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Synthesis and use of QCy7-derived modular probes for the detection and imaging of biologically relevant analytes

Nature Protocols volume 9, pages 27–36 (2014)Cite this article

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Abstract

This protocol describes the synthesis of modular turn-ON QCy7-based probes for the detection of biologically relevant analytes, such as hydrogen peroxide, ubiquitous sulfhydryl and β-galactosidase. The probes presented herein are prepared through a simple procedure that involves the preliminary alkylation of 4-hydroxy-isophthalaldehyde with a relevant analyte-responsive protecting group, followed by condensation of the resulting product with 2 equivalents of sulfo-indolium moieties. Evaluation of the turn-ON near-IR fluorescence response to their relevant analytes for the three different QCy7 probes is also reported. The preparation of a QCy7 diagnostic probe requires 1–2 d. Probes for other analytes can be prepared according to this modular procedure by incorporating a specific analyte-responsive group as a triggering substrate.

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Figure 1
Figure 2: Chemical structures and activation of three different QCy7-based probes.
Figure 3
Figure 4
Figure 5: Fluorescence turn-ON response of probe 1 to hydrogen peroxide, probe 2 to cysteine and probe 3 to β-galactosidase.
Figure 6: Intravital fluorescence imaging of turn-ON response of the probes.

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Acknowledgements

D.S. thanks the Israel Science Foundation (ISF), the Binational Science Foundation (BSF) and the German-Israeli Foundation (GIF) for financial support. This work was partially supported by grants from the Israeli National Nanotechnology Initiative (INNI), Focal Technology Area (FTA) program: Nanomedicine for Personalized Theranostics; and by The Leona M. and Harry B. Helmsley Nanotechnology Research Fund.

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Authors and Affiliations

  1. School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, Israel

    Orit Redy-Keisar, Einat Kisin-Finfer & Doron Shabat

  2. Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

    Shiran Ferber & Ronit Satchi-Fainaro

Authors
  1. Orit Redy-Keisar
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  2. Einat Kisin-Finfer
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  3. Shiran Ferber
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  4. Ronit Satchi-Fainaro
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  5. Doron Shabat
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Contributions

O.R., E.K.-F., S.F., R.S.-F. and D.S. designed the experiments; O.R., E.K.-F. and S.F. conducted the experiments; O.R., E.K.-F., S.F., R.S.-F. and D.S. analyzed the data; and O.R., E.K.-F., S.F., R.S.-F. and D.S. wrote the manuscript.

Corresponding authors

Correspondence to Ronit Satchi-Fainaro or Doron Shabat.

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Competing interests

A patent related to this work has been filed by Tel Aviv University (reference no. PCT/IB2012/051255). The title of the invention is: 'Activatable Fluorogenic Compounds And Uses Thereof As Near-Infrared Probes'.

Integrated supplementary information

Supplementary Figure 1 Activation mechanism of QCy7-based probe 1 by H2O2 to produce a fluorescent turn-ON response.

Supplementary Figure 2 Activation mechanism of a QCy7-based probe 2 by a ubiquitous thiol to produce a fluorescent turn-ON response.

Supplementary Figure 3 Activation mechanism of a QCy7-based probe 3 by β-galactosidase to produce a fluorescent turn-ON response.

Supplementary Figure 4 Synthesis of intermediate 2a.

Supplementary Figure 5 Synthesis of intermediate 3a.

Supplementary Figure 6 Fluorescence and absorption spectra of protected and unprotected QCy7.

Fluorescence a) and absorption b) spectra of protected (red) and unprotected (green) QCy7, [100 μM], 1%DMSO in 0.1 M PBS, pH=7.4, λex=590 nm, λem=720 nm.

Supplementary Figure 7 NIR fluorescence spectra of probe 1 in the presence or absence of H2O2.

a) NIR Fluorescence (λex=590 nm, λem=720 nm) emitted upon incubation of probe 1 [100 μM], in the presence (green) or absence (red) of H2O2 (5 equivalents) in 1%DMSO in 0.1 M PBS, pH=7.4. b) NIR Fluorescence (λex=590 nm, λem=720 nm) emitted upon incubation of probe 1 [100 μM] with 0, 10, 20, 30, 40, 70 and 100 μM H2O2, in 1%DMSO in 0.1 M PBS, pH=7.4.

Supplementary Figure 8 NIR fluorescence spectra of probe 2 in the presence or absence of cysteine.

a) NIR Fluorescence (λex=590 nm, λem=720 nm) emitted upon incubation of probe 2 [100 μM], in the presence (green) or absence (red) of cysteine (1 equivalent) in 1%DMSO in 0.1 M PBS, pH=7.4. b) NIR Fluorescence (λex=590 nm, λem=720 nm) emitted upon incubation of probe 2 [100 μM] with 0.1, 2.5, 5, 10, 15, 20 μM cysteine, in 1%DMSO in 0.1 M PBS, pH=7.4.

Supplementary Figure 9 NIR fluorescence spectra of probe 3 in the presence or absence of β-galactosidase.

a) NIR Fluorescence (λex=590 nm, λem=720 nm) emitted upon incubation of probe 3 [100 μM], in the presence (green) or absence (red) of β-galactosidase (1.37 units) in 1%DMSO in 0.1 M PBS, pH=7.4. b) NIR Fluorescence (λex=590 nm, λem=720 nm) emitted upon incubation of probe 3 [100 μM] with 1.37, 0.91, 0.45, 0.22, 0.11 or 0 enzyme units of β-galactosidase, in 1%DMSO in 0.1 M PBS, pH=7.4.

Supplementary information

Supplementary Figure 1

Activation mechanism of QCy7-based probe 1 by H2O2 to produce a fluorescent turn-ON response. (PDF 120 kb)

Supplementary Figure 2

Activation mechanism of a QCy7-based probe 2 by a ubiquitous thiol to produce a fluorescent turn-ON response. (PDF 93 kb)

Supplementary Figure 3

Activation mechanism of a QCy7-based probe 3 by β-galactosidase to produce a fluorescent turn-ON response. (PDF 106 kb)

Supplementary Figure 4

Synthesis of intermediate 2a. (PDF 141 kb)

Supplementary Figure 5

Synthesis of intermediate 3a. (PDF 138 kb)

Supplementary Figure 6

Fluorescence and absorption spectra of protected and unprotected QCy7. (PDF 206 kb)

Supplementary Figure 7

NIR fluorescence spectra of probe 1 in the presence or absence of H2O2. (PDF 254 kb)

Supplementary Figure 8

NIR fluorescence spectra of probe 2 in the presence or absence of cysteine. (PDF 249 kb)

Supplementary Figure 9

NIR fluorescence spectra of probe 3 in the presence or absence of β-galactosidase. (PDF 248 kb)

Supplementary Methods

Supplementary Methods (PDF 296 kb)

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Redy-Keisar, O., Kisin-Finfer, E., Ferber, S. et al. Synthesis and use of QCy7-derived modular probes for the detection and imaging of biologically relevant analytes. Nat Protoc 9, 27–36 (2014). https://doi.org/10.1038/nprot.2013.166

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