Azo-sulforhodamine dyes: A novel class of broad spectrum dark quenchers

Article abstract of DOI:10.1021/ol501753b

A rapid access to a novel class of water-soluble dark quencher dyes was achieved using an azo-coupling reaction between a fluorescent primary arylamine derived from a sulforhodamine 101 scaffold and a tertiary aniline equipped with different bioconjugatable groups. The thus obtained nonfluorescent azo-sulforhodamine hybrids display a broad quenching range spanning the visible to NIR regions. This was demonstrated through the preparation and enzymatic activation of FRET-based fluorogenic substrates of urokinase.

Full text of DOI:10.1021/ol501753b

Letter
pubs.acs.org/OrgLett
Azo-Sulforhodamine Dyes: A Novel Class of Broad Spectrum Dark
Quenchers
Arnaud Chevalier,† Pierre-Yves Renard,*,† and Anthony Romieu*,‡,§
†Normandie Universite,
Mont-Saint-Aignan Cedex, France
‡Institut de Chimie Molec
ulaire de l’Universite
21078 Dijon, France
́
COBRA UMR 6014 & FR 3038, UNIV Rouen, INSA Rouen, CNRS, IRCOF, 1 Rue Tesnier
̀
es, 76821
́
de Bourgogne, UMR CNRS 6302, Universite de Bourgogne, 9 Avenue Alain Savary,
́ ́
§Institut Universitaire de France, 103 Boulevard Saint-Michel, 75005 Paris, France
S
* Supporting Information
ABSTRACT: A rapid access to a novel class of water-soluble dark quencher
dyes was achieved using an azo-coupling reaction between a fluorescent
primary arylamine derived from a sulforhodamine 101 scaffold and a tertiary
aniline equipped with different bioconjugatable groups. The thus obtained
nonfluorescent azo-sulforhodamine hybrids display a broad quenching range
spanning the visible to NIR regions. This was demonstrated through the
preparation and enzymatic activation of FRET-based fluorogenic substrates of
urokinase.
uring the past few years, biomedical imaging has
undergone manifold and rapid developments which
the efficacy of energy transfer and hence also to the spectral
overlap between the donor emission and acceptor absorption,
the distance between them, and their relative orientation.
Research efforts devoted to the field of such “activatable”
probes have promoted the chemistry of nonfluorescent FRET
acceptors bearing a bioconjugatable handle and displaying a
specific dynamic quenching range fully compatible with the
selected fluorescent label (acting as FRET donor). Thus,
various dark quenchers mainly belonging to the family of azo or
cyanine dyes have been reported and/or commercialized. In
general, azo dyes are nonfluorescent owing to ultrafast
conformational change around the NN bond after photo-
excitation (e.g., BHQ dyes from Biosearch Technologies).5
Alternatively, the native fluorescence of cyanine dyes is readily
abolished by intramolecular charge transfer (ICT) or photo-
induced electron transfer (PeT) through the incorporation of
electron-donating and/or -withdrawing groups (typically N,N-
dialkylamino or nitro groups) within their core structures (e.g.,
CytoCy5S from GE Healthcare and IRDye QC-1 from LI-COR
Biosciences).6 All these compounds are commonly used in the
design of FRET-based probes for in vitro detection or in vivo
imaging of biologically relevant enzyme activities.7 Despite
these achievements, the quencher dyes do not necessarily have
ideal physicochemical and spectral properties, and overall
(photo)chemical stability (especially for the cyanine deriva-
D
make it particularly suited for visualization of physiological
structures, measurements of biological functions, and evaluation
of cellular and molecular events without requiring invasive
procedures.1 Compared to other imaging modalities, optical
imaging techniques (i.e., bioluminescence and fluorescence
intensity imaging) that refer to both sensitive and quantitative
detection of transmitted visible- or NIR-light photons are cost-
effective and widely available and do not involve any form of
ionization radiation.2 The successful implementation of
fluorescence intensity imaging generally involves the use of
“activatable” probes (also known as “smart” imaging probes)
which exhibit significant changes in their spectroscopic
properties (i.e., a turn-on emission increase or a shift in
excitation/emission profiles) upon interaction and/or subse-
quent reaction with the targeted bioanalyte (e.g., an enzyme, a
biomolecule, or a biologically relevant anion or cation).3 A
turn-on response gives a bright signal against a dark
background, which is the preferred way to achieve the high
signal-to-noise responses and to maximize spatial resolution.
