Synthesis of N,N-Dialkylamino-nor-Dihydroxanthene-Hemicyanine Fused Near-Infrared Fluorophores and Their First Water-Soluble and/or Bioconjugatable Analogues

Article abstract of DOI:10.1002/asia.201700176

The effective synthesis of extended conjugated N,N-dialkylamino-nor-dihydroxanthene-based fluorophores is described from diversely functionalized salicylic aldehydes. The access to these original fluorescent derivatives proceeded in two steps through a one-pot construction of the unusual nor-dihydroxanthene (nor-DHX) scaffold followed by a diversification step providing a wide variety of nor-DHX-hemicyanine fused dyes emitting in the range of 730–790 nm. The versatility of our approach has enabled a further extension to the late-stage introduction of negatively/positively charged polar groups onto their terminal nitrogen heterocyclic subunit, thereby giving access to the first water-soluble and/or bioconjugatable members of this emerging class of NIR fluorophores. Our water-solubilizing method is easily implementable, and the nor-DHX-hemicyanine skeleton maintains satisfying fluorescence quantum yields (5–20 %) under physiological conditions. Finally, the bioconjugation ability of fluorescent derivatives bearing a free carboxylic acid was demonstrated through the covalent labeling of a model protein, namely, bovine serum albumin.

Full text of DOI:10.1002/asia.201700176

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Synthesis of N,N-Dialkylamino-nor-Dihydroxanthene-
Hemicyanine Fused Near-Infrared Fluorophores and Their First
Water-Soluble and/or Bioconjugatable Analogues
Authors: Jean-Alexandre Richard, Michelle Jui Hsien Ong, Sylvain
Debieu, Mathieu Moreau, and Anthony Romieu
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Asian J. 10.1002/asia.201700176
Link to VoR: http://dx.doi.org/10.1002/asia.201700176
A Journal of
A sister journal of Angewandte Chemie
and Chemistry – A European Journal

10.1002/asia.201700176
Chemistry - An Asian Journal
Synthesis
of
N,N-Dialkylamino-nor-Dihydroxanthene-
Hemicyanine Fused Near-Infrared Fluorophores and Their First
Water-Soluble and/or Bioconjugatable Analogues
Michelle Jui Hsien Ong,[a] Sylvain Debieu,[b] Mathieu Moreau,[b] Anthony Romieu*[b],[c] and Jean-
Alexandre Richard*[a]
Abstract: The effective synthesis of extended conjugated N,N-
dialkylamino-nor-dihydroxanthene-based fluorophores is described
from diversely functionalized salicylic aldehydes. The access to
these original fluorescent derivatives proceeded in two steps through
a one-pot construction of the unusual nor-dihydroxanthene (nor-
DHX) scaffold followed by a diversification step providing a wide
variety of nor-DHX-hemicyanine fused dyes emitting in the range
730‒790 nm. The versatility of our approach has enabled a further
extension to the late-stage introduction of negatively/positively
charged polar groups onto their terminal nitrogen heterocyclic
subunit giving access to the first water-soluble and/or
they provide in biological systems because of the gain of
sensitivity (i.e., directly linked to low background fluorescence in
the therapeutic window 700–900 nm), safety profile compared to
dyes absorbing at more energetic wavelengths, and deeper
tissue penetration.[2] The families of NIR fluorophores emitting
above 700 nm[3] have so far been dominated by the cyanine[4],[5]
and (aza-)BODIPY[6],[7] dyes and despite intense research
interest for alternative scaffolds, new classes of high
performance NIR fluorescent organic dyes are slow to emerge.[8]
In this context, we became interested in the dihydroxanthene
(DHX) fluorescent scaffold first reported by Czerney and
bioconjugatable members of this emerging class of NIR fluorophores. Grummt in 1996.[9] This fluorescent core is a modified rhodamine
Our water-solubilizing method is easily-implementable and the nor-
DHX-hemicyanine skeleton keeps satisfying fluorescence quantum
yields (5‒20%) under physiological conditions. Finally, the
bioconjugation ability of fluorescent derivatives bearing a free
carboxylic acid was demonstrated through the covalent labeling of a
model protein namely bovine serum albumin.
skeleton where the C1’‒C2’ double bond is reduced and the π-
conjugated system extended at the position C4’, resulting in a
fluorescence emission above 700 nm. Since the rediscovery of
the DHX skeleton by Lin and co-workers in 2012,[10] particularly
through a fortuitous synthesis during the reaction between a
meso-chloro substituted heptamethine cyanine dye and
Introduction
Compared to the vast number of options offered for the choice of
organic-based fluorophores emitting in the UV and visible region
of the electromagnetic spectrum, near-infrared (NIR) families of
dyes are in much shorter supply.[1] The need for new classes of
NIR fluorophores is however apparent considering the benefits
[a]
[b]
M. J. H. Ong+, Dr. J.-A. Richard
Organic Chemistry
Institute of Chemical and Engineering Sciences (ICES), Agency for
Science, Technology and Research (A*STAR)
8 Biomedical Grove, Neuros, #07-01, Singapore 138665
E-mail: jean_alexandre@ices.a-star.edu.sg
S. Debieu+, Dr. M. Moreau, Prof. A. Romieu
ICMUB, UMR 6302, CNRS, Univ. Bourgogne Franche-Comté
9, Avenue Alain Savary, 21078 Dijon cedex, France
E-mail: anthony.romieu@u-bourgogne.fr
Homepage: http://www.icmub.fr
[c]
[+]
Prof. A. Romieu
Institut Universitaire de France
103, Boulevard Saint-Michel, 75005 Paris, France
These authors contributed equally to this work.
Figure 1. (A) and (B) Previous approaches to access dihydroxanthene-
hemicyanine fused NIR fluorophores; (C) Alternative de novo synthesis
allowing the easy access to structural analogues.
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/asia.201700176
Chemistry - An Asian Journal
Figure 2. Synthesis of N,N-dialkylamino-nor-dihydroxanthene-hemicyanine fused NIR fluorophores.
resorcinol,[11] an increasing number of publications reporting
their use in a wide range of biosensing/bioimaging applications
have demonstrated the growing interest for this family of
fluorophores,[12] especially those bearing an aniline/phenol
moiety in C6’ and acting as an effective fluorescence switch (i.e.,
fluorogenic center).[13] However, the two synthetic routes
reported so far to form the DHX core rely either on the use of
Fischer’s aldehyde precursors with limited diversification
potential (Figure 1A)[10] or on the degradation of structurally
advanced, valuable heptacarbocyanine dyes (Figure 1B).[11],[14]
These approaches haven’t given much leeway to access
analogues featuring improved photophysical properties or
provide site-specifically functionalized dyes. To streamline the
access to the DHX-hemicyanine fused dyes and study more in
handle(s) for late-stage diversification.[15] Using that approach,
we also synthesized the first nor-dihydroxanthene (nor-DHX)
scaffold, a more compact skeleton than its original sibling and
for which we have already reported N,N-dialkylamino-
dihydroxanthene-pyrylium conjugated fluorophores emitting
above 800 nm.[16] However, a systematic diversification of the
electron-pulling and electron-donating parts of the nor-DHX-
based dyes has not been explored yet. In order to fill this gap,
we considered the synthesis of a small library of such NIR
fluorophores through substituent variations for the N,N-
dialkylamino donor and by changing the nature of indolium
acceptor moiety. This library enabled us to conduct a structure-
fluorescence relationship (SFR) study providing valuable
information related to the fine-tuning of photophysical properties
of these unconventional DHX-type fluorophores. In this article,
we report the results of this ambitious study as well as a further
extension of the claimed synthetic methodology to the
preparation of the first water-soluble DHX-hemicyanine fused
depth their fluorescence properties, we developed
a
methodology allowing the one-pot access to DHX skeletons
featuring an aldehyde function at the position C4’ and/or a
bromine atom as C6’ substituent, both of them used as
Table 1. Photophysical properties of nor-DHX-hemicyanine fused dyes 6a‒h and 7a‒h at 25 °C. For structures, see Figure 2.
[a] Assigned to S0–S1 transition but vibronic and/or S0–S2 transitions are also observed (see Figure 3 for examples of Abs spectra). [b] PBS buffer
containing 5% BSA. [c] Excitation at 650 nm. [d] Determined at 25 °C by using ICG (ΦF = 10.6% in DMSO, λex = 650 nm) as standard (see the Supporting
Information).
This article is protected by copyright. All rights reserved.

