We report a mechanistic investigation of an isomeric series of β-galactosidase-activated magnetic resonance contrast agents. Our strategy focuses on the synthesis of macrocyclic caged-complexes that coordinatively saturate a chelated lanthanide. Enzyme cleavage of the complex results in an open coordination site available for water that creates a detectable MR contrast agent. The complexes consist of a DO3A Gd(III) chelator modified with a galactopyranose at the N-10 position of the macrocycle. We observed significant differences in relaxometric properties and coordination geometry that can be correlated to subtle variations of the linker between the macrocycle and the galactopyranose. After synthesis and purification of the R, S, and racemic mixtures of complexes 1 and 3 and measurement of the hydration number, water residence lifetime, and longitudinal relaxation rates, we propose mechanisms for water exclusion from the lanthanide in the precleavage state. While the stereochemistry of the linker does not influence the agents' properties, the mechanism of water exclusion for each isomer is significantly influenced by the position of modification. Data for one series with a methyl group substituted on the sugar-macrocycle linker at the R-position suggests a steric mechanism where the galactopyranose sugar blocks water from the Gd(III) center. In contrast, our observations for a second series with methyl substitution at the β position of the sugar-macrocycle linker are consistent with a mechanism in which a bidentate anion occupies two available coordination sites of Gd(III) in the precleavage state.

The preparation of beta-EgadMe and alpha-EgadMe, magnetic resonance (MR) contrast agents that can be used for in vivo detection of beta-galactosidase, is described. Diastereomerically pure beta-EgadMe can be synthesized by kinetically resolving the starting material as described in Step 1. The total time for the preparation of the racemic mixture of beta-EgadMe is about 8 d, and the total time for an diastereomerically resolved agent is about 9 d. The final metallated agent is stable at room temperature as a solid or in aqueous buffer (pH 5.5-10) indefinitely. Diastereomerically pure alpha-EgadMe can be prepared by beginning the synthesis with enantiomerically pure bromopropionic acid. The total time for the preparation of racemic alpha-EgadMe or diastereomerically pure alpha-EgadMe is about 8 d. The final metallated agent is stable at room temperature as a solid or in aqueous buffer (pH 5.5-10) indefinitely.