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Synthesis of a highly Mg2+-selective fluorescent probe and its application to quantifying and imaging total intracellular magnesium

Nature Protocols volume 12, pages 461–471 (2017)Cite this article

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Abstract

Magnesium plays a crucial role in many physiological functions and pathological states. Therefore, the evolution of specific and sensitive tools capable of detecting and quantifying this element in cells is a very desirable goal in biological and biomedical research. We developed a Mg2+-selective fluorescent dye that can be used to selectively detect and quantify the total magnesium pool in a number of cells that is two orders of magnitude smaller than that required by flame atomic absorption spectroscopy (F-AAS), the reference analytical method for the assessment of cellular total metal content. This protocol reports itemized steps for the synthesis of the fluorescent dye based on diaza-18-crown-6-hydroxyquinoline (DCHQ5). We also describe its application in the quantification of total intracellular magnesium in mammalian cells and the detection of this ion in vivo by confocal microscopy. The use of in vivo confocal microscopy enables the quantification of magnesium in different cellular compartments. As an example of the sensitivity of DCHQ5, we studied the involvement of Mg2+ in multidrug resistance in human colon adenocarcinoma cells sensitive (LoVo-S) and resistant (LoVo-R) to doxorubicin, and found that the concentration was higher in LoVo-R cells. The time frame for DCHQ5 synthesis is 1–2 d, whereas the use of this dye for total intracellular magnesium quantification takes 2.5 h and for ion bioimaging it takes 1–2 h.

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Figure 1: Synthetic route to DCHQ5 fluorescent dye.
Figure 2: Emission spectra (λex = 360 nm) of DCHQ5 (10 μM) in titration with Mg2+ in MeOH:MOPS upon addition of increasing MgSO4 concentrations (1, 2, 5, 10, 20, 40, 80, 160, 320 and 500 μM) reported in arbitrary units (a.u.).
Figure 3: Total magnesium quantification.
Figure 4: Magnesium concentrations in LoVo-S and LoVo-R.
Figure 5: Two-photon fluorescence microscopy image (λexc = 750 nm) of ROS rat osteosarcoma (left) and SaOS-2 human osteosarcoma (right) cells stained with 10 μM DCHQ5.

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Acknowledgements

This work was supported by the RFO (Ricerca Fondamentale Orientata) of the University of Bologna.

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Author notes

  1. Azzurra Sargenti and Giovanna Farruggia: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacy and Biotechnologies, University of Bologna, Bologna, Italy

    Azzurra Sargenti, Giovanna Farruggia, Concettina Cappadone, Emil Malucelli, Alessandra Procopio & Stefano Iotti

  2. National Institute of Biostructures and Biosystems, Rome, Italy

    Giovanna Farruggia & Stefano Iotti

  3. Department of Chemistry ′Giacomo Ciamician’, University of Bologna, Bologna, Italy

    Nelsi Zaccheroni, Massimo Sgarzi, Luca Prodi & Marco Lombardo

  4. Department of Transfusion Medicine, ASMN-IRCCS, Reggio Emilia, Italy

    Chiara Marraccini

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  1. Azzurra Sargenti
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Contributions

A.S., G.F., C.C., M.L., N.Z., E.M., A.P., C.M. and M.S. conducted the experiments; M.L., S.I., L.P. and G.F. designed the experiments; and A.S., M.L., G.F. N.Z. and S.I. wrote the paper.

Corresponding authors

Correspondence to Luca Prodi, Marco Lombardo or Stefano Iotti.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 1H NMR (400 MHz, CDCl3) of 5-chloro-8-tosyloxyquinoline (2).

Supplementary Figure 2 13C NMR (100 MHz, CDCl3) of 5-chloro-8-tosyloxyquinoline (2).

Supplementary Figure 3 MS-ESI of 5-chloro-8-tosyloxyquinoline (2).

Supplementary Figure 4 Purification of of 5-phenyl-8-tosyloxyquinoline (3).

TLC (cyclohexane:ethyl acetate 75:25) of flash-chromatography relative to the purification of the Suzuki cross-coupling reaction for the synthesis of 5-phenyl-8-tosyloxyquinoline 3; R = real purified product (3); 6-16 = eluted fractions. UV lamp, 254 nm.

Supplementary Figure 5 1H NMR (400 MHz, CDCl3) of 5-phenyl-8-tosyloxyquinoline (3).

Supplementary Figure 6 13C NMR (100 MHz, CDCl3) of 5-phenyl-8-tosyloxyquinoline (3).

Supplementary Figure 7 MS-ESI of 5-phenyl-8-tosyloxyquinoline (3).

Supplementary Figure 8 TLC check of the Suzuki cross-coupling reaction.

TLC (cyclohexane:ethyl acetate 75:25) of Suzuki cross-coupling for the synthesis of 5-phenyl-8-tosyloxyquinoline 3. S = starting material (5-chloro-8-tosyloxyquinoline, 2); P = crude reaction mixture; R = real purified product (3). UV lamp, 254 nm.

Supplementary Figure 9 TLC check of the hydrolysis reaction.

TLC (cyclohexane:ethyl acetate 75:25) of basic hydrolysis deprotection for the synthesis of 5-phenyl-8-hydroxyquinoline 4. S = starting material (5-phenyl-8-tosyloxyquinoline, 3); PE = crude extracted reaction mixture; R = real purified product (4). UV lamp, 254 nm.

Supplementary Figure 10 1H NMR (400 MHz, CDCl3) of 5-phenyl-8-hydroxyquinoline (4).

Supplementary Figure 11 13C NMR (100 MHz, CDCl3) of 5-phenyl-8-hydroxyquinoline (4).

Supplementary Figure 12 MS-ESI of 5-phenyl-8-hydroxyquinoline (4).

Supplementary Figure 13 TLC check of the Mannich reaction.

TLC check (dichloromethane:methanol 9:1 + 2.5% aqueous NH4OH) of the Mannich reaction for the synthesis of DCHQ5. A = 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (5); S = starting material (5-phenyl-8-hydroxyquinoline, 4); P = crude reaction mixture; R = real purified product (DCHQ5). Left) UV lamp, 254 nm, right) UV lamp, 310 nm.

Supplementary Figure 14 1H NMR (400 MHz, CDCl3) of DCHQ5.

Supplementary Figure 15 13C NMR (100 MHz, CDCl3) of DCHQ5.

Supplementary Figure 16 MS-ESI of 5-phenyl-8-hydroxyquinoline DCHQ5.

Supplementary information

Supplementary Figures and Text

Supplementary Table 1 and Supplementary Figures 1–16. (PDF 1108 kb)

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Sargenti, A., Farruggia, G., Zaccheroni, N. et al. Synthesis of a highly Mg2+-selective fluorescent probe and its application to quantifying and imaging total intracellular magnesium. Nat Protoc 12, 461–471 (2017). https://doi.org/10.1038/nprot.2016.183

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