10.1002/asia.201700176
Chemistry - An Asian Journal
Figure 3. Normalized absorption, fluorescence emission (excitation at 650 nm) and excitation (emission at 800, 820 or 830 nm) spectra of N,N-diethylamino-nor-
DHX NIR fluorophores 6b (a and b) and 7b (c and d) at 25 °C. Please note: absorption and fluorescence emission spectra were recorded with solutions whose
concentrations are in the range 10-5–10-6 M and 10-6–10-7 M respectively. All emission spectra (also those of standard ICG) are corrected until 850 nm, which
explains the artefact observed at this wavelength.
dyes (Figure 1C). Furthermore, the availability of a free
carboxylic acid moiety within the terminal indolic subunit of some
of these compounds has enabled us to use them in fluorescent
covalent labeling of proteins.
identified the 1,3,3-trimethyl-2-methyleneindoline unit (also
known as Fischer's base) and related derivatives as the ideal
structural moieties to maximize both absorption and emission of
the π-extended nor-DHX fluorophores in the NIR range. We
therefore chose two of such indolinium salts in order to produce
sixteen new NIR fluorophores upon condensation with 3a–h,
whose fluorescence properties would give us insight regarding
the SFR of this family of dyes. We chose 1,2,3,3-tetramethyl-3H-
indolium 4 and 1,1,2,3,-tetramethyl-1H-benz[e]indolium 5 (both
as iodide salt) as condensing partners because of their simplicity
and the availability of related precursors functionalized at
various positions of the indolinium or benzoindolinium skeleton.
The Knoevenagel condensation proceeded in good to excellent
yields (50–95%) under mild conditions (i.e., K2CO3, Ac2O). The
structures of these sixteen novel DHX-type fluorophores were
Results and Discussion
Synthesis of N,N-dialkylamino-nor-DHX-hemicyanine
fused NIR fluorophores
The access to a wide variety of fluorescent organic dyes could
be ensured at the outset by choosing an appropriate range of
salicylic aldehydes displaying diversity on the amino group
donor. We therefore sourced or prepared salicylic aldehyde
precursors featuring either an acyclic N,N-dialkylamino (1a–c), a
cyclic N,N-dialkylamino (1d–f) or a fused N,N-dialkylamino (1g–
h) moiety, the latter presenting the additional advantage to
potentially lead to fluorophores not accessible using our
previously reported late-stage amination strategy.[15b] The
construction of the nor-DHX scaffold could be secured using our
optimized conditions (i.e., Cs2CO3, DMF) where N,N-substituted
salicylic aldehydes condensed one-pot with 2-bromocyclopent-1-
ene-1-carbaldehyde 2 to form the formyl derivatives 3a–h in
yields ranging from 47% to 87%. With eight nor-DHX aldehyde
precursors 3a–h in hand, we then performed a diversification
step to vary the electron-acceptor moiety of the targeted NIR
fluorophores. In accordance with the recent results reported by
Yuan and co-workers,[17] the work conducted in our group
unambiguously
confirmed
by
ESI-HRMS
and
NMR
spectroscopic analyses (see the Supporting Information).
Photophysical properties of N,N-dialkylamino-nor-DHX-
hemicyanine fused NIR fluorophores
The photophysical properties of these novel DHX-based
fluorophores were evaluated in different media including
phosphate-buffered saline (PBS) with 5% (w/v) bovine serum
albumin (BSA) as simulated body fluid, EtOH, and CHCl3. These
spectroscopic data are gathered in Table 1 and selected
examples of electronic absorption, excitation and emission
This article is protected by copyright. All rights reserved.

10.1002/asia.201700176
Chemistry - An Asian Journal
Figure 4. Synthesis of water-soluble N,N-diethyllamino-nor-dihydroxanthene NIR fluorophores (compounds 13, 15, 16 and 18 were isolated as TFA salts; the
number of TFA molecules was determined by elemental analysis).
Table 2. Photophysical properties of water-soluble nor-DHX-hemicyanine fused dyes 8–18 at 25 °C. For structures, see Figure 4.
[a] Assigned to S0–S1 transition but vibronic and/or S0–S2 transitions are also observed (see Figure 5 and the Supporting Information for the corresponding
Abs spectra). [b] Determined at 25 °C by using ICG (ΦF = 10.6% in DMSO, λex = 650 nm) as standard (see the Supporting Information). [c] Only one
absorption maxima at 715 nm was observed at a lower 10−6–10−7 M concentration. [d] A non-linear relationship between absorption and concentration was
obtained due to the formation of H-type dimer aggregates.
This article is protected by copyright. All rights reserved.

10.1002/asia.201700176
Chemistry - An Asian Journal
spectra of 6b and 7b are available in Figure 3. As expected,
fluorophores 6a–h and 7a–h display absorption and
fluorescence emission peaks in the NIR region at 710–774 nm
and 728–791 nm depending on the substitution pattern of the
amino group and the medium. However, no significant
solvatochromism effect was observed for this class of DHX-
hemicyanine hybrids. The reduction of the dihydro ring size
has no real positive or negative impact on the spectral features
of these DHX-based fluorophores because values close to
those of cyclohexenyl-based congeners previously studied
have been obtained. Comparing the properties of the 6 and 7
series differing only by the additional aromatic ring on the
hemicyanine part of the dye showed a 13‒21 nm red-shift
between 6a–h and their π–extended analogues 7a–h. The
relative fluorescence quantum yields under physiological
conditions were better for the indolinium analogues 6a–h (8–
16%) than for benzoindolinium 7a–h (4–6%), and sufficient for
considering the further use of these NIR emitters in
biosensing/bioimaging applications. Furthermore, it is known
that the presence of BSA or related proteins in aq. media
enhances the emission of organic-based fluorophores owing to
a combination of rigidification, a reduction in the polarity of the
dye’s microenvironment (binding in the hydrophobic BSA
pocket), and deaggregation.[18] Interestingly, we noted that the
quantum yields of the nor-DHX indolinium analogues 6a–h were
notably superior compared to the values (4–11%) already
Figure 5. Normalized absorption, fluorescence emission (excitation at 650
nm) and excitation (emission at 820 nm) spectra of water-soluble N,N-
diethylamino-nor-DHX NIR fluorophores 8 (a) and 17 (b) in PBS at 25 °C.
Please note: absorption and fluorescence emission spectra were recorded
with solutions whose concentrations are in the range 10−5–10−6 M and 10−6
–
10−7 M respectively. All emission spectra (also those of standard ICG) are
corrected until 850 nm, which explains the artefact observed at this
wavelength. If the absorption spectrum of 8 in PBS is recorded in the range of
10−6–10−7 M, the red-shift band at 787 nm disappears and a single maximum
at 715 nm is observed.
reported for
a
similar subset of N,N-dialkylamino-
dihydroxanthene-based fluorophores.[15b] This may be explained
by the less flexibility of the 5-ring system that limits molecular
motions involved in the non-radiative decay pathways. The
nature of the N,N-dialkylamino electron-donating group
appeared to be also important to influence the brightness of the
nor-DHX fluorophores. For instance, it is worth highlighting the
superiority of dyes 6e and 6f featuring either a 3,4-dihydro
pyrrolidinyl or pyrrolidinyl over the other N,N-dialkylamino
donating moieties, including the azetidinyl ring which had
previously been reported as being the optimum electron-
donating group for brighter rhodamine-based fluorophores
namely Janelia Fluor™dyes (Table 1).[19]
alkylating agent. For this purpose, we set out to take advantage
of the divergency of our synthetic route to introduce one or two
polar groups negatively- and/or positively-charged at
physiological pH, to the skeleton of the fluorophore. The three
hydrophilic
moieties
(i.e, carboxylate,
sulfonate and
trimethylalkylammonium) selected to be added to the nor-DHX-
hemicyanine hybrid scaffold during the diversification step were
therefore positioned at various specific positions of the indolium
partner. Because of its convenient availability on gram scale, we
performed the water-solubilization step on nor-DHX aldehyde
precursor 3b (see Figure 4 as an illustration of the scope of our
investigations). Our previsouly established conditions for the
Knoevenagel condensation (i.e., K2CO3, Ac2O) proved to be
disappointing since only traces of mono-sulfonated fluorophore 8
could be identified in the reaction mixture. To our delight, we
realized that the presence of base was detrimental to the
formation of the dye and that by simply removing it (i.e., Ac2O,
conditions A), the Knoevenagel-type condensation between 3b
and a set of indolinium salts bearing either a single or two
sulfonates or a carboxylic acid functionality worked well. The
resulting hydrophilic derivatives were isolated by flash-column
chromatography on silica gel in good yields (53–88%). It is worth
noting the additional benefit of the carboxylic acid function in 11
Synthesis of water-soluble N,N-dialkylamino-nor-DHX-
hemicyanine fused NIR fluorophores
Capitalizing on the promising fluorescence properties obtained
for the nor-DHX- and DHX-based fluorophores in PBS + 5%
BSA, we were then interested in exploring the synthesis of the
first hydrophilic variations of these unusual fluorescent scaffolds
to make easier their applications in biological media. To the best
of our knowledge, no water-soluble DHX analogue has been
reported to date and we hypothesized that the hemicyanine part
could be functionalized through the site-specific introduction of
polar group(s) onto the phenyl ring and/or through the N-
quaternarization of (benzo)indole unit with
a hydrophilic
This article is protected by copyright. All rights reserved.

