation of the cyclic esters when the environmental condition is changed to acidic. The general formula of the reaction between boronic acids and cis-diol-containing compounds is illustrated in Fig. 1. This pH-dependent chemistry makes boronic acids excellent ligands for molecular recognition, which had led to the generation of boronic acid-based chemical sensing for saccharides [13] and the invention of BAC in the 1970s [14]. BAC exhibits several features. First, it has broadspectrum affinity, so one boronic-acid ligand can capture most, if not all, cis-diol biomolecules, making it very useful for global -omics analyses. Second, BAC relies on covalent reactions, so adsorption induced by non-specific interactions can be suppressed or eliminated by choosing appropriate conditions, giving high specificity. Third, the capture/release process can be manipulated easily through switching pH. Fourth, the desorption kinetics is usually fast, so analyte carry-over is usually limited. Finally, as the elution is carried out at moderate acidic conditions, this approach is very compatible with mass spectrometry (MS), facilitating applications in -omics analyses. Due to these merits, boronic-acid-functionalized materials have increasingly attracted attention in recent years, particularly in proteomics [15–18] and metabolomics [19,20].
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The properties of the boronate-affinity-separation media are detemined by both the boronic-acid ligands and the support materials. Generally, the boronic-acid ligands can be any aromatic boronic acids or their derivatives. The pKa value of a boronic acid is a key parameter that influences its performance in BAC. Usually, a lower pKa value results in a stronger affinity and requires a lower environmental pH for binding. The nature of support materials is another key factor in BAC. An inappropriate support material may greatly degrade performance, particularly the specificity of the resulting BAC column. Monolithic columns, pioneered by Hjerten [21], Svec [22] and Tanaka [23], are defined as ‘‘continuous stationary phases that form as a homogeneous column in a single piece and prepared in various dimensions with agglomeration-type or fibrous microstructures’’ [24]. Due to the advantages of ease of preparation, low back pressure and convective mass transfer, versatile surface chemistry for ligand attachment and ease of miniaturization in channels and capillaries, great attention has been paid on monolithic columns in recent decades. Especially, monolithic capillaries based high-performance liquid chromatography (HPLC) have the advantages of improved chromatographic resolution, higher efficiency, lower sample consumption, convenient online coupling to MS and improved mass-detection sensitivity, compared with classical column-based HPLC [25–30]. Capillary monolithic columns have been widely used in the -omics studies [31–34]. Monolithic column-based BAC has developed rapidly since its first appearance in 2006 [35]. A variety of boronate-affinity monolithic columns have been developed and the analytes investigated included nucleosides [36–42], glycoproteins [37–39,41,43,44], glycopeptides [38], and catechol [36,37,42]. Also, some boronateaffinity monolithic materials in the format of disks integrated in microchip systems for the determination [45] and extraction [46] of catecholamines have been developed, but they are not the focus of this review. The
Figure 1. The general interaction between boronic acids and cis-diol-containing compounds.
