A novel thiol-affinity micropipette tip method using zinc(II)–cyclen-attached agarose beads for enrichment of cysteine-containing molecules

• A novel thiol-affinity micropipette tip method was developed.
• Thiol compounds captured by the thiol-affinity micropipette tip are stable under air.
• The separation can be conducted by using aqueous solutions at room temperature.
• The operation time from sample addition to elution is less than 15 min.
• The thiol-affinity agarose beads are stable for multiple use and long-term storage.

Cysteine-containing biomolecules are attractive targets in the study of thiol biology. Here we introduce a novel method for the selective enrichment of thiol-containing molecules using a thiol-capture zinc(II) complex of 1,4,7,10-tetraazacyclododecane (Zn2+–cyclen). Recognition of N-acetylcysteine amide by Zn2+–cyclen has been studied by potentiometric pH titration, revealing formation of a 1:1 thiolate-bound Zn2+–cyclen complex with a large thiolate-affinity constant of 106.2 M−1 at 25 °C and I = 0.10 M (NaCl). The Zn2+-bound thiolate anion is unexpectedly stable in aqueous solution at pH 7.8 under atmospheric conditions for a few days. These findings have contributed to the development of a convenient method for separation of thiol compounds by using a micropipette tip. A 200 μL micropipette tip containing 10 μL of hydrophilic cross-linked agarose beads attached to Zn2+–cyclen moieties was prepared. All steps for thiol-affinity separation (binding, washing, and eluting) are conducted using aqueous buffers at room temperature. The entire separation protocol requires less than 15 min per sample. We demonstrate practical example separations of cysteine-containing molecules. This micropipette tip method would be used preferentially as an alternative to existing tools for reliable enrichment of thiol-containing molecules.