10.1002/asia.201700176
Chemistry - An Asian Journal
Figure 6. MALDI-TOF mass spectra of BSA (a) and fluorescent conjugates BSA-12 (b, labeling conditions: 15 eq. of NHS ester of nor-DHX-hemicyanine fused
dye 12 in phosphate buffer at pH 7.0), BSA-15 (c, labeling conditions: 15 eq. of NHS ester of nor-DHX-hemicyanine fused dye 15 in phosphate buffer at pH 7.0)
and BSA-18 (d, labeling conditions: 15 eq. of NHS ester of nor-DHX-hemicyanine fused dye 18 in phosphate buffer at pH 7.0) recorded in the positive mode
(matrix: sinapinic acid).
and 12 which can also be used as a reactive handle for further
functionalization through amidification reactions. As an
illustrative example, we performed an amide coupling between
12 and 2-aminoethane-1,1-disulfonic acid (DIEA salt)[20] and
mediated by uronium reagent HATU,[21] that allowed the post-
synthetic sulfonation of this DHX-hemicyanine fused dye 12
under very mild conditions. After purification by semi-preparative
RP-HPLC with aq. 0.1% TFA and CH3CN as eluents, compound
13 was obtained in pure form and in a satisfying 31% isolated
yield. Further extension of the Knoevenagel-type reaction with
more polar indolinium condensing partner failed due to solubility
issues even at higher reaction temperatures. Inspired by a
literature precedent[22], we found that the use of CH3OH as
solvent was not only able to solubilize all the starting materials
but also successfully led to the desired water-soluble DHX-
based fluorophores 14–18 in the presence of a trace amount of
a weak base (i.e., pyridine, CH3OH, conditions B). Isolation in a
pure form of 15, 16 and 18 was achieved by semi-preparative
RP-HPLC with aq. 0.1% TFA and CH3CN as eluents (isolated
yields: 10%, 14% and 37% respectively) whereas compounds
14 and 17 were purified by flash-column chromatography on
silica gel. It is important to underline that the preparation of the
indolium precursor involved in the synthesis of 15 was not trivial
because the N-quaternarization of 2,3,3-trimethyl-3H-indole-5-
carboxylic acid with an excess of 1,3-propanesultone (acting as
both reagent and solvent) at 120–145 °C also led to complete
esterification of the carboxylic acid (see the Supporting
Information for the preparation of such starting indolinium salt).
A further acidic hydrolysis (aq. 6 N HCl) followed by a
purification
by
semi-preparative
RP-HPLC
with
aq.
triethylammonium bicarbonate buffer (TEAB, 50 mM, pH 7.5)
and CH3CN as eluents were required to readily obtain this
starting material. Trimethylalkylammonium groups have recently
emerged as valuable water-solubilizing moieties to dramatically
improve the solubility of a wide range of organic-based
fluorophores under physiological conditions.[23] That approach,
mainly applied to heptamethine cyanine dyes, has led to better
performances in vivo in the context of NIR fluorescence
molecular imaging[5b, 5e, 5f, 5i, 24] and prompted us to introduce the
positively-charged trimethylpropylammonium group onto the nor-
DHX-hemicyanine hybrid scaffold. Fluorophores 17 and 18
bearing a positive net charge q = +2 and q = +1 at physiological
pH were therefore synthesized, the latter one featuring an
additional carboxylic acid group for further functionalization (vide
infra).
This article is protected by copyright. All rights reserved.