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separation mechanism in monolithic column-based BAC has been systematically investigated [36], and this work provided a theoretical guideline for the selection of binding buffers and the design of new monolithic capillaries. Further, attempts have been made to improve the specificity especially to glycoproteins [37,43] and to lower the binding pH [40–42]. For better understanding of monolithic column-based BAC and even general BAC, it is necessary to survey the common issues and concerns encountered in this area. In this review, we examine the recent advances in the development of boronate-affinity monolithic columns, focusing on common issues and challenging tasks. We also discuss possible solutions to these challenges and further development of BAC. 2. Preparation and characterization Generally, boronate-functionalized monolithic columns can be prepared by direct or indirect methods. In direct methods, boronic-acid ligands with active groups (e.g., vinyl and amino group) participate in the reactions and directly generate the boronate-affinity monoliths [36– 41,43,44]. In indirect methods, a base monolithic column, which initially contains no boronic-acid ligands but certain active functional groups (e.g., epoxy group), is first prepared for subsequent surface modification, and then an appropriate boronic-acid ligand is immobilized onto the surface of the base monolith by appropriate chemical reactions [35,42]. Fig. 2 shows the boronic-acid ligands, co-monomers and cross-linkers that have been used in the literature. 2.1. Preparation According to the nature of monolithic materials, existing boronate-affinity monolithic columns can be classified into organic polymer and organic-inorganic hybrid monolithic columns. Table 1 summarizes the co-monomers, the boronic acids and the porogenic systems used in the literature. Two kinds of in-situ polymerization have been used to prepare polymer-based monolithic stationary phases. The first is in-situ polymerization initiated by free radicals through heating or UV exposure. Generally, a monomer, a cross-linker and an initiator are dissolved in porogenic solvents and the resulting mixture is injected into a column (e.g., a fused-silica capillary). With two ends sealed, the column is heated in a water bath or exposed to UV light to initiate polymerization. After the reaction, a wash step is employed to remove the unreacted monomer, cross-linker and porogenic solvents. Most boronate-affinity monolithic capillaries were prepared by this kind of polymerization [35–38,41–44]. Generally, the porogenic solvents used in this method are organic solvents. However, novel materials can also
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be used as porogenic templates to generate macroporous materials. Yang et al. [44] first employed metal-organic gels (MOGs) as porogenic templates to fabricate a macroporous monolith. The resulting monolith showed lmsized pore distribution. This large pore-size distribution was essential for the analysis of macrobiomolecules (e.g., glycoproteins). The second kind of polymerization is based on condensation of multifunctional epoxy with di-amino or triamino compounds [23,47]. Our group [40] developed a novel approach, called ring-opening polymerization with synergistic co-monomers, to prepare boronate-functionalized monolithic columns. As shown in Fig. 3, this approach takes advantage of boron-nitrogen coordination between a regular boronic-acid ligand, m-aminophenylboronic acid (mAPBA), and a diamine, 1,6-hexamethylenediamine (HMDA). On being mixed together, mAPBA and HMDA can self-assemble into a relatively stable complex due to the boron–nitrogen coordination and the resultant complex can function as a bifunctional reagent to react with a multifunctional compound, tris(2,3-epoxypropyl)isocyanurate (TEPIC), to form a poly(TEPIC-co-(mAPBA-HMDA)) monolithic capillary. Relative to conventional silica monolithic columns, organic-inorganic hybrid monolithic columns are more hydrolytically stable [48]. Organic-inorganic hybrid monolithic columns were developed quickly in recent years. Initially, they were prepared by sol-gel technology based on the co-condensation of tetra-alkoxysilanes and organosiloxane precursors [49]. In this approach, the organosiloxane precursor is directly incorporated into the reaction mixture, so the desired functional monomer is directly introduced into the monolith obtained without additional surface modification [50–52]. However, the preparation procedure is time consuming. Recently, a novel ‘‘one-pot’’ approach was proposed by Zou et al. [53–55] for quick preparation of the organic-inorganic hybrid monolithic column. In this approach, vinylalkoxysilanes (as precursor) and an organic monomer with [53,55] or without [54] tetraalkoxysilane were mixed in one pot, along with an appropriate initiator. Condensation and polymerization occurred at the same time and the monolithic column could be obtained in about 24 h. By using this ‘‘one-pot’’ approach, Lin et al. [39] prepared an organic-inorganic boronateaffinity monolithic column using 4-vinylphenylboronic acid (VPBA) as a functional monomer. 2.2. Characterization Boronate-functionalized monolithic columns are usually characterized in terms of morphology, binding capacity and pore properties (e.g., pore-size distribution, specific surface area, average pore size and pore volume). The morphology of boronate-functionalized monolithic capillaries is usually characterized by scanning electron microscopy (SEM). The morphology of the pre-
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Figure 2. The structures of the boronic-acid ligands, monomers and cross-linkers used to prepare monolithic columns for boronate affinity chromatography (BAC).
