Yunchun Liu

Ring-Opening Polymerization with Synergistic Co-monomers: Access to a Boronate-Functionalized Polymeric Monolith for the Specific Capture of cis-Diol-Containing Biomolecules under Neutral Conditions†

Polymeric monoliths, which show fast convective mass transfer between the monolith bed and the surrounding solution, have already become important materials in separation science and (bio-)catalysis. Although some purely polymeric monoliths (such as acrylate-based monoliths) are excellent separation media, functionalization is of utmost importance in most situations. Conventional functionalization can be classified into two strategies: 1) copolymerization of functional monomers and 2) postpolymerization functionalization. In both cases, the designed functionalities depend on the structure and properties of functional monomers. To obtain a certain functionality, a high-purity monomer with appropriate structure and properties is indispensable. Boronate affinity chromatography (BAC) has been a useful means for the specific capture and separation of cisdiol-containing biomolecules, such as saccharides, nucleosides, and glycoproteins, since the early 1980s. The principle relies on reversible covalent complex formation/dissociation between boronic acids and cis diols in an alkaline/acidic aqueous solution. Recently, boronate-functionalized monoliths were synthesized by copolymerization and postpolymerization functionalization. However, as for other BACbased techniques, an apparent disadvantage is that the chromatography in aqueous solution has to be performed in alkaline media and can lead to the degradation of labile compounds. Thus, boronate-functionalized monoliths that function at neutral pH would be highly desirable for physiological samples. A conventional solution to this problem is to decrease the pKa value of the ligands by synthesizing novel boronic acids with exquisite structures through: 1) the introduction of an electron-withdrawing group, such as a sulfonyl group, into the ligand molecules, 2) the introduction of a neighboring amino group capable of B–N coordination into the ligand molecules (Wulff-type boronic acids), or 3) the replacement of intramolecular B–N coordination with intramolecular B–O coordination (improved Wulff-type boronic acids). According to these strategies, to make a boronate-functionalized monolith that is functional under neutral conditions, a boronic acid monomer with a low pKa value, if not commercially available, must be synthesized and purified first through tedious procedures. Herein, we present a new approach— ring-opening polymerization with synergistic co-monomers— for the preparation of a boronate-functionalized polymeric monolith that functions under neutral conditions, without the synthesis and purification of a single functional monomer. The main synthetic route is based on the ring-opening polymerization protocol established recently by Tanaka and co-workers. An epoxy resin, a diamine curing agent, and a porogenic solvent are required for the preparation of the monolith. To obviate the inconvenience of the synthesis of a new diamine monomer, we took advantage of the coordination of m-aminophenylboronic acid (mPBA, 1) with 1,6hexamethylenediamine (HMDA, 2) to form a stable complex 3 with a B N bond (Scheme 1). The B–N-coordinated complex was used as a diamine curing agent in a ring-opening polymerization reaction with the epoxy resin tris(2,3-epoxypropyl)isocyanurate (TEPIC, 4) to form a macroporous monolith. Since the conformation of the coordinated complex is “frozen” by the polymerization, the coordinated complex

Synthesis of hydrophilic boronate affinity monolithic capillary for specific capture of glycoproteins by capillary liquid chromatography.

Boronate affinity chromatography is an important tool for specific isolation of cis-diol-containing compounds such as glycoproteins, RNA and carbohydrates. Boronate functionalized monolithic capillaries have been recently developed for specific capture of cis-diol-containing small biomolecules, but the apparent hydrophobicity of the columns prevents them from specific capture of glycoproteins. In this paper, a hydrophilic boronate affinity monolithic capillary was prepared by in situ free radical polymerization, using 4-vinylphenylboronic acid (VPBA) and N, N-methylenebisacrylamide (MBAA) as functional monomer and cross-linker, respectively. The prepared poly(VPBA-co-MBAA) monolithic capillary exhibited uniform open channel network and high density of accessible boronic acid. Due to the utilization of hydrophilic cross-linker, the prepared column was hydrophilic, allowing for specific capture of glycoproteins.

Restricted access boronate affinity porous monolith as a protein A mimetic for the specific capture of immunoglobulin G

Antibodies are molecular workhorses in biological research, disease treatment and diagnostics. Purity is a critical prerequisite for antibody applications. Although protein A-based affinity chromatography has developed into the gold standard for antibody purification, protein A is associated with several apparent disadvantages, including high cost, poor stability and harsh product release conditions. Many attempts have been made towards molecular level biomimetics of protein A. However, practical substitutes have not yet been achieved. Here we present a novel functionalized material, called restricted access boronate affinity porous monolith, as a mimic of protein A for the specific capture of antibodies. This biomimetic relies on a novel strategy that combines the steric hindrance of the porous monolith with the chemical selectivity of boronic acid. This protein A biomimetic material demonstrated high specificity for antibodies. Meanwhile, original immunoaffinity and specificity of the captured antibodies were maintained. Compared with protein A, the monolithic biomimetic exhibited several significant advantages, including low cost, high stability and fast elution kinetics.

