have been developed to offer specific quenchers for differ-
ent emission ranges either in the UVꢀvis region or into the
NIR spectrum. Among the many different structures
reported in the literature and/or commercially available,
the most popular are undoubtedly (1) cyanine dyes whose
native fluorescence is abolished by intramolecular charge
transfer (ICT) or photoinduced 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., IRDye QC-1
from LI-COR Biosciences),4 (2) azo dyes5 whose none-
missive features are related to photochemical isomeriza-
tion of their azo bridge, either through a rotation
mechanism around the NdN double bond or an inversion
mechanism, in which a planar variation of one of the
CꢀNꢀN angles can exist in the excited state (e.g., BHQ
dyes from Biosearch Technologies),6 and (3) N,N0-diarylr-
hodamine derivatives developed by Invitrogen Molecular
Probes and known as the trademark QSY.7 However, as
displayed in Figure 1, no single compound belonging to
these dye classes is able to quench all fluorophores emitting
in the broad UV/NIR spectral range.
ranging from 460 to 730 nm, but its ability to quench UV
fluorophores was not investigated. A structurally simpler
quencher structure derived from an anthraquinone scaf-
fold (acide blue 40) was also identified and found to be
effective in almost the same visible/NIR spectral range.9
These two independent studies support the fact that it is
virtually impossible to design an UDQ only through
chemical modifications of a single quencher structure.
Thus, an alternative approach based on the covalent
association of several different nonfluorescent dyes through
high-yielding conjugation reactions, deserves to be
explored.
We report here the practical implementation of this new
promising strategyby using DABCYL, BHQ-1, and BHQ-
3 as the three azo dye components of the targeted UDQ 1
(see Scheme 3). Its effective quenching range is assessed
in the context of disulfide-based FRET pairs involving
a variety of fluorophores that range from UV-A
(naphthalene) to NIR emission (sulfoindocyanine dye
Cy 7.0).
The key issue for the use of this multiple azo dyes
assembly methodology for the synthesis of 1 is the avail-
ability of an heterotrifunctional quencher core. Since such
a diazo compound has never been described in the litera-
ture, we started from scratch, designing a DABCYL
analogue equipped with easily derivatizable terminal al-
kyne and thiol moieties for the grafting of BHQ-1 and
BHQ-3 dyes through copper-mediated azideꢀalkyne 1,3-
dipolar cycloaddition (CuAAC) and SN2 thio-alkylation
reactions, respectively.10 The third reactive group, namely
carboxylic acid, is required for (bio)conjugation purposes
involving the UDQ 1. Our choice for a relatively cheap
benzenic building block as starting material resulted in the
use of 3-amino-5-nitrobenzoic acid because we thought
that the presence of a nitro group within the resulting
DABCYL-like core structure should enhance its “native”
quenching ability linked to the photoinduced motions of
NdN double bond through the PeT process.
As depicted in Scheme 1, a practical seven-step synthetic
route to the key heterotrifunctional DABCYL analogue 6
was developed. First, alkylation of N-phenyldiethanola-
mine with propargyl bromide under phase-transfer condi-
tions has enabled us to differentiate its two identical
primary alcohols. Purification by flash column chromato-
graphy led to the monoterminal alkyne 2 in a good 72%
yield. The remaining hydroxyl group of 2 was then readily
converted into a primary amine using a sequence of
mesylation, azidation, and Staudinger reduction to give
the unsymmetrical N,N-disubstituted aniline 3. Thereafter,
amidification of 3 with 3,30-dithiodipropanoic acid
(DTDP) was achieved with BOP/DIEA in dry CH3CN
to provide the dianiline disulfide derivative 4. We found
that the disulfide bridge acts as a valuable protecting group
for the thiol moiety, especially to avoid undesired side
Figure 1. Wavelength range of commonly used commercially
available dark quenchers.
However, the availability of an universal dark quencher
(UDQ) that is effective over this ultrawide wavelength
range should simplify the construction of multicolored
families of fluorogenic substrates for multiplexed bioana-
lyses and high-throughput imaging assays. In this context,
the Kool group has recently reported an elegant molecular
approach to broaden the absorption spectrum (and thus
quenching efficiency) of popular azo dyes, namely DAB-
CYL and BHQ-2.8 The resulting multipath quenchers
(MPQ) have been designed to have multiple donor or
acceptor groups in their structure, allowing for a multi-
plicity of conjugation pathways of varied length. The lead
compound, namely MPQ6, possesses a valuable spectrum
width (full-width half-maximum, Δλ1/2 max) of 270 nm,
(4) Peng, X.; Chen, H.; Draney, D. R.; Volcheck, W.; Schutz-
Geschwender, A.; Olive, D. M. Anal. Biochem. 2009, 388, 220–228.
(5) Chevalier, A.; Massif, C.; Renard, P.-Y.; Romieu, A. Chem.;
Eur. J. 2013, 19, 1686–1699 and references cited therein.
(6) Cook, R. M.; Lyttle, M.; Dick, D. Dark quenchers for donorꢀ
acceptor energy transfer. Biosearch Technologies, Inc. WO 01/
86001 A1.
(7) Haugland, R. P.; Singer, V. L.; Yue, S. T. Xanthene dyes and their
application as luminescence quenching compounds. Molecular Probes,
Inc., US 6,399,392 B1.
(9) Jernigan, F. E.; Lawrence, D. S. Chem. Commun. 2013, 49, 6728–
6730.
(10) For comprehensive reviews about bioorthognal chemistry, see:
(a) Best, M. D. Biochemistry 2009, 48, 6571–6584. (b) Sletten, E. M.;
Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48, 6974–6998.
(8) Crisalli, P.; Kool, E. T. Bioconjugate Chem. 2011, 22, 2345–2354.
B
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