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Table 1. Summary of monolithic columns for boronate-affinity chromatography (BAC) Comonomer system
Boronate ligands
Porogenic systems
Analytes Nucleosides Catechol Nucleosides Glycoproteins Catechol Glycoproteins Catechol Nucleosides Glycoproteins Nucleosides Glycoproteins Glycopeptides Catechol Nucleoside Nucleosides Glycoproteins Catechol Nucleoside Nucleosides Glycoproteins
GMA/EDMA VPBA/EDMA
HPBA VPBA
Cyclohexanol, 1-Dodecanol Diethylene glycol, Ethylene glycol
VPBA/EDMA VPBA/MBAA
VPBA VPBA
Diethylene glycol, Ethylene glycol DMSO, DMF, H2O, Na2HPO4
AAPBA/EDMA
AAPBA
MOGs
AAPBA/EDMA
AAPBA
Methanol, PEG 20000
TEPIC/HMDA + mAPBA
HMDA + mAPBA
PEG 200
BSPBA/MBAA
BSPBA
DMSO, 1-Dodecanol
GMA/PEGDA
DMAMABA
DMSO, 1,4-butanediol
c-MAPS/TMOS/VPBA
VPBA
Diethylene glycol, PEG 10000
Binding pH
Ref.
9.0 8.5
[35] [36]
8.5 8.5
[43] [37]
8.5
[44]
9.2
[38]
P 7.0
[40]
P 7.0
[41]
P 5.5
[42]
8.5
[39]
Figure 3. The preparation of a monolithic column through the approach ring-opening polymerization with synergistic co-monomers. (I) B-N coordination and (II) ring-opening polymerization with synergistic co-monomers (Reprinted from [40] with permission).
pared monolithic columns is determined by not only the nature of the monomers and cross-linkers but also the properties and the amounts of porogenic solvents used in the polymerization system. For the control of the morphology of monolithic columns, readers can refer to an excellent article [56]. The pore properties of boronate-functionalized monolithic columns are usually measured by the Brunauer-Emmett-Teller (BET) method in terms of poresize distribution, specific surface area, average pore size and pore volume. The pore properties can be regulated by systematically changing the recipe of the pre-polymerization solutions and the temperature at which the polymerization is carried out. Binding capacity is an important performance indicator of boronate-functionalized monolithic columns. It is usually measured by frontal chromatography. The binding capacity of a boronate-functionalized monolithic column varies with the pH value of the binding buffer used.
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The pKa values of boronic-acid ligands are usually measured by the absorbance change at 268 nm that occurs upon conversion from the trigonal form (at low pH) to the tetrahedral form (at high pH) during titration of the boronic-acid ligands in free solution [57]. Gillespie et al. [58] reported a non-invasive approach for the determination of the pKa values of boronic-acid ligands on the monolith surface. Although the value measured by this method showed some deviation from that obtained by free-solution titration, the information offered by this method may reflect, to some extent, the basic property of a boronic-acid-functionalized monolithic column as a whole.
3. Separation mechanism and selectivity manipulation Specificity is an essential concern in BAC and is often a challenging task for macromolecules. To reach a pure
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Table 2. Strategies for selectivity manipulation in boronate-affinity chromatography (BAC) Interaction type Boronate-affinity interaction
Hydrophobic interaction Ionic interaction Hydrogen bonding interaction
Changing stationary phase Use B-N coordinated molecular team; use Wulff-type boronic acid; use improved Wulfftype boronic acid; increase ligand density Use hydrophilic monomer/cross-linker; use hydrophilic support material Use B-N coordinated molecular team; use Wulff-type boronic acid None
boronate-affinity-governed separation, a sound understanding of the separation mechanism is indispensable. The boronate-analyte interactions involved in regular BAC have been well documented in an excellent review [59]. In addition to boronate-affinity interaction, four secondary interactions, including hydrophobic, ionic, hydrogen bonding and coordination interactions, can occur in BAC. Our group [36] systematically investigated the retention mechanism in monolithic capillary-based BAC on a single monolithic capillary. It was observed that multiple intermolecular interactions can occur between the analytes and the boronateaffinity monolith, including boronate-affinity, hydrophobic, ionic and hydrogen-bonding interactions. Under certain conditions, the secondary interactions could generate significant undesirable secondary retention. In addition to the previous conclusion that unprotonated amines and carboxyl groups can serve as electron donors and thus can coordinate with boronic acids [59], the coordination effect of Lewis bases (e.g., fluoride ion) was confirmed, and can enhance the complexation between cis-diol-containing compounds with boronic acids. Knowledge of the separation mechanism has been used as a theoretical guide for how not only to manipulate the separation selectivity through choosing appropriate binding buffer composition on a certain column [36,37,41] but also to design new boronate-affinity monolithic columns [37,40–42]. Based on recent publications and our experience, we propose herein a set of strategies for manipulating selectivity in monolithic column-based BAC, as listed in Table 2. These strategies can be classified into two categories: (1) choosing/designing appropriate boronate-affinity stationary phases; and, (2) choosing appropriate binding buffer composition. As in common LC, the nature of the BAC stationary phase is a major selectivity-determining factor, while the composition of the binding buffer is an auxiliary factor that can tune the selectivity to a certain extent. For a given boronate-affinity column, the composition of an appropriate binding buffer changes, depending on the structure and the property of the analytes,
