sepharose and 3-aminobenzeneboronic-acid

Gambierdiscus spp. are epi-benthic dinoflagellates that have been associated with ciguatera poisoning. These microalgae can have complex secondary metabolite profiles including ciguatoxins, maitotoxins, and gambierones, with varying compositions and toxicities across species and strains. Given this chemical diversity there is a need to develop selective and sensitive methods for secondary metabolite profiling. In this study, we used a cultured Caribbean strain of Gambierdiscus silvae to develop sample preparation and analysis strategies for characterizing vic-diol containing secondary metabolites. A pooled cellular extract was first screened by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for ciguatoxin-related compounds, which resulted in the confirmation of gambierone (1) and a novel isomer of 44-methylgambierone (3). Treatment of the extract with periodate confirmed that the gambierones each contained one reactive vic-diol, which was exploited for the development of a selective extraction procedure using m-aminophenylboronic acid gel and the non-aqueous binding solvent chloroform. Using this non-traditional boronate affinity procedure, LC-HRMS also revealed the presence of additional sulfated polycyclic ethers in the gambierone-containing vic-diol fraction, while pigments and other contaminants were removed. The developed tools could be applied to screen collections of Gambierdiscus and other benthic algae to provide additional chemical characterization of gambierone-related compounds. The selective extraction procedure may also prove useful as a step in the isolation of these sulfated polyethers for structural, toxicological and biotransformation studies.

Topics: Boronic Acids; Chromatography, Liquid; Dinoflagellida; Ethers; Mass Spectrometry; Sepharose

This Letter describes a method for enriching C-terminal peptide of protein for C-terminal sequence analysis. This method employs endopeptidase ArgC digestion and C-terminal peptide enrichment using m-aminophenylboronic acid-agarose as an arginine-capture material. The selectively recovered C-terminal peptide incorporates no artificial derivatization. Therefore, the widely used functional groups (e.g. α-NH(2) and α-COOH) can be used for any necessary transformation. In this research, a TMPP mass tag was attached to the α-NH(2) group to clarify the amino acid sequence of the C-terminal peptide.

Topics: Aldehyde Oxidoreductases; Amino Acid Sequence; Bacteria; Bacterial Proteins; Boronic Acids; Peptides; Protein C; Protein Structure, Tertiary; Sepharose; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI TOFMS) combined with affinity chromatography on immobilized phenylboronic acid agarose gels was used for selective enrichment and detection of specifically modified proteins such as glycated proteins in complex biological samples. Physicochemical grafting of hydrophilic polymers on aluminum surface was developed to reduce nonspecific protein sorption and to create a proper support layer for a three-dimensional affinity hydrogel. Grafted agarose allowed the fixation of three-dimensional agarose hydrogel on the chip surface. Both pinched polymers and hydrogels were effectively derivatized. 3-Aminophenylboronic acid (mPBA) was covalently immobilized as an affinity ligand to achieve specific binding of glycated plasma proteins. Alternatively, the affinity sorbent was immersed into the hydrogel to increase binding capacity. MALDI TOFMS was used to evaluate binding efficiency and molecular mass changes of human serum albumin due to glycation. Glycated proteins were captured directly on the chip with high selectivity and efficacy, and low nonspecific binding. Thus they could easily be characterized by MALDI TOFMS.

Topics: Aluminum; Boronic Acids; Chromatography, Affinity; Ethanolamines; Fluoresceins; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Hydrogels; Polyvinyl Alcohol; Proteins; Sepharose; Serum Albumin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Cross-linked agarose particles (Sepharose CL-6B) and baker's yeast cells were found to adhere to siliceous supports end-grafted with boronate-containing copolymers (BCCs) of N,N-dimethylacrylamide at pH> or =7.5, due to boronate interactions with surface carbohydrates of the particles and the cells. These interactions were registered both on macroscopic and on molecular levels: the BCCs spontaneously adsorbed on the agarose gel at pH> or =7.5, with adsorption increasing with pH. Agarose particles and yeast cells stained with Procion Red HE-3B formed stable, monolayer-like structures at pH 8.0, whereas at pH 7.0-7.8 the structures on the copolymer-grafted supports were less stable and more random. At pH 9.0, 50 % saturation of the surface with adhering cells was attained in 2 min. Stained cells formed denser and more stable layers on the copolymer-grafted supports than they did on supports modified with self-assembled organosilane layers derivatized with low-molecular-weight boronate, presumably due to a higher reactivity of the grafted BCCs. Quantitative detachment of adhered particles and cells could be achieved by addition of 20 mM fructose--a strong competitor for binding to boronates--at pH 7.0-9.0. Regeneration of the grafted supports allowed several sequential adhesion and detachment cycles with stained yeast cells. Affinity adhesion of micron-sized carbohydrate particles to boronate-containing polymer brushes fixed on solid supports is discussed as a possible model system suggesting a new approach to isolation and separation of living cells.

