Development of novel artificial proteases and phosphatases

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Macrocyclic poly amines are a new class of multifunctional molecules (e.g., complexing agents for cations, anions and neutral molecules). Recently, a new insight into zinc enzyme chemistry has been achieved by studies of mononuclear and multinuclear zinc(II) complexes with macrocyclic polyamines. These complexes serve as both the structural and functional models for the active centers of zinc(II)-containing phosphatases (e.g., alkaline phosphatase, and protein serine/threonine phosphatase-1) and proteases (e.g., carboxypepetidase). Those study have given answers to some basic questions that surround the intrinsic properties of metal ions in those enzymes. On the bases of the finding from the zinc(II)-enzyme models, we synthesized novel artificial proteases and phosphatases (FW < 1000) and then characterized the compounds. In the course of an attempt to synthesize a new multinuclear zinc(II) complex for a phosphatase model, we incidentially isolated a new hexaaza cryptand dizinc(II) complex. The dinuclear zinc(II) complex (Phos-tag^<TM>) forms an extremely stable 1:1 phosphate complex in aqueous solution at physiological pH. We found that Phos-tag^<TM> recognizes phosphate monoester dianion (dissociation constat K_d = ca. 10^<-8> M) over 10^4 times selectively against carboxylate anions, which should facilitate further development of novel analytical methods for phospho-proteomics. As the first practical examples; we demonstrate the MADI-TOF mass spectrometry of phosphoryl-peptides by using Phos-tag^<TM> and a neutral matrix (e.g., THAP) at physiological pH. The single zinc-isotope Phos-tag^<TM> molecules (^<64>Zn and ^<68>Zn) enable the improvement of sensitivity and the analysis of phosphorylated residue.