Changing binding buffer composition Increase pH; add Lewis base
Add organic solvent; lower ionic strength Increase ionic strength Add urea
which may vary dramatically from small compounds to macromolecules. When adjusting the binding-buffer composition fails to provide satisfactory selectivity, changing to a more appropriate BAC column is the only solution. Boronate-affinity interaction can be generally enhanced by choosing boronic acids with low pKa values {e.g., the Wulff-type boronic acid [42] and electronwithdrawing group substituted boronic acid [41]} as the affinity ligands. However, for some specific analytes, such a general approach may be not true and other factors should be taken into account [57]. A boronnitrogen molecular team [40] (see later context for detailed explanation) is also an effective way to improve boronate affinity. Also, the density of the accessible affinity ligand on the surface of the stationary phase is an important factor that determines boronate-affinity retention. A higher density is favorable for boronateaffinity binding. However, it is difficult to predict the density of accessible affinity ligand under any selected route and procedure for monolith synthesis. A reasonable solution is to immobilize an appropriate boronicacid ligand onto a known base monolithic bed that exhibits high specific surface area and high density of accessible reactive functional. However, boronate-affinity retention can be enhanced by increasing the pH value of the binding buffer or adding a Lewis base (e.g., sodium fluoride) into the binding buffer. However, higher pH will make the affinity ligand more negatively charged and thereby cause increased ionic interaction if the analyte is charged. Moreover, too high concentration of Lewis base may result in increased hydrophobic interaction of proteins with the stationary phases. Hydrophobic interaction is one of the main secondary interactions that influence the selectivity of BAC. This interaction is due to the hydrophobicity of both the support materials and the boronate ligands. The boronate-affinity ligands are usually aromatic boronic acids, which can bring about hydrophobic interaction and aromatic p-p interaction. Hydrophobic interaction is undesirable because it can cause non-specific adsorption. Hydrophobic interaction can be reduced by choosing hydrophilic cross-linkers and/or hydrophilic-affinity li-
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Figure 4. Specific capture of glycoproteins on the poly(VPBA-co-MBAA) monolithic column and comparison with non-glycoproteins. Mobile phase: 250 mM ammonium acetate (pH 8.5), switched to 100 mM HOAc (pH 2.7) at 10 min; Sample: 0.5 mg/mL protein dissolved in 250 mM ammonium acetate (pH 8.5) (Reprinted from [37] with permission).
gands. For example, a poly(VPBA-co-EDMA) monolith [36], which exhibited significant hydrophobicity, failed to capture glycoproteins specifically when no organic solvent was added to the binding buffer (unpublished data). After replacing ethylene glycol dimethacrylate (EDMA) with a hydrophilic cross-linker, N,N 0 -methylenebiacrylamide (MBAA), reversed-phase retention on the resultant poly(VPBA-co-MBAA) monolith was reduced by 70% and the monolithic capillary exhibited specificity towards glycoproteins (see Fig. 4) [37]. Another example is that, by substituting the relatively hydrophobic boronic-acid VPBA with a relatively hydrophilic one, 3acrylamidophenylboronic acid (AAPBA) [38,44], the resultant poly(AAPBA-co-EDMA) monoliths exhibited specificity to glycoproteins. The poly(AAPBA-co-EDMA) monolith, especially, using MOGs as porogenic templates showed high specificity towards glycoproteins and a high analysis rate [44]. The combination of a hydrophilic cross-linker and a hydrophilic affinity ligand generated a more hydrophilic monolithic column {e.g., the poly(BSPBA-co-MBAA) monolithic column [41] exhibits a highly hydrophilic nature}. For monolithic columns prepared by post-modification approaches, the nature of the base monolith is an important factor that generates hydrophobic interaction. For example, poly(GMA-co-PEGDA) monolith was relatively hydrophobic, and a Wulff-type boronic-acidimmobilized poly(GMA-co-PEGDA) monolithic column [42] exhibited very poor specificity towards glycoproteins (unpublished data).
