A simple FRET-based modular design for diagnostic probes

Article abstract of DOI:10.1039/c1ob06667f

In recent years, there has been a massive effort to develop molecular probes with optical modes of action. Probes generally produce detectable signals based on changes in fluorescence properties. Here, we demonstrate the potential of self-immolative molecular adaptors as a platform for Turn-On probes based on the FRET technique. The probe is equipped with identical fluorophore pairs or a fluorophore/quencher FRET pair and a triggering substrate. Upon reaction of the analyte of interest with the triggering substrate, the self-immolative adaptor spontaneously releases the two dye molecules to break off the FRET effect. As a result, a new measurable fluorescent signal is generated. The fluorescence obtained can be used to quantify the analyte. The modular structure of the probe design will allow the preparation of various chemical probes based on the FRET activation technique.

Full text of DOI:10.1039/c1ob06667f

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A simple FRET-based modular design for diagnostic probes†
Orit Redy,‡ Einat Kisin-Finfer,‡ Eran Sella and Doron Shabat*
Received 2nd October 2011, Accepted 3rd November 2011
DOI: 10.1039/c1ob06667f
In recent years, there has been a massive effort to develop
molecular probes with optical modes of action. Probes gen-
erally produce detectable signals based on changes in fluores-
cence properties. Here, we demonstrate the potential of self-
immolative molecular adaptors as a platform for Turn-On
probes based on the FRET technique. The probe is equipped
with identical fluorophore pairs or a fluorophore/quencher
FRET pair and a triggering substrate. Upon reaction of
the analyte of interest with the triggering substrate, the
self-immolative adaptor spontaneously releases the two dye
molecules to break off the FRET effect. As a result, a new
measurable fluorescent signal is generated. The fluorescence
obtained can be used to quantify the analyte. The modular
structure of the probe design will allow the preparation
of various chemical probes based on the FRET activation
technique.
concept, we sought to develop a modular design for preparation
of Turn-On optical probes. A schematic representation of our
probe design is illustrated in Fig. 1. The head of an adaptor
molecule is attached to a protecting group that can be cleaved
by a specific analyte. The tails of the adaptor are attached to a
fluorophore and to a quencher unit (Fig. 1A). Removal of the
protecting group by the analyte of interest results in spontaneous
release of the fluorophore and the quencher from the molecular
adaptor. Consequently, the FRET process is no longer active and
the fluorescent signal is turned on. The same idea is illustrated
with two identical fluorophores, which quench each other through
a Homo-FRET interaction (Fig. 1B).
There are quite a few examples of small organic molecules
that can act as molecular adaptors with chemical reactivity
as illustrated in Fig. 1.7,8 Our group has reported the use of
several such adaptors for construction of self-immolative dendritic
molecules.9 These molecules can translate a single cleavage event
of a protective group at the head of the dendritic molecule into the
release of multiple tail-units from the periphery. The disassembly
mechanism of such an adaptor (molecule 1a) is depicted in Fig. 2.
In general any phenol or aniline derivative with substituents at the
ortho or para benzylic positions can be used to release tail-units.
Upon removal of a protective group from the head of the molecular
adaptor, a phenolate is obtained (molecule 1a). The latter can
undergo two consecutive quinone-methide-type eliminations
(molecules 1b and 1d) to release the two tail-units from the main
adaptor moiety. Thus, a single cleavage event at the head of the
adaptor is amplified with the release of two units from the tails.
Similar molecular reactivity can be achieved by using aniline or
aromatic thiol derivatives instead of the phenol. We have used
the structural properties of self-immolative dendritic adaptors to
demonstrate molecular amplification in the fields of drug delivery
and diagnostics.9–13
Molecular probes based on fluorescence signal generation are
widely used for various diagnostic applications. Several different
classes of Turn-On optical probes have been described in the liter-
ature for detection/imaging of chemical and biological factors.1–4
Among the methods used to obtain an emitted fluorescence signal
upon detection or imaging of a specific reactivity, Fo¨rster reso-
nance energy transfer (FRET), which is based on a fluorophore–
quencher interaction or fluorophores’ self-quenching, is one of
the most common and efficient options.5,6 Herein, we demonstrate
a simple modular approach for design of activatable fluorescent
probes using the FRET technique. One method for turning off
the fluorescence of a dye molecule is to force close proximity
of a quencher and a fluorophore through a covalent linkage.
