Synthesis and spectral properties of 6′-triazolyl-dihydroxanthene-hemicyanine fused near-infrared dyes

Article abstract of DOI:10.1039/d0nj01724h

We describe the synthesis of a range of 6′-triazolyl-dihydroxanthene-hemicyanine (DHX-hemicyanine) fused dyes through an effective copper-catalyzed azide-alkyne cycloaddition (CuAAC) "click"reaction, with the aim of providing molecular diversity and evaluating the spectral properties of these near-infrared (NIR)-active materials. This was implemented by reacting 15 different aliphatic and aromatic azides with a terminal alkynyl-based DHX-hemicyanine hybrid scaffold prepared in four steps and 35% overall yield from 4-bromosalicylaldehyde. The resulting triazole derivatives have been fully characterized and their optical properties determined both in organic solvents and under simulated physiological conditions (phosphate buffered saline containing 5% of bovine serum albumin protein). This systematic study is a first important step towards the development of NIR-I fluorogenic "click-on"dyes or related photoactive agents for light-based diagnostic and/or therapeutic applications.

Full text of DOI:10.1039/d0nj01724h

View Article Online
View Journal
NJC
New Journal of Chemistry
Accepted Manuscript
A journal for new directions in chemistry
This article can be cited before page numbers have been issued, to do this please use: J. Richard, G.
Lingyue, A. Romieu, R. Srinivasan and K. Renault, New J. Chem., 2020, DOI: 10.1039/D0NJ01724H.
Volume 42
This is an Accepted Manuscript, which has been through the
Number
6
21 March 2018
Pages 3963-4776
Royal Society of Chemistry peer review process and has been
accepted for publication.
NJC
New Journal of Chemistry
rsc.li/njc
A journal for new directions in chemistry
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 1144-2546
PAPER
Chechia Hu, Tzu-Jen Lin et al.
Yellowish and blue luminescent graphene oxide quantum dots
prepared via a microwave-assisted hydrothermal route using
H2O2 and KMnO4 as oxidizing agents
rsc.li/njc