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Silica-based monoliths should be excellent support materials for BAC, especially the organic-inorganic hybrid monolith, which combines the virtues of silica monolith and organic polymer monolith. Lin et al. [39] prepared an organic-inorganic hybrid monolithic column using VPBA as a functionalized organic monomer in a one-pot approach. The resultant hybrid boronate-affinity monolithic column showed high boronate affinity and basically eliminated the non-specific retention originating from hydrophobic interaction. However, hydrophobic interaction can also be suppressed by choosing an appropriate binding-buffer composition. For small cis-diol-containing compounds, adding adequate appropriate organic solvent [e.g., acetonitrile (ACN)] is effective. By adding 20% ACN to aqueous buffer to suppress the non-specific hydrophobic interactions, Lin et al. [43] improved the specificity of the hydrophobic poly (VPBA-co-EDMA) monolith towards glycoproteins. However, as the addition of too much organic solvent may increase the risk of protein precipitation, lowering the ionic strength is a possibility. Ionic interaction is another main secondary interaction that influences the selectivity of BAC. For regular boronic acids, the boron atom must have tetrahedral sp3 orbital hybridization status for strong binding, at which the boron is natively charged (see Fig. 1). Thereby, for charged analytes, ionic interaction always accompanies a boronate-affinity interaction. A solution to this issue seems to be the employment of Wulff-type boronic acid or a B-N coordinated molecular team because the B-N
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Figure 5. Separation of a mixture of myoglobin and HRP on the poly(BSPBA-co-MBAA) monolithic column. Sample, myoglobin and HRP, 1 mg/mL each, dissolved in 50 mM ammonium bicarbonate buffer (pH 7.4). Mobile phase: 50 mM ammonium bicarbonate (pH 7.4), switched to 100 mM HOAc (pH 2.7) containing 3 M urea at 15 min (Reprinted from [41] with permission).
coordination can reduce the charge of the boron. Although such a strategy has not been confirmed with boronate-affinity monolithic columns, it has been demonstrated that B-N coordinated molecular team-functionalized magnetic nanoparticles (MNPs) exhibit enhanced specificity toward adenosine monophosphate (negatively charged) as compared with MNPs functionalized with a regular boronic acid [60]. Ionic interaction in BAC can also be suppressed effectively by increasing the ionic strength of the binding buffer. The ionic strength becomes critical for the specificity towards significantly negatively charged glycoproteins (e.g., a1 acid glycoprotein). On a poly (BSPBA-co-MBAA) monolithic capillary, when the sodium-chloride concentration was less than 500 mM, a1 acid glycoprotein could not be captured, due to strong electrostatic repulsion [41]. Therefore, to suppress electrostatic attraction between non-glycoproteins with boronic-acid ligand, the presence of adequate salt in the binding buffer is a precondition. However too high ionic strength may increase the hydrophobic interaction between proteins and stationary phase. Although the presence of ionic interaction is an undesirable factor, this secondary interaction can be employed as a beneficial driving force for other purposes. For example, cationexchange separation of non-glycoproteins was realized by using boronate-affinity polymeric monolith as a cation-exchange column [61].
