Synthesis of γ-monodentate and β,γ-bidentate CrITP. Isolation and characterization of the two chelation isomers and the individual diastereomers

Abstract

The two chelation isomers of CrITP, γ-monodentate and β,γ-bidentate CrITP, as well as the diastereomers of β,γ-bidentate CrITP were synthesized, isolated, and characterized. Synthesis of these complexes was done using pH titration methods similar to that described by Cleland [W.W. Cleland, Methods Emzymol. 87, 159 (1982)], and separation of the two chelation isomers was accomplished with DEAE-sephadex A-25 using 0–0.3 N linear HC1 gradient. Diastereomer separation (analytical and preperative scales) of β,γ-bidentate CrITP using reverse-phase high-performance liquid chromatography, and then analysis of the diastereomers with circular dichroism spectroscopy, shows four diastereomers that exist as two pairs of mirror-image isomers, similar to the four diastereomers of β,γ-bidentate CrATP as presented by Dunaway-Mariano and Cleland [D. Dunaway-Mariano and W. W. Cleland, Biochemistry 19, 1496 (1980)]. Reverse-phase high-performance liquid chromatography analysis of γ-monodentate CrITP shows the presence of two major peaks, both of which convert to β, γ-bidentate CrITP upon incubation at pH 6.0 for 1 hr.