Historically, the most popular and convenient approach to
designing “smart” imaging probes is the one based on the
implementation of the Forster resonance energy transfer
̈
(FRET) mechanism through the attachment of a comple-
mentary fluorophore (donor) and quencher (acceptor) pair at
either side of a cleavable substrate chosen to react selectively
with the bioanalyte (mostly, an hydrolytic enzyme).4 The
performances of such dual labeled probes are closely linked to
Received: June 17, 2014
Published: July 18, 2014
© 2014 American Chemical Society
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Organic Letters
Letter
tives). For instance, the issue of water solubility (and cell
permability) of a quencher is not generally taken into account.
Furthermore, the vast majority of these nonfluorescent
chromophores display a strong spectral overlap only with a
limited number of fluorophores emitting in a specific range of
the UV−vis spectrum. With the aim of improving their
quenching ability and at converting them into water-soluble
derivatives with a wide range of bioconjugatable handles, some
azo dyes belonging to the BHQ family have been the focus of
several recent works from Kool et al. and our research group.8,9
Conversely, very little attention has been focused toward dark
quenchers based on a xanthene scaffold whereas they might
take advantage of superior properties that possess fluoresceins
and rhodamines: (1) high molar extinction coefficients, (2)
high chemical stability especially under harsh conditions of pH
and temperature, and (3) easy modulation of the quenching
ability through the chemical modification of their aniline or
phenol moiety. To the best of our knowledge, only three N,N′-
diarylrhodamine derivatives developed by Invitrogen Molecular
Probes (known as QSY 7, 9 and 21)10 are currently available. In
this context, it seems interesting to explore the feasibility of
designing a novel class of water-soluble nonfluorescent long-
wavelength rhodamines acting as broadly absorbing dark
quenchers. Two independent publications have recently
reported that the direct conjugation of an azo group to the
conjugated system of a rhodamine fluorophore is an effective
way to switch-off its fluorescence, which can be readily
recovered upon bioreduction.11,12 This feature has allowed
the design of fluorescent chemodosimeters, readily effective for
tumor hypoxia imaging through the detection of edogeneous
reductases. Interestingly, this fluorescence suppression strategy
was also applied to other aniline-based fluorophores such as 4-
amino-N-butyl-1,8-naphthalimide to obtain an effective colori-
metric and ratiometric probe for the detection of cyanide
ions,13 and 3,5,8-triaryl-2-amino BODIPY dyes14 (Figure 1).
hybrids is assessed in the context of a peptidyl-FRET substrate
for a serine protease namely the urokinase plasminogen
activator (uPA), known to play an important role in tumor-
associated proteolysis,15 involving a variety of fluorophores that
range from visible blue light (7-N,N-diethylaminocoumarin,
DEAC) to NIR emission (sulfoindocyanine dye Cy 7.0).
The synthetic strategy currently used to prepare azo dye
quenchers relies on the coupling reaction between a diazonium
compound and an electron-rich tertiary aniline. The use of
bench-stable, commercially available nitrosating agent, nitro-
sonium tetrafluoroborate (NOBF4), in a polar aprotic solvent
has recently emerged as valuable in performing this SEAr
reaction under mild conditions fully compatible with the
moderate stability of functionalized anilines.9,11 Thus, this diazo
coupling methodology appears to be well suited to the
conversion of a fluorescent aniline such as SR101-110 into
the corresponding azo-based quencher. Moreover, as shown in
Scheme 1, SR101-110 was readily obtained in good yield
Scheme 1. Synthesis of Azo-sulforhodamine Hybrids SR101-
Q-X
through a concise synthetic route based on the sequential
condensation of two different meta-aminophenols (i.e., 8-
hydroxyjulolidine and 3-hydroxyaniline) with 4-formylbenzene-
1,3-disulfonic acid in methanesulfonic acid heated at 150 °C.16
After isolation by RP-HPLC purification and freeze-drying, the
TFA salt of SR101-110 was reacted with NOBF4 in dry
CH3CN and in the presence of 1 equiv of TEA (aimed at
deprotonating its primary aniline). Surprisingly, the newly
formed diazonium salt intermediate was found to be unreactive
toward tertiary aniline 1 in CH3CN. The addition of acetate
buffer (pH 4.0) helped to solve this lack of reactivity, and a
good conversion into SR101-Q-CO2H was obtained. Purifica-
tion was achieved by semipreparative RP-HPLC to give SR101-
Q-CO2H in a good 52% isolated yield. Its structure was
unambiguously confirmed by detailed measurements including
NMR and ESI-HRMS analysis (see the Supporting Information
(SI)).