Please do not adjust margins
New Journal of Chemistry
Page 1 of 10
View Article Online
DOI: 10.1039/D0NJ01724H
1
2
3
4
5
6
7
8
ARTICLE
9
Synthesis and Spectral Properties of 6'-Triazolyl-Dihydroxanthene-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Hemicyanine Fused Near-Infrared Dyes†
Lingyue Gu,a Kevin Renault,b Anthony Romieu,*b Jean-Alexandre Richard*c and Rajavel Srinivasan*a
Received 00th January 20xx,
Accepted 00th January 20xx
We describe the synthesis of a range of 6’-triazolyl-dihydroxanthene-hemicyanine (DHX-hemicyanine) fused dyes through
an effective copper-catalyzed azide-alkyne cycloaddition (CuAAC) "click" reaction, with the aim of providing molecular
diversity and evaluating spectral properties of these near-infrared (NIR)-active materials. This was implemented by reacting
15 different aliphatic and aromatic azides with a terminal alkynyl-based-DHX-hemicyanine hybrid scaffold prepared in four
steps and 35% overall yield from 4-bromosalicylaldehyde. The resulting triazole derivatives have been fully characterized
and their optical properties determined both in organic solvents and simulated physiological conditions (phosphate buffered
saline containing 5% of bovine serum albumin protein). This systematic study is a first important step towards the
development of NIR-I fluorogenic "click-on" dyes or related photoactive agents for light-based diagnostic and/or therapeutic
applications.
DOI: 10.1039/x0xx00000x
the Huisgen 1,3-cycloaddition9 and reported a regioselective
Cu-catalyzed version (known as CuAAC for copper-catalyzed
azide-alkyne cycloaddition) which has become the most iconic
Introduction
The essence of "click chemistry" reactions lies in their simplicity,
speed and robustness even in aqueous media.1 While the
concept has found popularity in organic chemistry, material
science2 and drug discovery,3 it comes as no surprise that
biologists have also embraced it to perform chemistry in living
systems (known as in vivo chemistry).4 By doing so, a new field
coined bioorthogonal chemistry emerged and aims at
developing advanced chemistry tools for the rational
modification of biomolecules helping to a better understand of
biology.5 Of particular interest is the labelling of biomolecules
monitoring their activity and leading to the discovery and/or
deciphering of new biological mechanisms.
In this context, the association of reactions belonging to the
repertoire of "click chemistry" with organic-based fluorophores
has propulsed the field of bioorthogonal chemistry to an
indispensable tool for the optical imaging of biological systems.6
For instance, Meldal,7 Sharpless and co-workers8 rejuvenated
"click" reaction known so far.10 Wang and co-workers applied it
to fluorescent molecules and reported CuAAC reaction between
3-azidocoumarins and acetylenes.11 While the aryl azide
precursor did not display any fluorescence properties, the study
showed that some of the triazoles formed turned fluorescent.
In a similar fashion, Bertozzi's group used an azido aryl moiety
as C9-substituent of xanthene-based fluorophores.12 The initial
azido rosamine did not emit any light because of
a
photoinduced electron transfer (PeT)-mediated quenching
process. However, after evaluation of different positions of the
azido group and electronic density of the aromatic ring, the
fluorescence of the resulting triazole could be enhanced up to
58-fold (Scheme 1A).13 Kele's group also used aryl azide
precursors14 but the approach using these scaffolds as
fluorogenic precursors is limited because of their relative light
instability and propensity to form the corresponding nitrene. It
is possible to take advantage of this mechanism in the design of
photoreactive cross-linkers suitable for photoaffinity labeling15
but in the context of the development of fluorogenic probes
that instability is a serious limitation.16 One way around the
need for an aryl azide precursor is to use a more stable terminal
alkynyl-containing fluorophore. When the carbon-carbon triple
bond replaces electron-donating groups (typically, N,N-
dialkylamino moieties) it can turn the fluorophore to weakly or
not fluorescent. This strategy was demonstrated by Tung's
group on benzothiazoles17 and by Yao and co-workers on
rosamine- and xanthone-based fluorophores which displayed
an increase of fluorescence emission upon formation of the
triazole ring (Scheme 1B).18
a. G. Lingyue, Prof. R. Srinivasan
School of Pharmaceutical Science and Technology (SPST), Tianjin University,
Building 24, 92 Weijin Road, Nankai District, Tianjin, 300072 P. R. China
E-mail: rajavels@tju.edu.cn
b. Dr. K. Renault, Prof. A. Romieu
ICMUB, UMR 6302, CNRS, Univ. Bourgogne Franche-Comté
9, Avenue Alain Savary, 21000 Dijon, France
E-mail: anthony.romieu@u-bourgogne.fr
c. Dr. J.-A. Richard
Functional Molecules and Polymers
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
†Electronic Supplementary Informaꢀon (ESI) available: Protocols, NMR and HMRS
data as well as copies of NMR spectra, HPLC traces and selected UV and fluorescence
spectra. See DOI: 10.1039/x0xx00000x
Despite the advantages of longer-wavelength fluorescence
light, known to facilitate molecular imaging in complex
biological systems,19 such strategies have mostly been
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 2 of 10
View Article Online
DOI: 10.1039/D0NJ01724H
ARTICLE
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
implemented with fluorophores emitting in the visible spectral
range and little progress has been made so far on the
development of similar near-infrared (NIR)-emitting dyes
(specifically, within NIR-I optical window 650-900 nm). Over the
years, our respective groups have designed chemical methods
leading to the development of a novel class of NIR-I dyes based
on a molecular hybrid scaffold that combine both structural
features of xanthene and cyanine parent compounds.20 These
dihydroxanthene (DHX)-hemicyanine fused fluorophores are
now regarded as attractive alternatives to the popular and