10.1002/asia.201700176
Chemistry - An Asian Journal
Photophysical properties of water-soluble N,N-
fused
dialkylamino-nor-DHX-hemicyanine
fluorophores
NIR
The introduction of polar groups onto the skeleton of the nor-
DHX-based fluorophores gave us the opportunity to assess their
influence on the solubility and aggregation behavior in
physiological conditions. As
a
general trend, nor-DHX
derivatives bearing a single hydrophilic group are soluble in
water and related aq. buffers in the concentration range 1–100
µM, and the upper limit is 1–2 mM for the bis-sulfonated
derivatives 10, 13, 14 and 16, and carboxylic acids 15 and 18.
The photophysical properties were determined in pure PBS and
in PBS containing 5% (w/v) BSA and gathered in Table 2 (see
Figure 5 and the Supporting Information for the electronic
absorption, excitation and emission spectra). In the
concentration range 5–15 µM, we found that
a partial
aggregation occurred in PBS with fluorophores 8–16 featuring
carboxylate and/or sulfonate group(s) (i.e., negatively-charged at
physiological pH), which prevents an accurate determination of
their molar extinction coefficients at absorption maxima.
However, these compounds remained fluorescent and
satisfactory fluorescence quantum yields (3–9%) were obtained.
Such aggregation phenomenon in PBS could be completely
avoided through the introduction of the positively-charged
trimethylpropylammonium moiety. Indeed, for compounds 17
and 18, a good matching between the absorption and excitation
are accessible for conjugation.[26] Since the ε-amino groups of
lysine residues of BSA are known to form amides through
reaction with a wide range of activated esters in aqueous buffers,
the conversion of 12, 15 and 18 into their corresponding NHS
esters using uronium-based coupling agent TSTU[27] (1.1 eq.)
and DIEA (2–3 eq.) in DMSO was considered. BSA was labeled
after overnight incubation with either a 15- or 30-fold molar
excess of the NHS ester in phosphate buffer (pH 7.0 or pH 7.7)
at 4 °C. The nor-DHX-conjugated proteins BSA-12, BSA-15 and
BSA-18 were washed with an ultra-centrifugal filter device (30
kDa cut-off) to remove most of the excess of free unbound nor-
DHX-hemicyanine fused dye. Since the method of choice for
analyzing protein conjugates is mass spectrometry, all labeled
protein samples were subjected to matrix-assisted laser
desorption time-of-flight (MALDI-TOF) analyses and their
spectra were compared with that of the parent BSA (Figure 6
and Figures S1–S3 in the Supporting Information). In all of the
mass spectra, a main peak assigned to [M+H]+ (molecular ion)
was observed. The mass difference between molecular ions of
BSA and its fluorescent conjugates allowed to estimate labeling
densities, defined as the (average) number of fluorophore
molecules attached to a protein (F/P). The corresponding values
are gathered in Table 3. Moderate to high F/P values (4.9–14.4)
were obtained but a poor correlation was observed with those
determined by the UV-vis spectrophotometric method (see the
Supporting Information), mainly for fluorescent conjugates BSA-
15 and BSA-18. Even if there are no scientific certainties to
interpret these unexpected results, we assumed the
hetereogeneity of labeled protein samples containing residual
amounts of free nor-DHX-hemicyanine fused dye (monomeric
and/or aggregated forms) not removed by ultracentrifugation and
adsorbed on the protein. The latter hypothesis was supported by
gel electrophoresis and subsequent imaging of the gel by
fluorescence scanning with IVIS Lumina III in vivo imaging
system (Ex/Em filters 660/790 nm, bandwidth 20/40 nm, Figure
S4 in the Supporting Information) showing the presence of both
targeted protein-dye conjugates and free dye. Despite the
spectra was observed and
a linear relationship between
absorbance and concentration was obtained (see Figure 5b and
the Supporting Information). Compared to these measurements,
those achieved in PBS/BSA revealed a notable red-shift for the
absorption spectra, resulting in lower Stokes' shifts (7–26 nm,
130–480 cm−1 vs. 24–41 nm, 449–782 cm−1). Using the optical
properties of 6b as benchmark (i.e., Abs/Em λmax = 726/736 nm,
ε = 76 530 M−1 cm−1 and ΦF = 12% in PBS + 5% BSA) to
compare with those of more hydrophilic nor-DHX-hemicyanine
fused dyes 8–18 in PBS/BSA, we observed that the absorption
and emission maxima were in the range 722–740 nm and 738–
761 nm respectively, directly dependent on the presence of
ionized substituent(s) on N-alkyl arm and/or phenyl ring of
indolinium unit. Gratifyingly, a 50% increase of fluorescence
quantum yield was obtained with fluorophore 8, showing the
beneficial effect of such functionalization to obtain bright and
biocompatible DHX-hemicaynine fused dyes.
Water-soluble
N,N-dialkylamino-nor-DHX-hemicyanine
fused dyes as fluorescent covalent labeling reagents of
biomolecules
The marked solubility in aqueous media, the valuable optical
properties, and the availability of a free carboxylic acid group on
their core structure, are positive features for the use of DHX
derivatives 12, 15 and 18 in fluorescent bio-labeling
applications.[25] To demonstrate their ability both to readily react
with proteins under mild conditions and to give fluorescent
bioconjugates, the labeling of BSA as a model protein was
explored. BSA contains 59 lysine residues, and more than thirty
This article is protected by copyright. All rights reserved.

10.1002/asia.201700176
Chemistry - An Asian Journal
presence of unbound fluorophore molecules in the labeled
soluble fluorogenic probes (also known as fluorescent
chemodosimeters) for various analytes.[32]
protein
samples,
the
spectroscopic
features
(absorption/emission maxima and quantum yields) of the BSA
conjugates were determined (see Table 3 and Figure S5 in the Experimental Section
Supporting Information for selected examples of electronic
absorption, excitation and emission spectra). The absorption
spectrum of fluorescent protein conjugates in PBS revealed two
distinct peaks at 658–682 nm and 716–731 nm. The dual
absorption, the breadth of the absorption bands and the blue-
shift of one of them to 35–50 nm (compared to the absorption
maximum of 12, 15 or 18) support the formation of non-emissive
aggregates (i.e., H-type homodimers).[28] The low values of the
measured quantum yields (1–2% vs. 5–6% for the corresponding
free nor-DHX-hemicyanine fused dyes) may be attributed to a
combination of several effects, including (1) H-dimer formation,
(2) autoquenching induced by interactions between fluorophores
and aromatic rings on the side chains of amino acids such as
tryptophan and phenylalanine.[29] These results are mixed but
the use of DHX-based fluorophores as fluorescent labeling
reagents of amine-containing biomolecules was demonstrate
dual absorptiond for the first time.
See the Supporting Information for the details about sections
"General", "Instruments and methods", "HPLC separations" and
all experimental data associated with synthesized compounds
and fluorescent BSA conjugates.
Procedure for the synthesis of nor-DHX aldehydes 3a–h
To a solution of salicylic aldehydes 1a–d, 1g or 1h in dry DMF at
25 °C were added Cs2CO3 (3 eq.) and crude 2-bromocyclopent-
1-ene-1-carbaldehyde 2 (2 eq.) in solution in dry DMF. The
resulting reaction mixture was stirred for 48 h at 25 °C to reveal
an intense yellow spot (TLC hexane/EtOAc, 8:2, v/v). The
insoluble material was then filtered on a pad of silica gel and the
filtrate was concentrated under vacuum. The resulting residue
was dissolved in CH2Cl2 and washed with deionized water. The
organic layer was dried over anhydrous Na2SO4, filtered and
concentrated
in
vacuo. Purification
by flash-column
chromatography on silica gel (CH2Cl2/EtOAc, 9:1, v/v) provided
the desired aldehydes 3a–d, 3g or 3h as deep orange solids.
Conclusions
Salicylic aldehydes 3e and 3f were prepared as following: to a
solution of aldehyde 3c (37 mg, 0.13 mmol) in CH2Cl2 (4 mL)
were added 1st generation Grubbs’ catalyst (5.2 mg, 0.0063
mmol, 0.05 eq.) and p-benzoquinone (1.4 mg, 0.013 mmol, 0.1
eq.). The reaction mixture was stirred at 25 °C for 1 h before it
was filtered over a short pad of silica gel. The filtrate was
concentrated under vacuum and provided 3e which was used
directly in the next step, either for the formation of dyes 6e and
7e (vide infra) or hydrogenated in toluene for 1 h in the presence
of Adam's catalyst (PtO2 • H2O). After removing the catalyst on a
short pad of silica gel, the filtrate was concentrated and cleanly
afforded aldehyde 3f which was used without further purification
in the formation of nor-DHX dyes 6f and 7f.
In summary, we developed a new family of organic NIR
fluorophores based on the unusual nor-dihydroxanthene scaffold.
We designed a synthetic route allowing the expedient formation
of the nor-DHX scaffold which was suitably functionalized for a
subsequent diversification step allowing the formation of wide
range of DHX-hemicyanine fused dyes. We applied our strategy
to the rapid synthesis of nor-DHX-based fluorophores featuring
either a N,N-dialkylamino, a cyclic amino or a fused (bi)cyclic
amino group as electron-donating moiety and identified 5-
membered-ring heterocyclic amines as being optimum
substituent to maximize the fluorescence emission (fluorescence
quantum yields 15–20%). A further and easily-implementable
extension of this methodology has led to the first water-soluble
nor-DHX-based fluorophores through the introduction of
hydrophilic groups performed on the hemicyanine part of the
molecule. Despite the "anisotropic" character of this water-
solubilizing methodology known to favor the formation of non-
fluorescent micellar aggregates,[30] we managed to identify the
positively-charged trimethylpropylammonium group as an
effective moiety to readily water-solubilize the DHX-hemicyanine
fused dyes as well as to avoid their aggregation at physiological
pH. However, to improve the brightness of these water-soluble
DHX-based fluorophores, further work is in progress in our
General procedure for the synthesis of nor-DHX-
hemicyanine fused dyes 6a–h and 7a–h
To nor-DHX aldehydes 3a–h in anhydrous Ac2O (0.025–0.05 M)
were added 1,2,3,3-tetramethyl-3H-indolium iodide 4 or 1,1,2,3,-
tetramethyl-1H-benz[e]indolium iodide 5 (1.2 eq.) along with
K2CO3 (2 eq.) and the mixture was stirred at 25 °C for 16 h to
reveal an intense green spot (TLC CH2Cl2/CH3OH, 9:1, v/v). The
reaction mixture was concentrated and the resulting residue
dissolved in CH2Cl2 and washed with deionized water. The
organic layer was dried over anhydrous Na2SO4, filtered and
concentrated
in
vacuo. Purification
by flash-column
chromatography on silica gel (step gradient of CH3OH in CH2Cl2
from 0% to 3%) afforded NIR nor-DHX-based fluorophores 6a–h
and 7a–h.
laboratories to devise
a
complementary functionalization
approach of the DHX core through the introduction of polar
subtituent(s) on the donor part of the fluorophore skeleton.
Finally, we demonstrated the bioconjugation ability of nor-DHX-
hemicyanine fused dyes bearing a free carboxylic acid by
performing the fluorescent labeling of the BSA protein. Thus, this
contribution paves the way for a further use of DHX-based NIR
fluorophores in the rational design of either molecular imaging
agents (mainly, antibody-NIR dye conjugates)[31] or water-
General procedure for the synthesis of water-soluble
nor-DHX-hemicyanine fused dyes 8–12
To nor-DHX aldehyde 3b in anhydrous Ac2O (0.025–0.1 M) were
added the corresponding indolinium salt (1.2 eq.) and the
mixture was stirred at 25 °C for 16 h to reveal an intense green
spot (TLC CH2Cl2/CH3OH, 9:1, v/v). The reaction mixture was
concentrated and directly loaded on silica gel and purified (step
This article is protected by copyright. All rights reserved.

