Boronate affinity chromatography (BAC) is a unique means for selective separation and enrichment of cis-diol-containing compounds. Cis-diol-containing biomolecules are an important class of compounds, including glycoproteins, glycopeptides, ribonucleosides, ribonucleotides, saccharides, and catecholamines. These biomolecules play essential roles in many life-related processes. Because cis-diol-containing biomolecules are important target molecules in current research frontiers such as proteomics, metabolomics, and glycomics, BAC and boronate affinity materials have gained rapid development and found increasing applications in recent decades.

BAC is a unique mode of affinity chromatography, in which a boronic acid is used as the ligand for the selective isolation and enrichment of cis diol-containing compounds. The retention mechanism mainly relies on the pH-controlled reversible covalent interactions between cis-diol groups and the boronic acid ligand. As compared to other affinity chromatographic techniques, BAC exhibits several significant features, including broad-spectrum selectivity, reversible covalent binding, pH-controlled binding/release, and fast association/desorption kinetics. Owing to these merits, BAC is of great value in a variety of fields such as affinity separation, proteomic analysis, and metabolomics analysis.

 

HISTORICAL DEVELOPMENT

The history of BAC can be simply divided into three different periods: early development period before 1970, approach-forming period 1970–2005, and new development period since 2006.

 

PRINCIPLE AND BINDING PH

BAC principle relies on the reversible covalent reaction between cis-diol-containing compounds and boronic acid ligands. Figure 1 shows a general formula for the interaction between boronic acid and a cis diol-containing compound. When the surrounding pH is greater than the pKa value of the boronic acid, hydrolysis of the boronic acid occurs, resulting in a hybridization status change from trigonal coplanar shape to tetragonal boronate anion (from sp2 to sp3). The obtained tetragonal boronate anion can react with cis-diols and form five or six-membered cyclic esters. When the pH of the surrounding solution is switched to acidic, the boronic acid-cis-diol complex dissociates, because the binding strength between boronic acids in trigonal form and cis diol-containing compounds is very weak. Owing to the pH-controlled reversible covalent reaction, elution of captured analytes in BAC is very simple, just needing an acidic solution as the eluting buffer. Alternatively, the release of the captured analytes by the boronic acid ligands can be realized through adding excessive amounts of competing for cis-diol-containing molecules such as sorbitol into the loading buffer.

 

BORONATE AFFINITY CHROMATOGRAPHY

Figure 1 Schematic diagram showing the interaction between boronic acids and cis-diol-containing compounds.

INTERACTION MECHANISM AND SELECTIVITY MANIPULATION

Selectivity is an essential concern in BAC. It is relatively easy to obtain good selectivity for small cis-diol-containing molecules. However, it is often a challenging task for macromolecules, particularly glycoproteins. To reach a pure BAC separation, a sound understanding of the interaction mechanism is indispensable. In addition to boronate affinity interaction, four secondary interactions, including hydrophobic, ionic, hydrogen bonding, and coordination interactions, can occur in BAC. Under certain conditions, secondary interactions may result in significantly undesirable secondary retention. For example, unprotonated amines and carboxyl groups can serve as electron donors and thus can coordinate with boronic acids, which may reduce selectivity. A set of strategies for selectivity manipulation in BAC.  These strategies can be classified into two categories: choosing or designing appropriate stationary phases and choosing appropriate binding buffer composition. The strategies for manipulating the selectivity and related information are illustrated in Figure 2.

Figure 2 Selectivity manipulation and factor-affecting performance of BAC. Green arrows mean favorable interaction while cyan arrows mean unfavorable interactions. A red-up arrow means that the interaction can be enhanced by the factors specified, while blue arrows mean that the interactions can be suppressed by the specified factors.

APPLICATIONS

Although BAC appeared as early as 1970, BAC had not found wide applications until recently. The most important application before 2006 was the selective isolation of glycated hemoglobin for the clinical diagnosis of diabetes mellitus. Several fundamental issues, including selectivity, binding pH, and binding affinity, have been well solved with the rapid and deep development of BAC in recent decades. Thus, BAC has found more and more important applications. So far, the applications can be classified into four major aspects. 1. Selective enrichment of cis-diol-containing small molecules; 2. Selective enrichment of glycoproteins; 3. Specific detection of glycoprotein disease biomarkers; 4. Selective enrichment of digested glycopeptides.