Preparation of organic-silica hybrid boronate affinity monolithic column for the specific capture and separation of cis-diol containing compounds

A new boronate-silica hybrid monolithic column was prepared using a one-pot approach with 3-acrylamidophenylboronic acid (AAPBA) as the boronate affinity ligand. The AAPBA-silica monolith exhibited several attractive advantages. First, it is highly hydrophilic, providing excellent specificity and avoiding the presence of organic solvent in the mobile phase. Second, due to its large surface area, it exhibited a high binding capacity, 49.5 μmol/mL, the highest among the boronate affinity monolithic columns appeared in the literature. Third, the monolith can bind with cis-diol containing compounds at pH as low as 6.5, which not only avoids the use of basic pH conditions at which the silica monolith may hydrolysis but also facilitates the applications to wider sample range. Finally, the hybrid monolithic column exhibited apparent secondary separation capability, which allows for two-dimensional (2D) separation of cis-diol compounds in a single column. Due to these merits, the AAPBA-silica hybrid monolithic column can be a promising separation medium for the analysis of cis-diol containing compounds.

On-line coupling of in-tube boronate affinity solid phase microextraction with high performance liquid chromatography-electrospray ionization tandem mass spectrometry for the determination of cis-diol biomolecules.

Boronate affinity solid phase microextraction (BA-SPME) is a new format appeared recently with great potential for specific extraction of cis-diol-containing compounds. Unlike conventional SPME, BA-SPME relies on covalent interactions and thereby features with specific selectivity, eliminated matrix effect and manipulable capture/release. However, only on-fiber BA-SPME and its off-line combination with high performance liquid chromatography (HPLC) have been reported so far. In this study, we report on-line coupling of in-tube BA-SPME with HPLC-electrospray ionization tandem mass spectroscopy (in-tube BA-SPME-HPLC-ESI-MS/MS) for the specific and sensitive determination of cis-diol-containing biomolecules. A boronate affinity extraction phase was prepared onto the inner surface of the capillary by copolymerization of vinylphenylboronic acid (VPBA) and ethylene glycol dimethacrylate (EDMA). The extraction conditions were optimized by choosing appropriate extraction/desorption solutions and extraction time. The extraction capacity, linear range, reproducibility and life-time were investigated. The developed method was successfully applied for the determination of dopamine in urine samples. Since many cis-diol-containing compounds are of great biological importance, the in-tube BA-SPME-HPLC method can be a promising tool.

Weak anion exchange chromatographic profiling of glycoprotein isoforms on a polymer monolithic capillary

High resolution separation of intact glycoproteins, which is essential for many aspects such as finger-print profiling, represents a great challenge because one glycoprotein can exhibit many isoforms with close physicochemical properties. Monolithic columns are important separation media for the separation of intact proteins due to its significant advantages such as easy preparation, high column efficiency and high permeability. However, there are few reports on high resolution profiling of intact glycoproteins. Herein, we presented a polymeric weak anion exchange (WAX) monolithic capillary for high resolution separation of glycoprotein isoforms. A base monolith was first prepared through ring-opening polymerization between tris(2,3-epoxypropyl)isocyanurate and tri(2-aminoethyl), and then modified through reacting with ammonia aqueous solution to convert the unreacted epoxide moieties into primary amino groups. The prepared monolithic capillary was characterized in terms of morphology, pore size, hydrophilicity and reproducibility. The obtained WAX monolithic capillary exhibited desired through-pores and mesopore size, stable skeleton and hydrophilic nature. The performance of the capillary was evaluated using several typical glycoproteins such as α(1)-acid glycoprotein (AGP) as mode analytes. Effects of the experimental parameters on the glycoform resolution were investigated. Under the optimized separation conditions, the tested glycoproteins were all resolved into distinct glycoforms. A comparative investigation with capillary zone electrophoresis (CZE) revealed that this WAX column provided better selectivity as more isoforms were observed, although the resolution of some glycoprotein isoforms decreased.

Development of poly((3-acrylamidophenyl)boronic acid-co-N,N-methylenebisacrylamide) monolithic capillary for the selective capture of cis-diol biomolecules

A new hydrophilic boronate affinity monolithic capillary was synthesized for the selective capture of cis-diol biomolecules including glycoproteins and nucleosides, using (3-acrylamidophenyl)boronic acid (APBA) and N,N-methylenebisacrylamide (MBAA) as the functional monomer and cross-linker, respectively. A comparison with other compositionally similar monolithic capillaries was carried out in terms of reversed-phase retention, selectivity, binding capacity and binding pH. The poly(APBA-co-MBAA) monolithic capillary exhibited notably different properties. As both the functional monomer and the cross-linker are hydrophilic, the monolith capillary showed suppressed reversed-phase retention and as a result excellent selectivity toward both nucleosides and glycoproteins was observed. It exhibited the highest binding capacity as compared with other compositionally similar monolithic capillaries. Besides, because of the lower pKa value of APBA, the monolithic capillary was able to selectively capture nucleosides at near-physiological pH.