10.1002/asia.201700176
Chemistry - An Asian Journal
gradient of CH3OH in CH2Cl2 from 0% to 10‒15%) to afford nor-
DHX-based fluorophores 8–12.
gradient of CH3OH in CH2Cl2 from 0% to 50%). N-
(Trimethylammonio)propyl nor-DHX-hemicyanine NIR dye 17
was obtained as a green solid (44 mg, yield 33%).
Synthesis of nor-DHX-hemicyanine fused dye (13)
Isolation of dye 18: The crude product was dissolved in a (1 :
1) mixture of aq. 0.1% TFA and CH3CN (ca. 5 mL) and purified
by semi-preparative RP-HPLC (for more detail see the
Supporting Information). The product-containing fractions were
lyophilized to give the TFA salt (3.5 TFA) of compound 18, as a
green amorphous powder (60 mg, yield 37%).
Carboxylic acid-functionalized nor-DHX-hemicyanine NIR dye 12
(60 mg, 0.1 mmol, 1 eq.) was dissolved in dry DMF (2 mL). DIEA
(34 µL, 0.2 mmol, 2 eq.) and HATU (43 mg, 0.11 mmol, 1.1 eq.)
were sequentially added. The resulting reaction mixture was
stirred at 25 °C for 15 min. The resulting crude HOAt activated
ester was added dropwise to a pre-cooled solution of 2-
aminoethane-1,1-disulfonic acid (DIEA salt) in dry DMF (0.19 M
in DMF, 1 mmol, 10 eq.) and DIEA (85 µL, 0.5 mmol, 5 eq.) and
the resulting mixture was stirred at 25 °C for 1 h. The reaction
was checked for completion by TLC (CH2Cl2/CH3OH, 9:1, v/v),
quenched by adding glacial AcOH (50 µL) and finally evaporated
under reduced pressure. The resulting residue was dissolved in
a (1:1, v/v) mixture of aq. 0.1% TFA and CH3CN (ca. 5 mL) and
purified by semi-preparative RP-HPLC (for more detail see the
Supporting Information). The product-containing fractions were
lyophilized to give the TFA salt (1.5 TFA) of compound 13, as a
green amorphous powder (26 mg, yield 31%).
Preparation of fluorescent BSA conjugates
(a) Synthesis of NHS esters: carboxylic acid-functionalized nor-
DHX-hemicyanine NIR dye 12, 15 or 18 (1.2‒2.0 µmole, 1 eq.,
weighed in a 0.5 mL "eppendorf"-type microtube) was dissolved
in dry DMSO (final concentration 25 mM). 1.1 eq. of TSTU (7.6‒
9.0 µL of a 180 mM solution in DMSO) and 2 eq. (or 3 eq. for 18)
of DIEA (1.47‒2.0 µL of a 2.0 M solution in NMP) were
sequentially added and the resulting mixture was periodically
vortexed for 1 h. The reaction was checked for completion by
ESI-MS. The resulting NHS esters were used in the next BSA
labeling step without purification.
General procedure for the synthesis of nor-DHX-
hemicyanine fused dyes 14–18
(b) Fluorescent labeling of BSA: The solution of NHS ester (see
above, 15- or 30-fold excess according to protein) was added to
a solution of BSA (500 µL, 1.8 mg/mL, 13.5 nmol) in phosphate
buffer (pH 7.05 or 7.70). The resulting mixture was protected
from light and periodically vortexed. The reaction was left at 4 °C
overnight and further 2 h at 20 °C. Thereafter, the mixture was
diluted with phosphate buffer (1 mL), centrifugated to remove
insoluble materials (excess of NHS ester and/or starting dye).
Thereafter, the solution was transferred to an ultra centrifugal
filter device (Amicon Ultra 2 mL, Ultracel cut-off 30 kDa from
Merck Millipore, ref. UFC203024) and centrifugated at 4000 rpm
for 15 min. For each fluorescent BSA conjugate, 50‒100 µL of
solution was recovered. Confirmation of conjugation to the
protein was achieved by MALDI-TOF mass spectrometry.
To nor-DHX aldehyde 3b in HPLC-grade CH3OH (0.025–0.05 M)
were added the corresponding indolinium or benzoindolinium
salt (1.2 eq.) and dry pyridine (one or two drops). The resulting
reaction mixture was stirred under reflux (except for 18, stirring
at 25 °C) for 2 h and at 25 °C for 16 h (please note: the color
gradually changed to green). The reaction was checked for
completion by TLC (CH2Cl2/CH3OH, 8:2, v/v), evaporated under
reduced pressure and purified by flash-column chromatography
on silica gel or by semi-preparative RP-HPLC.
Isolation of dye 14: The crude product was purified by flash-
column chromatography over silica gel (dry loading, step
gradient of CH3OH in CH2Cl2 from 0% to 20%). Bis-sulfonated
nor-DHX-hemicyanine NIR dye 14 was obtained as a green solid
(42 mg, yield 47%).
Acknowledgements
Financial support for this work was provided by ICES, A*STAR
(Singapore), Institut Universitaire de France (IUF), the Burgundy
Isolation of dye 15: The crude product was dissolved in a (1 :
1) mixture of aq. 0.1% TFA and CH3CN (ca. 5 mL) and purified
by semi-preparative RP-HPLC (for more detail see the
Supporting Information). The product-containing fractions were
lyophilized to give the TFA salt (1.5 TFA) of compound 15, as a
green amorphous powder (22 mg, yield 10%). Please note: the
low isolated yield was explained by partial degradation of nor-
DHX-hemicyanine fused dye in the crude reaction mixture due to
too prolonged heating at reflux.
region ("FABER" programme, PARI Action 6, SSTIC
6
"Imagerie, instrumentation, chimie et applications biomédicales"),
and a PHC Merlion grant (2015, 5.04.15). J.-A.R. thanks Ms
Doris Tan (ICES) for high resolution mass spectrometric (HRMS)
assistance. S.D. thanks the Burgundy region for his Ph. D. grant.
The authors thanks the "Plateforme d’Analyse Chimique et de
Synthèse Moléculaire de l’Université de Bourgogne" (PACSMUB,
http://www.wpcm.fr) and the plateform "Dispositif Interrégional
en Imagerie Cellulaire" (DImaCell, http://www.dimacell.fr) for
access to spectroscopy instrumentation. The authors also thank
Dr. Pascale Winckler (DImaCell PIMS, UMR PAM, AgroSup
Isolation of dye 16: The crude product was purified by flash-
column chromatography over silica gel (dry loading, bed size 20
× 170 mm, step gradient of CH3OH in CH2Cl2 from 0% to 30%).
Carboxylic acid 3-sulfonatopropyl ester-functionalized mono-
sulfonated nor-DHX-hemicyanine NIR dye 16 was obtained as a
green solid which was submitted to a further purification by
semi-preparative RP-HPLC (for more detail see the Supporting
Information). The product-containing fractions were lyophilized
to give the TFA salt (1.25 TFA) of compound 16, as a green
amorphous powder (17 mg, yield 14%).
Dijon)
for
technical
assistance
during
fluorescence
measurements, Marie-José Penouilh and Marcel Soustelle
(University of Burgundy, ICMUB, UMR CNRS 6302) for HRMS
measurements and elemental analysis respectively.
Keywords: nor-dihydroxanthene • fluorescence • NIR dyes •
Isolation of dye 17: The crude product was purified by flash-
column chromatography over silica gel (dry loading, step
protein labeling • water solubility
This article is protected by copyright. All rights reserved.