Under such circumstance, the excited fluorophore can transfer its
excitation energy to the nearby quencher-chromophore in a non-
radiative fashion through long rang dipole–dipole interactions.
Disconnection of the linkage results in diffusion of the fluorophore
away from the quencher and generation of a measurable fluores-
cent signal. Similar effects can be obtained by self-quenching of
two identical fluorophores (Homo-FRET). Based on this known
Initially, we sought to explore the capability of an AB2 self-
immolative dendritic adaptor to serve as a platform for a Homo-
FRET with the design presented in Fig. 1B. Probe 2 is based on an
aniline AB2 self-immolative adaptor with substituents at the ortho
or para benzylic positions (Fig. 3). The probe is equipped with a
protecting group (phenylacetamide) that is a substrate for cleavage
by the protease penicillin-G-amidase (PGA). We have used two
molecules of a Cy5 dye as the fluorophore.14 This dye molecule
has a small Stokes shift and thus can significantly quench the
fluorescence of a neighboring molecule.15 Removal of the pheny-
lacetamide group by PGA followed by 1,6 azaquinonemethide
elimination results in formation of aniline 2a. The latter can
Department of Organic Chemistry, School of Chemistry, Raymond and
Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv
69978, Israel. E-mail: chdoron@post.tau.ac.il; Fax: +972 (0) 3 640 9293;
Tel: +972 (0) 3 640 8340
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob06667f
‡ O.R. and E.K.F. contributed equally to this work.
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Fig. 1 Modular design of a FRET-based optical probe. (A) Modular probe with a fluorophore–quencher pair. (B) Modular probe with
fluorophore–fluorophore pair.
Fig. 2 Disassembly pathway of an AB2 self-immolative dendritic adaptor upon removal of a protective group.
undergo consecutive 1,6 and 1,4 eliminations to release the two
Cy5 molecules.
whereas emission remained at background levels in absence of
PGA.
The synthesis of probe 2 was performed as shown in Fig. 4. Com-
pound 3 was prepared as previously described.16 Activation of the
hydroxybenzyl groups of diol 3 with 4-nitrophenyl-chloroformate
afforded dicarbonate 3a. The latter was reacted with 2 eq. of Cy5-
amine derivative 3b to afford probe 2.
The fluorescence spectrum of probe 2 was measured and
compared to that of free Cy5 (see ESI†). Expectedly, the Homo-
FRET quenching effect was observed. To evaluate the capability of
probe 2 for detection of the protease PGA, the probe was incubated
in PBS 7.4 with and without the enzyme, and the fluorescence
emission was monitored with a spectrofluorometer (Fig. 5). In the
presence of PGA, a significantfluorescenceemission was observed,
Next, we prepared two additional probes based on the FRET-
pair design presented in Fig. 1A. These probes were equipped
with a triggering group, phenyl-boronic ester, which is known
to undergo cleavage by hydrogen peroxide.17–20 The protecting
group was linked to a phenol derivative of an AB2 self-immolative
adaptor with two substituents at the ortho benzylic positions.
Probe 4 contained fluorescein dye (4a) as a fluorophore and
disperse red 1 (DR1) dye as a quencher (Fig. 6).21 Probe 5
contained Cy5 dye as a fluorophore and compound 5a as a
quencher (Fig. 7).22 Both probes are designed to release both
fluorophore and quencher upon removal of the protecting group,
similarly to the reaction shown in Fig. 2. The phenol obtained
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Fig. 3 Activation of a Homo-FRET probe by PGA to release Cy5 fluorophores.
Fig. 4 Chemical synthesis of probe 2.
The synthesis of probe 4 was achieved as depicted in Fig. 8.
4-Hydroxybenzoic acid 6a was coupled with allyl bromide to
protect the carboxylic acid in the form of allyl ester 6b. The
latter was reacted with formaldehyde to generate dibenzylalcohol
6c, which was then protected with two equivalents of t-
butyldimethylsilylchloride to give phenol derivative 6d. The
phenol was alkylated with iodide 6e to give ether 6f. Deprotection
of 6f with p-toluene sulfonic acid afforded diol 6g. Activation
of diol 6g with two equivalents of 4-nitrophenyl-chloroformate
gave dicarbonate 6h, which was selectively reacted with
one equivalent of disperse red 1 to yield compound 6i.