Hydrogen binding is an inevitable secondary interaction, since boronic acids always carry hydroxyl group(s). The effect of hydrogen bonding is usually negligible. However, in some special circumstances, hydrogen bonding can be strong enough to become the main mechanism for retention on a boronate-affinity column. An example is the apparent secondary separation capability of the poly(BSPBA-co-MBAA) monolithic column [41], on which glycoprotein horseradish peroxidase (HRP) was separated into two sharp peaks under acidic elution (see Fig. 5) and similar secondary separation of nucleosides was also observed. The mechanism for this secondary separation was attributed to hydrogen-bonding interaction. Such a finding opened a new opportunity for the separation of hydrophilic compounds, since effective columns are still lacking for high-resolution separation of hydrophilic compounds. This boronateaffinity column enables two-dimensional separation on a single column. However, as the separation capability was still poor, further development is necessary to make hydrogen-bonding-driven separation of practical use. Based on the above discussion, it is clear that appropriate boronate-affinity columns are the key to highperformance BAC, though appropriate binding-buffer composition is very important for the specificity. It should be noted that the operational parameters of binding-buffer composition do not work independently and compromises among several factors are often
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encountered. To obtain high specificity towards cis-diol compounds, especially glycoprotein and RNA, all the factors that govern the selectivity must be taken into account.
4. Binding pH The binding pH of BAC is generally determined by the pKa value of the boronic-acid ligands, although some other factors can make optimal binding pH deviate from the pKa-determining rule {e.g., the structure of analytes [57] and the species and the concentrations of the ions in the binding buffers [62,63]}. Generally, boronic-acid ligands of lower pKa values generate stronger affinity towards the cis-diol-containing compounds. However, most of the commercially-available boronic acids are weak acids with a pKa 8–9, so a basic medium is required to form stable cyclic esters between boronic-acid ligands and cis-diol-containing compounds. However, the use of basic pH gives rise to not only the inconvenience of pH adjustment but also the risk of degradation of labile biomolecules [64,65]. In order to reduce the binding pH, novel boronic-acid ligands and innovative methods are required. Although this issue has not been satisfactorily solved in conventional BAC, the binding pH has been reduced by a great extent in monolithic column-based BAC in recent years.
So far, there are four main strategies to lower the pKa values of boronic-acid ligands: (1) introduction of an electron-withdrawing group (e.g., nitro and sulfonyl) into the phenyl ring [66,67]; (2) incorporation of an amino group adjacent to the boron atom of a boronic acid to form an intramolecular B-N coordination (Wulff-type boronic acids) [68,69]; (3) introduction of intramolecular B-O coordination into the boronic-acid molecules (improved Wulfftype boronic acids) [70,71]; and, (4) ‘‘molecular team’’ or ‘‘teamed boronate-affinity (TBA)’’ proposed by our group [40,60]. 4-(3-Butenylsulfonyl) phenylboronic acid (BSPBA), which carries a strong electron-withdrawing group in the phenyl ring, exhibited a pKa value of 7.0 [67]. Using this boronic acid as the affinity ligand, the poly(BSPBAco-MBAA) monolithic column exhibited strong affinity to both cis-diol small molecules and glycoproteins at neutral pH conditions [41]. An additional advantage of this ligand is that the strong electron-withdrawing sulfonyl group greatly enhanced the hydrophilicity of the ligand, so the monolithic column exhibited excellent specificity towards both cis-diol small molecules and glycoproteins. Among the four strategies mentioned above, Wulfftype boronic acids exhibit the lowest pKa, which is as
Figure 6. The chromatographic retention of deoxyadenosine (5) and adenosine (6) on Wulff-type boronate modified (a–g) and non-modified poly(GMA-co-PEGDA) monolithic column (h). Mobile phase: (a) 10 mM sodium phosphate buffer (pH 7.4); (b) 100 mM HOAc (pH 2.7); (c–h) 10 mM sodium phosphate buffer at pH 7.0, 6.0, 5.5, 5.0, 5.0 and 7.4, respectively, switched to 100 mM HOAc (pH 2.7) at 30 min. Sample: (a) 0.5 mg/mL deoxyadenosine dissolved in the loading buffer; (b–f, h) 0.1 mg/mL adenosine dissolved in corresponding loading buffers; (g) the loading buffer at pH 5.0 as a blank control (Reprinted from [42] with permission).
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Figure 7. Intermolecular synergistic action of a boronic-acid molecular team for the reversible capture/release of cis-diol-containing compounds at neutral conditions (Reprinted from [40] with permission).