10.1002/asia.201700176
Chemistry - An Asian Journal
[1] a) L. D. Lavis, R. T. Raines, ACS Chem. Biol. 2008, 3, 142−155; b) L. D.
Lavis, R. T. Raines, ACS Chem. Biol. 2014, 9, 855−866.
[2] a) R. Weissleder, U. Mahmood, Radiology 2001, 219, 316−333; b) J. V.
Frangioni, Curr. Opin. Chem. Biol. 2003, 7, 626−634; c) Martinić, I.;
Eliseeva, S. V.; Petoud, S. J. Lumin. 2017, in press, see DOI:
http://dx.doi.org/10.1016/j.jlumin.2016.09.058.
6545; c) S. Zhu, W. Lin, L. Yuan, Anal. Methods 2013, 5, 3450−3453; d)
L. Li, Z. Li, W. Shi, X. Li, H. Ma, Anal. Chem. 2014, 86, 6115−6120; e) H.
Chen, B. Dong, Y. Tang, W. Lin, Chem. Eur. J. 2015, 21, 11696−11700;
f) Z. Li, X. He, Z. Wang, R. Yang, W. Shi, H. Ma, Biosens. Bioelectron.
2015, 63, 112−116; g) J. Zhang, L. Ning, J. Liu, J. Wang, B. Yu, X. Liu, X.
Yao, Z. Zhang, H. Zhang, Anal. Chem. 2015, 87, 9101−9107; h) J.
Zhang, J. Wang, J. Liu, L. Ning, X. Zhu, B. Yu, X. Liu, X. Yao, H. Zhang,
Anal. Chem. 2015, 87, 4856−4863; i) Y. Tan, R. Liu, H. Zhang, R. Peltier,
Y.-W. Lam, Q. Zhu, Y. Hu, H. Sun, Sci. Rep. 2015, 5, 16979; j) Y. Li, Y.
Wang, S. Yang, Y. Zhao, L. Yuan, J. Zheng, R. Yang, Anal. Chem. 2015,
87, 2495−2503; k) C. Han, H. Yang, M. Chen, Q. Su, W. Feng, F. Li,
ACS Appl. Mater. Interfaces 2015, 7, 27968−27975; l) S. Chen, C. Ma,
M.-S. Yuan, W. Wang, D.-E. Wang, S.-W. Chen, J. Wang, RSC Adv.
2016, 6, 85529−85537; m) B. Dong, K. Zheng, Y. Tang, W. Lin, J. Mater.
Chem. B 2016, 4, 1263−1269; n) Q. Wan, S. Chen, W. Shi, L. Li, H. Ma,
Angew. Chem. Int. Ed. 2014, 53, 10916−10920; o) X. Gong, X.-F. Yang,
Y. Zhong, Y. Chen, Z. Li, Dyes Pigm. 2016, 131, 24−32; p) Q. Hu, C. Yu,
X. Xia, F. Zeng, S. Wu, Biosens. Bioelectron. 2016, 81, 341−348; q) F.
Kong, L. Ge, X. Pan, K. Xu, X. Liu, B. Tang, Chem. Sci. 2016, 7, 1051−
1056; r) F. Kong, Z. Liang, D. Luan, X. Liu, K. Xu, B. Tang, Anal. Chem.
2016, 88, 6450−6456; s) L. Li, W. Shi, X. Wu, Q. Gong, X. Li, H. Ma,
Biosens. Bioelectron. 2016, 81, 395−400; t) F. Xu, H. Li, Q. Yao, J. Fan,
J. Wang, X. Peng, J. Mater. Chem. B 2016, 4, 7363−7367; u) J. Zhang,
C. Li, R. Zhang, F. Zhang, W. Liu, X. Liu, S. M.-Y. Lee, H. Zhang, Chem.
Commun. 2016, 52, 2679−2682; v) X. Wu, L. Li, W. Shi, Q. Gong, H. Ma,
Angew. Chem. Int. Ed. 2016, 55, 14728−14732; w) L. Zhang, X. E.
Zheng, F. Zou, Y. Shang, W. Meng, E. Lai, Z. Xu, Y. Liu, J. Zhao, Sci.
Rep. 2016, 6, 18868; x) P. Qi, D. Zhang, Y. Sun, Y. Wan, Anal. Methods
2016, 8, 3339−3344; y) W. Su, B. Gu, X. Hu, X. Duan, Y. Zhang, H. Li, S.
Yao, Dyes Pigm. 2017, 137, 293−298; z) J. Xu, J. Pan, Y. Zhang, J. Liu,
L. Zeng, X. Liu, Sens. Actuator B-Chem. 2017, 238, 58−65; aa) Y. Pan,
T.-B. Ren, D. Cheng, Z.-B. Zeng, L. Yuan, X.-B. Zhang, Chem. Asian J.
2016, 11, 3575−3582.
[3] K. Umezawa, D. Citterio, K. Suzuki, Anal. Sci. 2014, 30, 327−349.
[4] For comprehensive reviews, see: a) A. Mishra, R. K. Behera, P. K.
Behera, B. K. Mishra, G. B. Behera, Chem. Rev. 2000, 100, 1973−2012;
b) B. Ballou, L. A. Ernst, A. S. Waggoner, Curr. Med. Chem. 2005, 12,
795−805; c) M. Panigrahi, S. Dash, S. Patel, B. K. Mishra, Tetrahedron
2012, 68, 781−805; d) M. Henary, A. Levitz, Dyes Pigm. 2013, 99, 1107−
1116; e) A. P. Gorka, R. R. Nani, M. J. Schnermann, Org. Biomol. Chem.
2015, 13, 7584−7598; f) W. Sun, S. Guo, C. Hu, J. Fan, X. Peng, Chem.
Rev. 2016, 116, 7768−7817.
[5] For selected examples, see: a) A. Samanta, M. Vendrell, R. Das, Y.-T.
Chang, Chem. Commun. 2010, 46, 7406−7408; b) H. S. Choi, K. Nasr, S.
Alyabyev, D. Feith, J. H. Lee, S. H. Kim, Y. Ashitate, H. Hyun, G.
Patonay, L. Strekowski, M. Henary, J. V. Frangioni, Angew. Chem. Int.
Ed. 2011, 50, 6258−6263; c) R. K. Das, A. Samanta, H.-H. Ha, Y.-T.
Chang, RSC Adv. 2011, 1, 573−575; d) N. Karton-Lifshin, L. Albertazzi,
M. Bendikov, P. S. Baran, D. Shabat, J. Am. Chem. Soc. 2012, 134,
20412−20420; e) H. S. Choi, S. L. Gibbs, J. H. Lee, S. H. Kim, Y.
Ashitate, F. Liu, H. Hyun, G. Park, Y. Xie, S. Bae, M. Henary, J. V.
Frangioni, Nat. Biotechnol. 2013, 31, 148−153; f) H. Hyun, E. A. Owens,
L. Narayana, H. Wada, J. Gravier, K. Bao, J. V. Frangioni, H. S. Choi, M.
Henary, RSC Adv. 2014, 4, 58762−58768; g) H. Hyun, H. Wada, K. Bao,
J. Gravier, Y. Yadav, M. Laramie, M. Henary, J. V. Frangioni, H. S. Choi,
Angew. Chem. Int. Ed. 2014, 53, 10668−10672; h) C. N. Njiojob, E. A.
Owens, L. Narayana, H. Hyun, H. S. Choi, M. Henary, J. Med. Chem.
2015, 58, 2845−2854; i) D. Su, C. L. Teoh, A. Samanta, N.-Y. Kang, S.-J.
Park, Y.-T. Chang, Chem. Commun. 2015, 51, 3989−3992; j) H. Hyun, M.
Henary, T. Gao, L. Narayana, E. A. Owens, J. H. Lee, G. Park, H. Wada,
Y. Ashitate, J. V. Frangioni, H. S. Choi, Mol. Imaging Biol. 2016, 18, 52−
61.
[14] H. Chen, W. Lin, H. Cui, W. Jiang, Chem. Eur. J. 2015, 21, 733−745.
[15] a) J.-A. Richard, Org. Biomol. Chem. 2015, 13, 8169−8172; b) M. J. H.
Ong, R. Srinivasan, A. Romieu, J.-A. Richard, Org. Lett. 2016, 18, 5122−
5125.
[16] A. Romieu, J.-A. Richard, Tetrahedron Lett. 2016, 57, 317−320.
[17] Y. Wei, D. Cheng, T. Ren, Y. Li, Z. Zeng, L. Yuan, Anal. Chem. 2016, 88,
1842−1849.
[6] For comprehensive reviews, see: a) H. Lu, J. Mack, Y. Yang, Z. Shen,
Chem. Soc. Rev. 2014, 43, 4778−4823; b) Y. Ni, J. Wu, Org. Biomol.
