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Synthesis and characterization of BODIPY-alpha-tocopherol: a fluorescent form of vitamin E.

ABSTRACT: Fluorescent nitrobenzoxadiazole analogues of alpha-tocopherol (NBD-alpha-Tocs; lambda(ex) = 468 nm, lambda(em) = 527 nm) have been made previously to aid study of the intracellular location and transfer of vitamin E. However, these analogues are susceptible to photobleaching while under illumination for confocal microscopy as well as in in vitro FRET transfer assays. Here we report the synthesis of three fluorescent analogues of alpha-tocopherol incorporating the more robust dipyrrometheneboron difluoride (BODIPY) fluorophore. A BODIPY-linked chromanol should have no intervening polar functional groups that might interfere with binding to the hydrophobic binding site of the tocopherol transfer protein (alpha-TTP). A key step in bringing the two ring systems together was a metathesis reaction of vinyl chromanol and an alkenyl BODIPY. An o-tolyl containing second generation Grubbs catalyst was identified as the best catalyst for effecting the metathesis without detectable alkene isomerization, which when it occurred produced a mixture of chain lengths in the alkyl linker. C8-BODIPY-alpha-Toc 10c (lambda(ex) = 507 nm, lambda(em) = 511 nm, epsilon(507) = 83,000 M(-1) cm(-1)) having an eight-carbon chain between the chromanol and fluorophore, had the highest affinity for alpha-TTP (K(d) = 94 +/- 3 nM) and bound specifically as it could not be displaced with cholesterol.

SUBMITTER: West R

PROVIDER: S-EPMC2891523 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Synthesis and characterization of BODIPY-alpha-tocopherol: a fluorescent form of vitamin E.

West Ryan R Panagabko Candace C Atkinson Jeffrey J

The Journal of organic chemistry 20100501 9

Fluorescent nitrobenzoxadiazole analogues of alpha-tocopherol (NBD-alpha-Tocs; lambda(ex) = 468 nm, lambda(em) = 527 nm) have been made previously to aid study of the intracellular location and transfer of vitamin E. However, these analogues are susceptible to photobleaching while under illumination for confocal microscopy as well as in in vitro FRET transfer assays. Here we report the synthesis of three fluorescent analogues of alpha-tocopherol incorporating the more robust dipyrrometheneboron ...[more]

PMID: 20387845

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Project description:Background: Norbormide (NRB) is a selective rat toxicant endowed with vasoconstrictor activity confined to the rat peripheral arteries. In a recent work we used a fluorescent derivative of NRB (NRB-AF12), obtained by coupling the NBD fluorophore to the parent molecule via a linker, in order to gain information about the possible site of action of the unlabeled compound. We found that NRB-AF12 labeled intracellular organelles in both NRB-sensitive and -insensitive cells and we accordingly proposed its use as a scaffold for the development of a new class of fluorescent probes. In this study, we examined the fluorescent properties of a BODIPY FL-conjugated NRB probe (MC009) developed: (A) to verify if NRB distribution could be influenced by the attached fluorophore; (B) to improve the fluorescent performance of NRB-AF12. Methods: MC009 characteristics were investigated by confocal fluorescence microscopy, in freshly isolated rat caudal artery myocytes (FIRCAM) and in LX2 cells, representative of NRB-sensitive and insensitive cells, respectively. Main results: In both FIRCAM and LX2 cells MC009 stained endoplasmic reticulum, mitochondria, Golgi apparatus and lipid droplets, revealing the same intracellular distribution as NRB-AF12, and, at the same time, had both improved photostability and gave a more intense fluorescent signal at lower concentrations than was possible with NRB-AF12, which resulted in a better and finer visualization of intracellular structures. Furthermore, MC009 was effective in cellular labeling in both living and fixed cells. At the concentration used to stain the cells, MC009 did not show any cytotoxic effect and did not affect the regular progression of cell cycle and division. Conclusions: This study demonstrates that the distribution of fluorescently labeled NRB is not affected by the type of fluorophore attached to the parent compound, supporting the idea that the localization of the fluorescent derivatives may reasonably reflect that of the parent compound. In addition, we observed a marked improvement in the fluorescent properties of BODIPY FL-conjugated NRB (MC009) over its NBD-derived counterpart (NRB-AF12), confirming NRB as a scaffold for the development of new, high performance, non-toxic fluorescent probes for the labeling of intracellular structures in both living and fixed cells.

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Synthesis and characterization of BODIPY-alpha-tocopherol: a fluorescent form of vitamin E.

Publications

Synthesis and characterization of BODIPY-alpha-tocopherol: a fluorescent form of vitamin E.

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