Synthesis and Characterization of Novel lacZ Gene Reporter Molecules: Detection of β-Galactosidase Activity by 19F Nuclear Magnetic Resonance of Polyglycosylated Fluorinated Vitamin B6

Abstract

Gene therapy has emerged as a promising strategy for treatment of various diseases. However, widespread implementation is hampered by difficulties in assessing the success of transfection, in particular, the spatial extent of expression in the target tissue and the longevity of expression. Thus, the development of noninvasive reporter techniques based on appropriate molecules and imaging modalities may help to assay gene expression. We have previously demonstrated the ability to detect beta-galactosidase (beta-gal) activity on the basis of 19F NMR chemical shift associated with release of fluorophenyl aglycons from galactopyranoside conjugates. Use of fluoropyridoxol as the aglycon provides a potential less toxic alternative and we now report the design, synthesis, and structural analysis of a series of novel polyglycosylated fluorinated vitamin B6 derivatives as 19F NMR-sensitive aglycons for detection of lacZ gene expression. In particular, we report the activity of 3,alpha4,alpha5-tri-O-(beta-D-galactopyranosyl)-6-fluoropyridoxol 4, 3-O-(beta-D-galactopyranosyl)-alpha4,alpha5-di-O-(beta-D-glucopyranosyl)-6-fluoropyridoxol 12, and 3-O-(beta-D-galactopyranosyl)-alpha4,alpha5-di-O-(alpha-D-mannopyranosyl)-6-fluoropyridoxol 13. Compounds 4, 12, and 13 all show promising characteristics including highly sensitive 19F NMR response to beta-gal activity (Deltadelta=9.0 approximately 9.4 ppm), minimal toxicity for substrate or aglycon, and good water solubility. However, the differential glycosylation of 12 and 13 appears more advantageous for assessing lacZ gene expression in vivo.