Biotime’s opposition in Boronate affinity chromatography

Profound product Affinity A1c Analyzer has been recently introduced by Xiamen Biotime Biotechnology Co., Ltd., which is a detection system based on reflective colorimetric analysis technology. It is used with glycosylated hemoglobin reagent (hereinafter referred to as reagent), by measuring the concentration of the marker corresponding to the reagent, combined with medicine. The reference value gives quantitative results, which have the characteristics of accurate detection, fast detection speed, portable use, and so on. Affinity A1c analyzer is mainly composed of the host.

CONCLUSION AND FUTURE PROSPECTS

This article gives a brief introduction to BAC. Here we reviewed the basic mechanism of separation and selectivity employed in BAC. The binding pH, selectivity, and specificity of BAC are reviewed in detail. A comprehensive understanding of the interaction mechanism is helpful for better utilization of BAC and boronate affinity materials. As to future development, we believe the combination of boronate ligands with structural features will be an important direction. Boronate affinity molecular imprinting is an example in this direction. We foresee that BAC and boronate affinity materials will find more and more important applications in the future.

 

REFERENCES

1. Q. Li, C. Lu, H. Li, Y. Liu, H. Wang, X. Wang, Z. Liu, ‘Preparation of Organic-Silica Hybrid Boronate Affinity Monolithic Column for the Specific Capture and Separation of Cis-Diol Containing Compounds’, J. Chromatogr. A, 1256, 114–120 (2012).

2. R.J. Carvalho, J. Woo, M.R. Aires-Barros, S.M. Cramer, A.M. Azevedo, ‘Phenyl boronate Chromatography Selectively Separates Glycoproteins through the Manipulation of Electrostatic, Charge Transfer, and Cis-diol Interactions’, Biotechnol. J., 9, 1250–1258 (2014). 3. Z. Bie, Y. Chen, H. Li, R. Wu, Z. Liu, ‘Off-line Hyphenation of Boronate Affinity Monolith-based Extraction with Matrix-assisted Laser Desorption/ionization Time-of-flight Mass Spectrometry for Efficient Analysis of Glycoproteins/Glycopeptides’, Anal. Chim. Acta, 834, 1–8 (2014).

4. Q. Zhang, N. Tang, J.W. Brock, H.M. Mottaz, J.M. Ames, J.W. Baynes, R.D. Smith, T.O. Metz, ‘Enrichment and Analysis of Nonenzymatically Glycated Peptides: Boronate Affinity Chromatography Coupled with electron-transfer Dissociation Mass Spectrometry’, J. Proteome Res., 6, 2323–2330 (2007).

5. H. Li, Y. Liu, J. Liu, Z. Liu, ‘A Wulff-type Boronate for Boronate Affinity Capture of Cis-diol Compounds at Medium Acidic pH Condition’, Chem. Commun., 47, 8169–8171 (2011).

6. Y. Jiang, Y. Ma, ‘A Fast Capillary Electrophoresis Method for Separation and Quantification of Modified Nucleosides in Urinary Samples’, Anal. Chem., 81, 6474–6480 (2009).

7. X.C. Liu, ‘Boronic acids as Ligands for Affinity Chromatography’, Chin. J. Chromatogr., 24, 73–80 (2006).

8. H. Li, Z. Liu, ‘Recent Advances in Monolithic Columnbased Boronate-affinity Chromatography’, Trac-Trend. Anal. Chem., 37, 148–161 (2012).

9. E. Bisse, H.Wieland, ‘Coupling of m-Amino phenyl boronic Acid to S-Triazine-Activated Sephacryl-Use in the Affinity-Chromatography of Glycated Hemoglobins’, J. Chromatogr. B, 575, 223–228 (1992).

10. D.C. Klenk, G.T. Hermanson, R.I. Krohn, E.K. Fujimoto, A.K. Mallia, P.K. Smith, J.D. England, H.M. Wiedmeyer, R.R. Little, D.E. Goldstein, ‘Determination of Glycosylated Hemoglobin by Affinity Chromatography: Comparison with Colorimetric and Ion-exchange Methods, and Effects of Common Interferences’, Clin. Chem., 28, 2088–2094 (1982).