Chem. 2014, 12, 3774−3791; c) Y. Ge, D. F. O'Shea, Chem. Soc. Rev.
2016, 45, 3846−3864; d) J. Xin-Dong, L. Shuang, G. Jian, F. Tao, L. Xin,
X. Lin-Jiu, Curr. Org. Chem. 2016, 20, 1736−1744.
[7] For selected examples, see: a) W. Zhao, E. M. Carreira, Angew. Chem.
Int. Ed. 2005, 44, 1677−1679; b) W. Zhao, E. M. Carreira, Chem. Eur. J.
2006, 12, 7254−7263; c) K. Umezawa, Y. Nakamura, H. Makino, D.
Citterio, K. Suzuki, J. Am. Chem. Soc. 2008, 130, 1550−1551; d) K.
Umezawa, A. Matsui, Y. Nakamura, D. Citterio, K. Suzuki, Chem. Eur. J.
2009, 15, 1096−1106; e) X.-D. Jiang, D. Xi, C.-l. Sun, J. Guan, M. He,
L.-J. Xiao, Tetrahedron Lett. 2015, 56, 4868−4870.
[8] a) S. Achilefu, Angew. Chem. Int. Ed. 2010, 49, 9816−9818; b) M.
Ptaszek, Prog. Mol. Biol. Transl. Sci. 2013, 113, 59−108.
[9] P. Czerney, U.-W. Grummt, J. Chem. Res., Synop. 1996, 4, 173.
[10] L. Yuan, W. Lin, Y. Yang, H. Chen, J. Am. Chem. Soc. 2012, 134, 1200−
1211.
[18] J. Pauli, M. Grabolle, R. Brehm, M. Spieles, F. M. Hamann, M. Wenzel, I.
Hilger, U. Resch-Genger, Bioconjugate Chem. 2011, 22, 1298−1308.
[19] a) J. B. Grimm, B. P. English, J. Chen, J. P. Slaughter, Z. Zhang, A.
Revyakin, R. Patel, J. J. Macklin, D. Normanno, R. H. Singer, T. Lionnet,
L. D. Lavis, Nat. Methods 2015, 12, 244−250; b) J. B. Grimm, B. P.
English, H. Choi, A. K. Muthusamy, B. P. Mehl, P. Dong, T. A. Brown, J.
Lippincott-Schwartz, Z. Liu, T. Lionnet, L. D. Lavis, Nat. Meth. 2016, 13,
985-988.
[20] A. Romieu, D. Tavernier-Lohr, S. Pellet-Rostaing, M. Lemaire, P.-Y.
Renard, Tetrahedron Lett. 2010, 51, 3304−3308.
[21] A. El-Faham, F. Albericio, Chem. Rev. 2011, 111, 6557−6602.
[22] J.-T. Miao, C. Fan, R. Sun, Y.-J. Xu, J.-F. Ge, J. Mater. Chem. B 2014, 2,
7065−7072.
[11] L. Yuan, W. Lin, S. Zhao, W. Gao, B. Chen, L. He, S. Zhu, J. Am. Chem.
Soc. 2012, 134, 13510−13523.
[23] a) H. Wang, Z. Lu, S. J. Lord, W. E. Moerner, R. J. Twieg, Tetrahedron
Lett. 2007, 48, 3471−3474; b) C. Ruzié, M. Krayer, T. Balasubramanian,
J. S. Lindsey, J. Org. Chem. 2008, 73, 5806−5820; c) F. He, L. Liu, L. Li,
Adv. Funct. Mater. 2011, 21, 3143−3149; d) E. L. Cole, E. Arunkumar, S.
Xiao, B. A. Smith, B. D. Smith, Org. Biomol. Chem. 2012, 10, 5769−
5773; e) H. Itoi, T. Kambe, N. Kano, T. Kawashima, Inorg. Chim. Acta
2012, 381, 117−123; f) J. Bartelmess, W. W. Weare, Dyes Pigm. 2013,
97, 1−8; g) S. Lin, S. Liu, F. Ye, L. Xu, W. Zeng, L. Wang, L. Li, R.
Beuerman, D. Cao, Sens. Actuator B-Chem. 2013, 182, 176−183; h) Y.
Hang, L. Yang, Y. Qu, J. Hua, Tetrahedron Lett. 2014, 55, 6998−7001; i)
A. W. Thomas, C. Catania, L. E. Garner, G. C. Bazan, Chem. Commun.
2015, 51, 9294−9297; j) L. Wang, L. Yang, D. Cao, Sens. Actuator B-
Chem. 2015, 221, 155−166; k) J. Jiang, E. Yang, K. R. Reddy, D. M.
Niedzwiedzki, C. Kirmaier, D. F. Bocian, D. Holten, J. S. Lindsey, New J.
Chem. 2015, 39, 5694−5714; l) A. Jacquemet, S. Rihn, G. Ulrich, P.-Y.
Renard, A. Romieu, R. Ziessel, Eur. J. Org. Chem. 2015, 1664−1669; m)
B. Roubinet, L. Bailly, E. Petit, P.-Y. Renard, A. Romieu, Tetrahedron
Lett. 2015, 56, 1015−1019; n) B. Roubinet, A. Chevalier, P.-Y. Renard, A.
Romieu, Eur. J. Org. Chem. 2015, 166−182; o) S.-J. Woo, S. Park, J.-E.
Jeong, Y. Hong, M. Ku, B. Y. Kim, I. H. Jang, S. C. Heo, T. Wang, K. H.
Kim, J. Yang, J. H. Kim, H. Y. Woo, ACS Appl. Mater. Interfaces 2016, 8,
15937−15947; p) L. Yan, L. Shen, H. Zhou, C. Wu, Y. Zhao, L. Wang, X.
Fang, G. Zhang, J. Xu, W. Yang, Tetrahedron 2016, 72, 8578−8583; q)
M. Sun, K. Müllen, M. Yin, Chem. Soc. Rev. 2016, 45, 1513-1528.
[12] a) T. Chen, Y. Lai, S. Huang, Bull. Korean Chem. Soc. 2013, 34, 2937−
2941; b) L. Yuan, W. Lin, H. Chen, Biomaterials 2013, 34, 9566−9571; c)
G. K. Vegesna, J. Janjanam, J. Bi, F.-T. Luo, J. Zhang, C. Olds, A.
Tiwari, H. Liu, J. Mater. Chem. B 2014, 2, 4500−4508; d) A. T. Wrobel, T.
C. Johnstone, A. D. Liang, S. J. Lippard, P. Rivera-Fuentes, J. Am.
Chem. Soc. 2014, 136, 4697−4705; e) M. Homma, Y. Takei, A. Murata,
T. Inoue, S. Takeoka, Chem. Commun. 2015, 51, 6194−6197; f) X. Wu,
M. Yu, B. Lin, H. Xing, J. Han, S. Han, Chem. Sci. 2015, 6, 798−803; g)
B.-L. Wang, C. Jiang, K. Li, Y.-H. Liu, Y. Xie, X.-Q. Yu, Analyst 2015,
140, 4608−4615; h) Y. Liu, Q. Su, X. Zou, M. Chen, W. Feng, Y. Shi, F.
Li, Chem. Commun. 2016, 52, 7466−7469; i) J.-Y. Xie, C.-Y. Li, Y.-F. Li,
J. Fei, F. Xu, J. Ou-Yang, J. Liu, Anal. Chem. 2016, 88, 9746−9752; j) K.
Liu, H. Shang, X. Kong, M. Ren, J.-Y. Wang, Y. Liu, W. Lin, Biomaterials
2016, 100, 162−171; k) X. Song, M. Hu, C. Wang, Y. Xiao, RSC Adv.
2016, 6, 69641−69646; l) S. Zhang, R. Adhikari, M. Fang, N. Dorh, C. Li,
M. Jaishi, J. Zhang, A. Tiwari, R. Pati, F.-T. Luo, H. Liu, ACS Sensors
2016, 1, 1408−1415; m) P. Wang, J. Huang, Y. Gu, RSC Adv. 2016, 6,
95708−95714; n) J.-W. Xiao, W.-J. Zhu, R. Sun, Y.-J. Xu, J.-F. Ge, RSC
Adv. 2016, 6, 98985−98993; o) J.-Y. Xie, C.-Y. Li, Y.-F. Li, Y.-J. Fu, S.-X.
Nie, H.-Y. Tan, Dyes Pigm. 2017, 136, 817−824.
[13] a) H. Chen, W. Lin, L. Yuan, Org. Biomol. Chem. 2013, 11, 1938−1941;
b) X. Xiong, F. Song, G. Chen, W. Sun, J. Wang, P. Gao, Y. Zhang, B.
Qiao, W. Li, S. Sun, J. Fan, X. Peng, Chem. Eur. J. 2013, 19, 6538−
This article is protected by copyright. All rights reserved.