To expand the scope of azo-sulforhodamine dyes in
biolabeling applications involving popular reactions such as
copper-catalyzed azide−alkyne cycloaddition (CuAAC), Stau-
dinger ligation, or thiol-alkylation (Michael addition),17 we
have next explored the diazo coupling between SR101-110 and
four different N,N-dialkyl-substituted anilines terminated with
azido, alkyne, maleimide, and amino groups, respectively.
Figure 1. Nonfluorescent azo dyes derived from fluorescent anilines
and recently reported in the literature.
Thus, the conversion of the single primary aniline moiety of an
unsymmetrical rhodamine dye into π-extended azobenzene
derivatives should enable novel dark quenchers with valuable
properties to be rapidly obtained.
We report here the practical implementation of this synthetic
strategy by using an unsymmetrical derivative of sulforhod-
amine 101 namely SR101-110 and functionalized tertiary
anilines to introduce five distinct bioconjugatable groups
(alkyne, amino, azido, carboxylic acid and maleimide) onto
the diaryl-azo scaffold. Indeed, SR101-110 exhibits structural
features (i.e., two sulfonate groups on the meso phenyl ring and
a positively charged julolidine unit) that are valuable to keep
within the core structure of a targeted quencher, aimed at
improving water solubility and shifting absorption to the red
region as compared to conventional BHQ dyes. The effective
quenching range of these bioconjugatable azo-sulforhodamine
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Organic Letters
Letter
Anilines 2, 3, and 5 were prepared according to literature
procedures, and maleimide-terminated derivative 4 was
synthesized from N-methyl-N-phenylpropane-1,3-diamine (see
the SI). The use of previously optimized conditions for the azo
coupling has enabled us to readily obtain four additional
bioconjugatable azo-sulforhodamine dyes (Scheme 1). These
compounds were isolated in a pure form by semipreparative
RP-HPLC and characterized by NMR and ESI-HRMS (see the
SI). As illustrated in Figure 2 (and table portion in figure),
Scheme 2. Synthesis of FRET-Based Fluorogenic uPA
Substrates
phase peptide synthesis techniques (Fmoc/tBu strategy) and
subsequent TFA deprotection. Acylation of its N-terminal α-
amino group with HOBt active esters of SR101-Q-CO2H
(formed in situ with PyBOP/DIEA) in NMP/DIEA has led to
the monolabeled peptide. The removal of the Dde protecting
group from the lysine residue was achieved by treatment with
an excess of hydroxylamine and imidazole to afford 7.18 These
mild deprotection conditions were preferred to the standard
use of hydrazine, since the sensitivity of some fluorophores
(especially cyanine and rhodamine dyes) to such an α-
nucleophile by either the reduction of ethylenic insaturations
(mediated by diimide resulting from slow oxidation of
hydrazine) or Michael-type addition to the xanthene core has
already been observed.19 Finally, amidification of the lysine side
chain with the selected fluorophore carboxylic acid provided
the uPA fluorogenic substrates. These dual-labeled peptides
were purified by semipreparative RP-HPLC (overall yields 25−
50%, purity >95%), and their structures were unambiguously
confirmed by ESI-MS analyses (see the SI).
Quenching Efficiency (QE) was measured as the difference
in fluorescence (area under the emission curves) of the FRET
probes before and after enzymatic digestion with uPA, and
expressed as a percentage (QE = 100 × (1 − I0(em)/I(em)).
Data displayed in the table portion of Figure 3 show that SRQ
has an excellent quenching efficiency (>95%) for all selected
fluorescent organic dyes, except for NIR-emitting cyanine dye
Cy 7.0 (92.7%). Due to the lack of significant spectral overlap
between emission curves of sulfoindocyanine dyes Cy 5.0, Cy
5.5, and Cy7.0 and absorption curve of SRQ (see Figure 3),
static quenching (also known as contact quenching) wherein
the fluorophore and the quencher form an intramolecular
complex20 is likely to occur in these three probes instead of
conventional FRET quenching. This was supported by the
shape of UV−vis absorption spectra which are not the simple
sum of absorption curves of cyanine and SRQ dyes (see the SI).
