Novel Fe³⁺‐Based ¹H MRI β‐Galactosidase Reporter Molecules

Abstract

There is increasing interest in the development of reporter agents to reveal enzyme activity in vivo using small animal imaging. We have previously demonstrated the feasibility of detecting lacZ gene activity using the commercially available 3,4-cyclohexenoesculetin-β-D-galactopyranoside (S-Gal™) as a 1H MRI reporter. Specifically, β-galactosidase (β-gal) releases the aglycone, which forms an MR contrast-inducing paramagnetic precipitate in the presence of Fe3+. Contrast was primarily T2-weighted signal loss, but T1 effects were also observed. Since T1-contrast generally provides signal enhancement as opposed to loss, it appeared attractive to explore whether analogues could be generated with enhanced characteristics. We now report the design and successful synthesis of novel analogues together with characterization of 1H MRI contrast based on both T1 and T2 response to β-gal activity in vitro for the lead agent.

6-Trifluoromethylpyridoxine: Novel 19F NMR pH Indicator for in Vivo Detection

“…[1a,b] Previously, we successfully enhanced the solubility of 19 F-NMR and 1 H-MRI β-galactosidase reporters by conjugating them with carbohydrates. [16] Prompted by these results, we designed another novel molecule 17 with two D -glucoses coupled to the 4- and 5-methylenehydroxyl moieties of 6-trifluoromethyl pyridoxine 8 (Figure 3). …”

Novel S-Gal® analogs as 1H MRI reporters for in vivo detection of β-galactosidase

“…This can be attributed to the need for two reactions on the di-galactopyranoside before liberating the aglycone to form the iron chelate. The mono and di-galactopyranosides display a variety of hydrolytic rate constants all of which lead to reaction completion times that are greater than the 30 min incubation time used here (even after accounting for the higher enzyme amount used in [26]). Thus, differences between various analogs may reflect differences in product generation at the 30 min time point apart from the relaxivities of the chelation complexes.…”

“…Monogalactopryanosides 7- O -(β- d -galactopyranosyl)-8-hydroxy-3, 4-cyclohexenocoumarin (C1-MGD), 7- O -(β- d -galactopyranosyl)-8-hydroxy-4-methylcoumarin (C2-MGD), 7- O -(β- d -galactopyranosyl)-6-hydroxy-4-methylcoumarin (C3-MGD), 7- O -(β- d -galactopyranosyl)-8-hydroxy-6-methoxycoumarin (C4-MGD) and digalactopryanosides 7,8-di- O -(β- d -galactopyranosyl)-3, 4-cyclohexenocoumarin (C1-GD), 7,8-di- O -(β- d -galactopyranosyl)-4-methylcoumarin (C2-GD), 6,7-di- O -(β- d -galactopyranosyl)-4-methylcoumarin (C3-GD), 7,8-di- O -(β- d -galactopyranosyl)-6-methoxycoumarin (C4-GD) (Fig. 2) were synthesized by us as described previously [26]. All MR studies were performed on a Varian INOVA 4.7 T horizontal-bore MR system (200 MHz for 1 H) equipped with actively shielded gradients.…”

“…In a recent study, we demonstrated the ability to detect β-gal activity in lacZ -transfected breast cancer cells (MCF7) in vitro and in vivo using the commercially available S-Gal ® by 1 H MRI [25]. We also demonstrated the synthesis of several S-Gal ® analogs for improved response to β-gal activity [26]. In both cases, β-gal cleaves the substrate molecule at the C-O bond between β- d -galactopyranoside and aglycone, to release the aglycone, which forms a paramagnetic chelate in the presence of Fe 3+ generating T1/T2/T 2 * -weighted 1 H MRI contrast.…”

New frontiers and developing applications in 19F NMR

“…Noting that 1 H MRI provides much greater signal, we recently demonstrated detection of β-gal using T 2 *-weighted MRI of S-Gal ® [319], as well as several analogs providing enhanced T 1 and T 2 * contrast [320]. A potential problem with proton MRI is differentiating enzyme/reporter indicator contrast from the natural heterogeneity of tumors.…”