10.1002/asia.201700176
Chemistry - An Asian Journal
[24] a) E. A. Owens, H. Hyun, S. H. Kim, J. H. Lee, G. L. Park, Y. Ashitate, J.
Choi, G. H. Hong, S. Alyabyev, S. J. Lee, G. Khang, M. Henary, H. S.
Choi, Biomed. Mater. 2013, 8, 014109, 014109 pp; b) K. Sato, T.
Nagaya, Y. Nakamura, T. Harada, R. R. Nani, J. B. Shaum, A. P. Gorka,
I. Kim, C. H. Paik, P. L. Choyke, M. J. Schnermann, H. Kobayashi, Mol.
Pharm. 2015, 12, 3303−3311; c) K. Sato, A. P. Gorka, T. Nagaya, M. S.
Michie, R. R. Nani, Y. Nakamura, V. L. Coble, O. V. Vasalatiy, R. E.
Swenson, P. L. Choyke, M. J. Schnermann, H. Kobayashi, Bioconjugate
Chem. 2016, 27, 404−413.
[25] For a review about fluorescent labeling of biomolecules, see: M. S. T.
Gonçalves, Chem. Rev. 2009, 109, 190−212.
[26] C. C. Goodno, H. E. Swaisgood, G. L. Catignani, Anal. Biochem. 1981,
115, 203−211.
[27] a) R. Knorr, A. Trzeciak, W. Bannwarth, D. Gillessen, Tetrahedron Lett.
1989, 30, 1927−1930; b) W. Bannwarth, R. Knorr, Tetrahedron Lett.
1991, 32, 1157−1160; c) A. Barré, M.-L. Ţînţaş, V. Levacher, C.
Papamicaël, V. Gembus, Synthesis 2017, 49, 472−483.
[28] M. Ogawa, N. Kosaka, P. L. Choyke, H. Kobayashi, ACS Chem. Biol.
2009, 4, 535−546.
[29] H. Kobayashi, P. L. Choyke, Acc. Chem. Res. 2011, 44, 83−90.
[30] S. L. Niu, G. Ulrich, R. Ziessel, A. Kiss, P.-Y. Renard, A. Romieu, Org.
Lett. 2009, 11, 2049−2052.
[31] a) M. Mitsunaga, H. Tajiri, P. L. Choyke, H. Kobayashi, Therapeutic
Delivery 2013, 4, 523−525; b) H. Kobayashi, P. L. Choyke, M. Ogawa,
Curr. Opin. Chem. Biol. 2016, 33, 32−38.
[32] a) Y. Yang, Q. Zhao, W. Feng, F. Li, Chem. Rev. 2013, 113, 192−270; b)
X. Li, X. Gao, W. Shi, H. Ma, Chem. Rev. 2014, 114, 590−659.
This article is protected by copyright. All rights reserved.

10.1002/asia.201700176
Chemistry - An Asian Journal
Entry for the Table of Contents (Please choose one layout)
Layout 1:
FULL PAPER
Like a fish to water! A concise and
efficient synthetic route toward nor-
dihydroxanthene-hemicyanine hybrids
has been developed. A facile further
extension to water-soluble derivatives
has led to a novel class of NIR
fluorescent dyes (see figure) with
valuable spectral features for
applications in biosensing and
bioimaging.
M. J. H. Ong, S. Debieu, M. Moreau, A.
Romieu*, J.-A. Richard*
Page No. – Page No.
Synthesis of N,N-Dialkylamino-nor-
Dihydroxanthene-Hemicyanine Fused
Near-Infrared Fluorophores and Their
First
Water-Soluble
and/or
Bioconjugatable Analogues
This article is protected by copyright. All rights reserved.