Figure 8. Chromatographic retention of quinol (6) and catechol (7) on the poly(TEPIC-co-(mAPBA-HMDA)) monolithic column. Mobile phase: (a) 100 mM HOAc (pH 2.7); (b–e) 10 mM sodium phosphate buffer at pH 6.0, 6.5, 7.0, and 7.5, respectively; the mobile phase was changed to 100 mM HOAc (pH 2.7) at 30 min. Sample: Quinol and catechol were dissolved in the mobile phase at a concentration of 1 mg/mL each (Reprinted from [40] with permission).
low as 5.2 [68]. Thus, Wulff-type boronic-acid ligands may greatly facilitate the application of boronate-affinity techniques to biological samples of acidic pH (e.g., urine, tears and saliva). Although Wulff-type boronic acids have been widely employed in biological sensing [72– 74], the available Wulff-type boronic acids do not usually carry a reactive moiety for immobilization onto support materials, and this limited the use of Wulff-type boronic acid in BAC.
Our group [42] synthesized a novel Wulff-type boronate, 3-(dimethylaminomethyl) aniline-4-pinacol boronate (DMAMAPB), which possesses an amino group in the phenyl ring for its attachment to various support materials. The synthesized boronate was attached to a poly(GMAco-PEGDA) monolith, and the resulting boronate-affinity monolithic column could specifically capture cis-diol-containing compounds at or above pH 5.5 (See Fig. 6). This is the lowest binding pH in BAC reported in the literature so
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far. Such a Wulff-type boronate can greatly expand the application range of BAC-based approaches to samples of medium acidic pH (e.g., urine and saliva). In strategies (1)–(3) mentioned above, multi-step reaction routes are required to synthesize appropriate boronic-acid ligands (e.g., a four-step reaction route is needed to synthesize the DMAMAPB). To overcome this problem, our group [40] presented a new approach, called ring-opening polymerization with synergistic comonomers, for the preparation of a boronate-affinity monolith that can bind cis-diol compounds under neutral conditions. The principle of this approach is based on a core concept – ‘‘molecular team’’ or ‘‘molecular teamwork’’. A boronic-acid molecular team comprises a regular boronic acid (e.g., mAPBA) and a neighboring diamine (e.g., HMDA) (see Fig. 7). By virtue of boronnitrogen coordination between the boronic acid and the diamine, the two compounds can form a complex or a molecular team. Through ring-opening polymerization, the molecular team was fixed in the polymer, and the resulting monolith can function as a single Wulff-type boronic-acid ligand to enable the specific capture of cisdiol-containing compounds under neutral conditions. When the medium is made more acidic, the amine group is protonated, and B–N coordination is broken, which results in the release of the cis-diol compound from the monolith. As shown in Fig. 8, the binding pH was reduced to 7.0. Recently, our group [60] proposed the concept of molecular team and coined a term ‘‘teamed boronate-
affinity (TBA)’’, which means the boronate-affinity originated from a boronic-acid molecular team, instead of a single boronic acid in the conventional way. The concept of TBA opened new possibilities for the preparation of boronate-affinity functionalized materials of lower binding pH. In addition to the ring-open polymerization approach, other chemical-processing approaches, which can fix the team members to proper positions, can also be used to build a molecular team. For example, molecular self-assembly on a gold surface was employed as the driving-force to build TBA-MNPs and TBA-functionalized matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) target-plate analysis. Both exhibited enhanced affinity towards cis-diol-containing biomolecules [60].
5. Applications So far, published works on monolithic column-based BAC have mainly focused on fundamental aspects, especially how to achieve high specificity and how to reduce the binding pH. A few impressive applications have been demonstrated. Feng et al. [38] successfully employed a poly(AAPBA-co-EDMA) monolith as a boronate-affinity sorbent for selective enrichment of glycopeptides. MALDI-TOF-MS analysis indicated that glycopeptides from the tryptic digests of HRP were specifically en-
Figure 9. MEKC analysis of (a) urine sample and (b) extracted components by the Wulff-type monolithic column for boronate-affinity chromatography (BAC). Peak identity: 5, cytidine; 12, guanosine; other, unknown (Reprinted from [42] with permission).
