Novel ¹⁹ F-MRS β-galactosidase reporter molecules incorporated nitrogen mustard analogues

In this study, we propose a novel molecular platform-integrated fluorinated antitumor nitrogen mustards for ¹⁹ F-MRS assay of β-galactosidase (β-gal) activity. Following this idea, we have designed, synthesized, and characterized 2-fluoro-4-[bis(2'-chloroethyl)amino]phenyl β-D-galactopyranoside 5, 2-fluoro-4-{bis[2'-O-(β-D-galactopyranosyl)ethyl]amino}phenyl β-D-galactopyranoside 8, 2-fluoro-4-{bis[[1″-(β-D-galactopyranosyl)-1″, 2″, 3″-triazol-4″-yl]methyl] amino}phenyl β-D-galactopyranoside 14 and 2-fluoro-4-{bis[[1″-(β-D-glucopyranosyl)-1″, 2″, 3″-triazol-4″-yl]methyl]amino}phenyl β-D-galactopyranoside 15 through glycosylation and click reaction strategies, and their structures were confirmed by NMR and HRMS or elemental analysis data. Among them, 2-fluoro-4-[bis(2'-chloroethyl)amino]phenyl β-D-galacto-pyranoside 5 was found very sensitive to β-gal (E801A) in PBS at 37°C with big Δδ_F response. Here, we demonstrated the feasibility of this platform for assessing β-gal activity in solution, and in vitro with lacZ-transfected human MCF7 breast and PC3 prostate tumor cells, by the characterization of β-gal-responsive ¹⁹ F-chemical shift changes Δδ_F and hydrolytic kinetics.