Topics: Acrylamides; Boronic Acids; Carbohydrates; Cell Adhesion; Glass; Hydrogen-Ion Concentration; Osmolar Concentration; Polymers; Saccharomyces cerevisiae; Sepharose; Silanes; Surface Properties

We examined capacity related properties of "Glyco-Gel" (Pierce), a boronate agarose gel for separating and measuring glycated proteins by affinity chromatography. Our data indicate linear capacity to as much as 20 mg as applied hemoglobin or almost 10 mg as bound hemoglobin and 26 mg as applied serum proteins or a minimum of 2.5 mg as bound serum protein for each mL of gel. The capacity and affinity of the support for glycated proteins becomes optimum only after four regeneration cycles. The support matrix appears to have a small concentration of nonspecific binding sites equivalent to 0.09 to 0.18 mg as serum protein for each mL of gel. These sites do not bind hemoglobin. They lead to an overestimation of glycated protein that can cause large errors when the proportion of glycated protein is determined with small column loads. If near capacity loads are applied, the samples must be dialyzed or diluted to avoid decreased analytical recovery resulting from competitive and eluting properties of endogenous sugars.

Topics: Boronic Acids; Chromatography, Affinity; Proteins; Sepharose

This paper (which is part of a series of articles on high-performance liquid chromatographic separations on agarose columns) describes the chromatographic behaviour of catecholamines, nucleosides, aminoacylated and non-aminoacylated tRNA, and glycosylated and non-glycosylated hemoglobins on agarose matrices derivatized with m-aminophenylboronic acid. These matrices primarily bind molecules with two vicinal hydroxy groups in the cis-configuration. The possible existence of secondary electrostatic and hydrophobic interactions has been studied with the aid of diagrams showing retention time as a function of pH and buffer concentration.

Topics: Boronic Acids; Catecholamines; Chromatography, High Pressure Liquid; Glycated Hemoglobin; Humans; Nucleosides; RNA, Transfer; RNA, Transfer, Amino Acyl; Sepharose

The use of m-aminophenylboronic acid immobilized on agarose as the affinity matrix for the separation and quantitation of Glyco Hb has been investigated. The glyco fraction isolated from the affinity columns contained about 10% Hb A1a + b, 52% Hb A1c, and 38% Hb A0-like glyco components. The nonglyco fraction had major portions of Hb A1a + b and Hb A0 and a small amount of Hb A1c (also contained Hb F). In normals and diabetic patients, the comparison of the affinity method with the ion-exchange chromatographic, fluorometric, and colorimetric methods showed good correlation. The affinity method also showed acceptable precision and reproducibility. The presence of labile aldimine did not influence the affinity method because it seems that only the stable ketoamine was bound to the affinity gel and thus separated by this method. Moreover, this method was less sensitive to variations in column temperature and sample age. Glyco Hb levels were determined in newborn infants and in adults with various hemoglobinopathies. The results indicated that the presence of Hb F, Hb S, and Hb C did not affect the glyco Hb determinations. Thus the affinity chromatographic approach has wider application than ion-exchange chromatography and is, at the same time, much simpler and faster than the chemical methods.

Topics: Adult; Boronic Acids; Chromatography, Affinity; Chromatography, Ion Exchange; Colorimetry; Diabetes Mellitus; Female; Fluorometry; Glycated Hemoglobin; Hemoglobinopathies; Humans; Imines; Infant, Newborn; Male; Sepharose

The synthesis of 3-nitro-4-(6-aminohexylamido)phenylboronic acid is described. The properties of two novel forms of immobilized phenylboronate agarose adsorbents [m-aminophenylboronic acid-Matrex Gel and 3-nitro-4-(6-aminohexylamido)phenylboronic acid-Sepharose CL-6B] were investigated. Both gels bind and selectively retard the glycoprotein alpha-glucosidase from yeast. The retardation is affected by following parameters: (i) pH, (ii) presence of sugar, (iii) concentration of sugar and (iv) buffer species (especially triethanolamine). Five sugars were studied, namely sorbitol, fructose, ribose, glucose and maltose. The concentration of sugar required to produce significant retardation increased in the above order, whereas the ability of sugar to form a complex with boron decreases in the same order. These effects were observed with crude as well as pure enzyme. Since alpha-glucosidase is a glycoprotein, it is proposed that this protein is mainly bound to these immobilized phenylboronates via sugar (glyco) residues. Displacement of the enzyme from the column is effected by the sugar in the buffer (or in a preincubation mixture). However, the marked pH-dependence (this retardation effect could only be observed at pH 7.4) suggests that these results are not due solely to hydrophobic or ionic mechanisms and are more complex than simple sugar-phenylboronic acid interactions.

Topics: alpha-Glucosidases; Boronic Acids; Chemical Phenomena; Chemistry; Chromatography, Affinity; Glucosidases; Ligands; Maltose; Monosaccharides; Saccharomyces cerevisiae; Sepharose; Sorbitol