Simple enrichment of thiol-containing biomolecules by using zinc(II)-cyclen-functionalized magnetic beads.

A simple and efficient method based on magnetic-bead technology has been developed for the enrichment of thiol-containing biomolecules, such as l-glutathione and cysteine-containing peptides. The thiol-binding site on the bead is a mononuclear complex of zinc(II) with 1,4,7,10-tetraazacyclododecane (cyclen); this is linked to a hydrophilic cross-linked agarose coating on a particle that has a magnetic core. All steps for the thiol-affinity separation are conducted in aqueous buffers with 0.10 mL of the magnetic beads in a 1.5 mL microtube. The entire separation protocol for thiol-containing compounds, from addition to elution, requires less than one hour per sample, provided the buffers and the zinc(II)-cyclen-functionalized magnetic beads have been prepared in advance. The thiol-affinity magnetic beads are reusable at least 15 times without a decrease in their thiol-binding ability, and they are stable for six months at room temperature.

Phos-tag-based micropipette-tip method for analysis of phosphomonoester-type impurities in synthetic oligonucleotides.

Various chromatographic techniques, combined with mass spectrometry, have been developed for the analysis of impurities in oligonucleotide drugs, but those methods have generally been less focused on possible phosphomonoester-type compounds. Here, we introduce a simple method for separating terminally phosphorylated impurities from parent oligonucleotides by using a phosphate-affinity micropipette tip (Phos-tag tip). All steps for the phosphate-affinity separation (binding, washing, and elution) are conducted in aqueous buffers at neutral pH. The entire separation protocol requires less than 30 min per sample. In practical examples, we demonstrated that phosphorylated impurities in natural-type and chemically modified oligonucleotides can be efficiently separated by the Phos-tag tip method and subsequently characterized by using ion-pairing reversed-phase liquid chromatography mass spectrometry (IP-RPLC-MS). Thus, a combination of the Phos-tag tip method and IP-RPLC-MS is useful for characterizing and identifying phosphomonoester-type impurities in oligonucleotide drugs.

A simple method for determining the ligand affinity toward a zinc-enzyme model by using a TAMRA/TAMRA interaction.

Thiolate coordination to zinc(ii) ions occurs widely in such functional biomolecules as zinc enzymes or zinc finger proteins. Here, we introduce a simple method for determining the affinity of ligands toward the zinc-enzyme active-center model tetramethylrhodamine (TAMRA)-labeled 1,4,7,10-tetraazacyclododecane (cyclen)-zinc(ii) complex (TAMRA-ZnL). The 1 : 1 complexation of TAMRA-labeled cysteine (TAMRA-Cys) with TAMRA-ZnL (each at 2.5 µM), in which the TAMRA moieties approach one another closely, induces remarkable changes in the visible absorption and fluorescence spectra at pH 7.4 and 25 °C. The 1 : 1 complex formation constant (K = [thiolate-bound zinc(ii) complex]/[uncomplexed TAMRA-ZnL][uncomplexed TAMRA-Cys], M-1) was determined to be 106.7 M-1 from a Job's plot of the absorbances at 552 nm. By a ligand-competition method with the 1 : 1 complexation equilibrium, analogous K values for thiol-containing ligands, such as N-acetyl-l-cysteine, l-glutathione, and N-acetyl-l-cysteinamide, were evaluated to have similar values of about 104 M-1. As a result of the ligand affinities to TAMRA-ZnL, nonlabeled zinc(ii)-cyclen induced remarkable stabilization of the reduced form of l-glutathione and a cysteine-containing enolase peptide to aerial oxidation in aqueous solution at pH 7.4 and 25 °C.

A Phos-tag-based magnetic-bead method for rapid and selective separation of phosphorylated biomolecules.

A simple and efficient method based on magnetic-bead technology has been developed for the separation of phosphorylated and nonphosphorylated low-molecular-weight biomolecules, such as nucleotides, phosphorylated amino acids, or phosphopeptides. The phosphate-binding site on the bead is an alkoxide-bridged dinuclear zinc(II) complex with 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag), which is linked to a hydrophilic cross-linked agarose coating on a magnetic core particle. All steps for the phosphate-affinity separation are conducted in buffers of neutral pH with 50 µL of the magnetic beads in a 1.5-mL microtube. The entire separation protocol for phosphomonoester-type compounds, from addition to elution, requires less than 12 min per sample if the buffers and the zinc(II)-bound Phos-tag magnetic beads have been prepared in advance. The phosphate-affinity magnetic beads are reusable at least 15 times without a decrease in their phosphate-binding ability and they are stable for three months in propan-2-ol.