In summary, we have developed an original approach to
easily access a novel family of rhodamine-based quenchers that
are structurally compact, water-soluble, and bioconjugatable.
Contrary to commercially available QSY dyes whose synthesis
is tedious and time-consuming, these nonfluorescent derivatives
are readily obtained through a single-step protocol based on an
azo coupling reaction involving a sulforhodamine as one of the
Figure 2. Absorption data of SR101-Q-X in PBS at 25 °C. a Isolated
yields after purification by semipreparative RP-HPLC. Each quencher
compound was recovered with a purity up to 99%.
SR101-Q-X exhibit a far broader absorption spectrum than that
of parent fluorophore SR101-110, which covers the full visible
spectrum, and was characterized by a maximum centered at
582−592 nm (according to the substitution pattern of aniline)
and good Δλ1/2 max values (142−161 nm).
As expected, sulfonate moieties onto the meso-phenyl ring
make these azo-sulforhodamine hybrids significantly soluble in
water and related aq buffers, over a concentration range (1.0
μM to 1.0 mM) full-compatible with bioanalytical applications
involving FRET probes.
To assess the quenching range of the azo-sulforhodamine
101 scaffold and to confirm the bioconjugation ability of
SR101-Q-CO2H, we have chosen to prepare different uPA-
sensitive fluorescence quenched probes (“SRQ-peptide-Fluo”)
using a wide range of fluorophores associated with this azo-
based quencher. Heptapeptide H-Ser-Gly-Arg-Ser-Ala-Asn-Ala-
OH (also known as SGRSANA) has been reported to be a
highly potent substrate of uPA (cleavage site between Arg and
Ser residues);15 therefore, we decided to label this peptide with
a FRET pair composed of an organic fluorophore (emitting in
the visible or NIR spectral region) and SR101-Q-CO2H,
abbreviated SRQ (Scheme 2). To achieve chemoselective
labeling reactions through aminolysis of activated esters, a
lysine residue whose ε-primary amine is masked with a
trifluoroacetic acid (TFA)-stable protecting group (Dde) was
incorporated at the C-terminus. Peptide H-Ser-Gly-Arg-Ser-
Ala-Asn-Ala-Lys(Dde)-NH2 6 was obtained by standard solid-
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Organic Letters
Letter
“Plateforme d’Analyse Chimique et de Synthes
l’Universite
̀
e Molec
́
ulaire de
́
de Bourgogne (PACSMUB, http://www.wpcm.fr)
for access to the Bruker Avance III 500 spectrometer and
Bruker Alpha FT-IR spectrometer. We also warmly thank B.
Roubinet (University of Rouen, laboratory COBRA, UMR
CNRS 6014) for the synthesis and determination of
fluorescence quantum yields of DEAC-3-carboxylic acid and
its carboxamide derivative BR020 and Dr. Cyrille Sabot
(laboratory COBRA, UMR CNRS 6014) for technical
assistance during LC-MS analyses (see the SI), P. Martel
(University of Rouen, laboratory COBRA, UMR CNRS 6014)
for IR analyses, A. Marcual (laboratory COBRA, UMR CNRS
6014) for HRMS analyses, and Dr. R. Ziessel (LCOSA,
ICPEES, UMR 7515 CNRS, ECPM Strasbourg) for the gift of
the green-emitting BODIPY dye.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures and analytical data reported herein. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(16) Chevalier, A.; Renard, P.-Y.; Romieu, A. Chem.Eur. J. 2014,
AUTHOR INFORMATION
Corresponding Authors
■
20, 8330−8337.
(17) For comprehensive reviews about bioorthogonal chemistry, see:
(a) Best, M. D. Biochemistry 2009, 48, 6571. (b) Sletten, E. M.;
Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48, 6974.
*E-mail: pierre-yves.renard@univ-rouen.fr.
*E-mail: anthony.romieu@u-bourgogne.fr.
Notes
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ACKNOWLEDGMENTS
This work has been partially supported by INSA Rouen, Rouen
University, CNRS, Labex SynOrg (ANR-11-LABX-0029) and
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■
́
region Haute-Normandie (CRUNCh network). Financial
support from FEDER (TRIPODE, No. 33883) for the Ph.D.
grant of A.C. is greatly acknowledged. A.R. thanks the Institut
Universitaire de France (IUF) for financial support and the
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