Mechanistic Investigation of β-Galactosidase-Activated MR Contrast Agents

Abstract

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.

Lanthanide Complexes with Heteroditopic Ligands as Fluorescent Zinc Sensors

“…For instance, ligands such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (H 4 DOTA) and 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (H 3 DO3A) form lanthanide complexes of exceptionally high thermodynamic and kinetic stability. [15] Furthermore, the heptadentate ligand H 3 DO3A can be easily functionalized on the secondary amine nitrogen atom, a property that has been exploited to prepare a wide range of Ln 3+ -DO3A derivatives containing biologically, [16] chemically [17] or photochemically [18] active moieties.…”

Recent advances in the development of synthetic chemical probes for glycosidase enzymes

“…Further mechanistic investigations were reported which demonstrated that varying substitutions patterns on the linker between the galactopyranose and cyclen had a profound effect on the MR image produced. 138 Structural isomers of EGadMe 32-a and the mechanism for enzyme activation in both the presence and absence of carbonate was explored. It was determined that the position of the methyl group (32-a or 32-b) had a significant impact on the mechanism of water exclusion prior to enzymatic cleavage with the stereochemistry of the methyl group appearing to play no significant role.…”

Molecular Imaging of In Vivo Gene Expression

“…This caged complex has all nine coordination sites of Gd(III) saturated, limiting access of water to the paramagnetic ion. Upon cleavage of the galactopyranose moiety from the chelate by β-gal, water access is restored resulting in an increase in relaxivity and signal enhancement on a T 1 -weighted image (Figure 3) [107, 108]. Enzymatic processing of the agent modulates the T 1, and in Xenopus laevis the detection of LacZ was shown in vivo [25].…”

“…Further modifications of this class of β-galactosidase activated probes have allowed for the determination of the mechanism that modulates this change in the coordination environment of the Gd(III) ion [108]. A series of isomers were synthesized with a methyl group substituted at the α or β position of the linker (α-EgadMe and β-EgadMe respectively).…”

“…The structure of the two isomers is dramatically different and employs two different mechanisms of activation by β-gal. With the cleavage of the galactopyranose by β-gal the hydoxyl group is sufficient to displace a coordinated carbonate ion (Figure 3) [108]. The galactopyranose in α-EgadMe is positioned over the Gd(III) ion and functions to sterically block water (Figure 3) [108].…”