Synthesis and characterization of novel lacZ gene reporter molecules

Detection of β-galactosidase activity by 19F nuclear magnetic resonance of polyglycosylated fluorinated vitamin B6

Jianxin Yu, Ralph P. Mason

Research output: Contribution to journalArticle

24 Citations (Scopus)

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 β-galactosidase (β-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,α45-tri-O-(β- D-galactopyranosyl)-6-fluoropyridoxol 4, 3-O-(β-D-galactopyranosyl)- α45-di-O-(β-D-glucopyranosyl) -6-fluoropyridoxol 12, and 3-O-(β-D-galactopyranosyl)-α4, α5-di-O-(α-D-mannopyranosyl)-6-fluoropyridoxol 13. Compounds 4, 12, and 13 all show promising characteristics including highly sensitive 19F NMR response to β-gal activity (Δδ = 9.0 ∼ 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.

Original languageEnglish (US)
Pages (from-to)1991-1999
Number of pages9
JournalJournal of Medicinal Chemistry
Volume49
Issue number6
DOIs
StatePublished - Mar 23 2006

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Galactosidases
Vitamin B 6
Lac Operon
Gene expression
Magnetic Resonance Spectroscopy
Genes
Nuclear magnetic resonance
Gene Expression
Molecules
Glycosylation
Gene therapy
Poisons
Chemical shift
Galactose
Structural analysis
Genetic Therapy
Solubility
Transfection
Toxicity
Assays

ASJC Scopus subject areas

  • Organic Chemistry

Cite this

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title = "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 β-galactosidase (β-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,α4,α5-tri-O-(β- D-galactopyranosyl)-6-fluoropyridoxol 4, 3-O-(β-D-galactopyranosyl)- α4,α5-di-O-(β-D-glucopyranosyl) -6-fluoropyridoxol 12, and 3-O-(β-D-galactopyranosyl)-α4, α5-di-O-(α-D-mannopyranosyl)-6-fluoropyridoxol 13. Compounds 4, 12, and 13 all show promising characteristics including highly sensitive 19F NMR response to β-gal activity (Δδ = 9.0 ∼ 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.",
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AU - Yu, Jianxin

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AB - 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 β-galactosidase (β-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,α4,α5-tri-O-(β- D-galactopyranosyl)-6-fluoropyridoxol 4, 3-O-(β-D-galactopyranosyl)- α4,α5-di-O-(β-D-glucopyranosyl) -6-fluoropyridoxol 12, and 3-O-(β-D-galactopyranosyl)-α4, α5-di-O-(α-D-mannopyranosyl)-6-fluoropyridoxol 13. Compounds 4, 12, and 13 all show promising characteristics including highly sensitive 19F NMR response to β-gal activity (Δδ = 9.0 ∼ 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.

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