Reaction of 6i with fluorescein derivative 6 (see ESI†) generated
compound 6j, which was deprotected using tetrakis(triphenylpho-
sphine)palladium
probe 4.
The synthesis of probe 5 was performed according to the scheme
shown in Fig. 9. Compound 7a was synthesized as previously
described.9 Alkylation of phenol 7a with iodide 6e afforded ether
7b, which was then deprotected with p-toluene sulfonic acid to give
and
tributyltin
hydride
to
afford
Fig. 5 NIR fluorescence (lEx = 630 nm, lEm = 670 nm) emitted upon
incubation of probe 2 [10 mM] in the presence (red) or absence (blue) of
PGA [0.1 mg ml-1] in PBS, pH 7.4.
after cleavage of the phenyl-boronic ester is known to release its
end-units through double ortho-quinone-methide elimination.9
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Fig. 6 Activation of a FRET-pair probe by hydrogen peroxide.
Fig. 7 Activation of a FRET-pair probe by hydrogen peroxide.
diol 7c. Activation of diol 7c with two equivalents of 4-nitrophenyl-
chloroformate gave compound 7d. Reaction of one equivalent of
Cy5 amine derivative 7f with dicarbonate 7d gave compound 7e,
which was further reacted with quencher 5a (see ESI†) to afford
probe 5.
The fluorescence spectra of probes 4 and 5 compared to those of
the free fluorophores confirmed the FRET quenching effect (see
ESI†). Both probes were incubated with and without hydrogen
peroxide in PBS pH 7.2 for probe 4, and pH 8.3 for probe
5, and the emission was monitored using a spectrofluorometer.
Fig. 10 shows emission from probe 4 as a function of time
after addition of hydrogen peroxide. A significant increase of the
emitted fluorescence was observed within minutes after addition
of hydrogen peroxide, whereas no change in fluorescence was
observed in the absence of hydrogen peroxide. Similar results were
obtained when probe 5 was activated with hydrogen peroxide (Fig.
11).
is most suitable for fluorophores with relatively small Stokes
shifts. The second design was based on a fluorescent molecule
and a quenching dye. Activation of the probe dissociated the two
moieties and resulted in measurable fluorescent signal. These two
options were tested in a probe system constructed with an AB2
self-immolative dendritic adaptor.
Usually FRET-based probes are composed of FRET-pair dyes
tethered through a peptide linker. Cleavage of the linker between
the dye molecules, by a protease for example, results in generation
of fluorescence.23 In this design, the dyes must be linked through
the terminal functional groups of the peptide substrate. To achieve
a quenching effect in the probe, the absorption spectrum of the
quencher must partially overlap with the fluorescent spectrum of
the fluorophore. Therefore, DR1 (absorption band between 400–
600 nm) was selected for probe 4 and quencher 5a (absorption
band between 600–900 nm) for probe 5.
The self-immolative dendritic adaptor is linked to the substrate
through one side only, while the other remains open. This feature
increases the flexibility of probe design and allows use of various
molecules as substrates for detection of either enzyme or chemical
In this study, we evaluated two designs of Turn-On probes
activated by the FRET technique. One was based on separation of
two identical fluorophores that quench each other. This option
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Fig. 8 Chemical synthesis of probe 4.
Fig. 9 Chemical synthesis of probe 5.
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simply by installing a specific protecting group as an activator.
The modular concept presented in this report should be useful for
preparation of many other chemical probes based on the FRET
activation technique.
Acknowledgements
D.S. thanks the Israel Science Foundation (ISF), the Binational
Science Foundation (BSF) and the German-Israeli Foundation
(GIF) for financial support.
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dendritic adaptor that releases two end-groups upon a single
activation event at the adaptor’s head by the analyte of interest. In
a self-quenching approach, the probes were equipped with two
identical fluorophores. In second design, a fluorophore and a
quencher were attached to the probe. The modular structure may
be adapted to incorporate a variety of fluorophores and various
protecting groups as triggering substrates. This design allows the
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