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riched. Without enrichment, the glycopeptides from HRP digests were hardly detected. With enrichment, the glycopeptides were all readily detected because the target molecules were enriched while the interfering non-glycopeptides were removed. We demonstrated [42] the application potential of a Wulff-type boronate-functionalized monolithic column for specific enrichment of modified nucleosides directly from a human-urine sample. Urinary-modified nucleosides have been suggested as biomarkers for early cancer diagnostics [4–7]. Urine samples without and with extraction using the boronate-affinity column were analyzed by micellar electrokinetic chromatography (MEKC). The results revealed that nucleosides (e.g., cytidine and guanosine) were significantly enriched (see Fig. 9). Compared with conventional boronate-affinity absorbents, the Wulff-type boronate-functionalized monolithic column has an apparent advantage that sample-pH adjustment prior to the enrichment was avoided. In addition to these applications, approaches to the preparation of boronate-affinity monolithic columns can be used to prepare open tubular boronate-affinity capillaries for on-line extraction through slight modification of the procedures. For example, our group [75] reported on-line coupling of open tubular capillary-based boronate-affinity microextraction with HPLC-electrospray ionization (ESI)-tandem MS for the specific, sensitive determination of dopamine in urine samples.
6. Conclusion and outlook Boronate-affinity monolithic columns have developed quickly in recent years, due to their potential as affinity platforms for the specific enrichment of cis-diol-containing target molecules. A variety of monolithic columns have been successfully developed. To lower the binding pH, most existing strategies have been carried out and new strategies have been proposed. The appearance of Wulff-type boronate-functionalized monolithic columns widely expanded the application range of BAC, while TBA provided new access to boronate-affinity functionalized materials for applications to physiological samples. Previous understanding of the separation mechanism has guided the selection of appropriate experimental conditions and the design of monolithic columns. Based on recent practices and experience in this area, more comprehensive understanding has been established. A set of strategies has been suggested for further development and application of boronate-affinity monolithic columns. All these advances have paved a solid way for further in-depth development and wider applications. Although boronic acids exhibited attractive features, they also showed some weaknesses. A major weakness comes from the relatively weak binding strength of
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boronic acids with cis-diol-containing compounds. The binding constants of regular boronic acids are in the range 101–104/M, depending on the structure of the cisdiol-containing compounds [57], so boronic-acid functionalized materials will fail the specific capture of target molecules of very low abundance. Another disadvantage is that specificity and affinity greatly depend on the structure and the properties of the cis-diol biomolecules under investigation. This will cause different enrichment efficiency for different target molecules under the binding conditions used. Apparently, boronate-affinity monolithic columns inherit these disadvantages. As to future development trends, we anticipate that several aspects will be the focus in this area. One is the development of high-performance boronate-affinity monolithic columns. Here, high performance means high affinity, high specificity and high capacity towards small and big cis-diol biomolecules. Wulff-type boronic acids have exhibited improved affinity at medium acidic pH. Another type of excellent boronic-acid ligand will probably be benzoboroxoles (improved Wulff-type boronic acids), which have shown excellent water solubility and improved sugar-binding capacity, even superior to Wulff-type boronic acids, in neutral water [70,71]. We predict that MBAA or similar cross-linkers and silicaorganic hybrid monolithic columns will get more attention due to their hydrophilic nature. A second aspect is the development of novel boronateaffinity-based monolithic columns (e.g., molecularly imprinted monolithic columns), in which boronate affinity will play important roles. A third aspect is to develop boronate-affinity-based separation techniques. So far, high-resolution separation of hydrophilic compounds, particularly glycoprotein isoforms, is still challenging. Boronate affinity could be a useful driving force if it is used appropriately. Meanwhile, hydrogen-binding interaction could provide a new driving force, as its potential has been demonstrated. Finally, more important applications will appear in near future. In particular, boronate-affinity monolithic columns would be promising on-line and off-line platforms for coupling with MALDI-TOF-MS and ESI-MS for analysis and identification of cis-diol biomolecules in omics. The chemistry of boronic acids binding with cisdiol compounds has been recognized over 160 years, and BAC appeared more than 40 years ago. Can one teach old dogs new tricks? Our answer is YES!
Acknowledgements This work was supported by the General Grant (No. 21075063) from the National Natural Science Foundation of China, the Fundamental Research Funds (No. 1093020506) and the New Century Excellent Talents Program (No. NCET-08-0270) from the Ministry of
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