commonly used polymethine-cyanine dyes, as illustrated by
numerous and valuable achievements in the fields of
biosensing, bioimaging and theranostics.21 Attracted by the
modularity and ease of chemical modification of DHX-
hemicyanine hybrid dyes, we set out to develop the access to a
DHX precursor allowing the facile "click" formation of triazoles.
In a first attempt toward this goal, the introduction of an
azido group on the 6'-position of the DHX skeleton proved to be
a dead-end because of the marked photosensitivity of the aryl
Results and discussion
Synthesis of a library of 6'-triazolyl-dihydroxanthene-hemicyanine
fused dyes
We started our study with the preparation of the alkyne "click"
partner
bifunctional arylbrominated aldehyde
DHX-hemicyanine precursor was prepared in two steps from 3-
bromophenol through (1) Casnati-Skattebøl ortho-
formylation (MgCl2, (HCHO)n, NEt3)23 that provided 4-
bromosalicylaldehyde in 69% yield, and its subsequent (2)
one-pot reaction with enal
aryl bromide moiety
trimethylsilylacetylene
1
which we readily obtained in two steps from
6
(Scheme 2). This latter
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
4
5
to form the DHX skeleton
was then reacted
6
. The
with
7
under Sonogashira reaction conditions
(Pd(PPh3)4, CuI, 79% yield).22 After removal of the TMS group
under conventional conditions (K2CO3, CH3OH), the formyl
group was reacted with 1,2,3,3-tetramethyl-3H-indol-1-ium
azide obtained. Instead, we took inspiration from our previous iodide (Fisher's base) 10 to yield the desired alkynyl-based DHX-
work to prepare an alkynyl-based DHX precursor from the
1
hemicyanine fused dye
1 in 88% yield over two steps. The
corresponding aryl bromide.22 We then explored CuAAC
reaction with aliphatic and aromatic azides. Finally, we
determined the spectral properties of the resulting "clicked"
fluorophores 2a-2o to assess the influence of both triazole
moiety and its substitution pattern on NIR absorption/emission
ability of fused DHX-hemicyanine scaffold (Scheme 1B). Details
of these investigations and their possible extensions are
discussed in the present Article.
structure was proven by NMR and ESI mass analyses and the
high level of purity (>94% whatever the wavelength used for the
UV-vis detection) was confirmed by RP-HPLC-based analytical
control (see ESI†).
Scheme 2 Preparation of the alkynyl-based DHX-hemicyanine fused dye 1 in five steps
from 3-bromophenol 3.
We next explored reaction conditions allowing the
formation of the desired triazole ring at the 6' position of the
DHX-hemicyanine fused dye (Scheme 3A). To this end, we
optimized the reaction conditions at room temperature in the
presence of (3-azidopropyl)benzene 11a as azido "click"
partner, sodium ascorbate and various copper sources. While
the use of copper(II) chloride and copper(II) sulfate
pentahydrate provided some product (10% and 13% yield,
respectively, Scheme 3A, entries 1-2), copper(II) acetate,
copper(II) triflate and copper(I) chloride were much less
successful (Scheme 3A, entries 3-6). Other usual "click"
conditions using copper(I) sources without sodium ascorbate
didn't provide any promising result either (Scheme 3A, entries
6 and 9). However, switching from the commonly used tert-
butanol/water mixture of solvent to pure water in the presence
Scheme 1 CuAAC reaction for the development of fluorogenic "click-on" dyes. A)
Reaction of 3-azidocoumarin and Si-rosamine fluorogenic dyes with terminal alkynes; B)
Reaction of alkynyl-based xanthone and DHX fluorogenic dyes with azido compounds.
2 |New J. Chem., 2020, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 3 of 10
View Article Online
DOI: 10.1039/D0NJ01724H
Journal Name
New Journal of Chemistry
1
2
3
4
5
of sodium ascorbate improved the yield significantly from 13% 81% and 61% yields, respectively.24 The availability of triazole-
to 38% (Scheme 3A, entries 2 and 7). Ultimately, a mixture of based fluorophores bearing an extra functional group for
DCM and water (Scheme 3A, entry 8) and 10 h reaction time structural tuning or covalent conjugation to other
appeared to be optimal (Scheme 3A, entries 10-12). With an (bio)molecular partners is of great interest for biological
interesting 50% yield (Scheme 3A, entry 8) it allowed us to applications. The CuAAC reaction was therefore achieved with
6
7
8
9
explore the scope of the reaction with a wide range of alkyl and 11g
aryl azides 11b-o (see ESI for their synthesis).
, 11i, 11m and 11n to introduce either a latent carboxylic
†
acid (masked as methyl ester) or an aryloxysulfonyl fluoride
moiety (this latter one being identified as an effective reactive
partner in "click" reaction sulfur(VI) fluoride exchange
(SuFEx)25). The possible influence of an electron-donating or -
withdrawing substituent introduced on triazole moiety, over
spectral properties of the resulting "clicked" DHX-based dyes,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
was also addressed through the successful synthesis of 2h-2l
.
Finally, the presence of an ortho-substituted sulfonic acid group
in 11o provided a simple way to introduce a polar group
facilitating the water-solubilization of the resulting DHX-
hemicyanine fused dye 2o (Scheme 3B). The structures of these
15 novel triazole-based DHX-hemicyanine fused dyes were
unambiguously confirmed by ESI-HRMS and NMR spectroscopic
analyses (see ESI†). Their purity was checked by RP-HPLC and
found to be above 95%, value usually required to achieve
relevant photophysical measurements.
Photophysical
hemicyanine fused dyes
properties of
6'-triazolyl-dihydroxanthene-
To assess changes in photophysical properties after triazole
formation, we next studied the spectral behavior of our library of 15
DHX-hemicaynine fused dyes 2a-2o and alkynyl precursor 1 in
different media including phosphate buffered saline (PBS) with 5%
(w/v) bovine serum albumin (BSA) as simulated body fluid, EtOH, and
CHCl3 (Table 1 and Fig. 1 for the Abs/Ex/Em spectra of 1 and 2o in
CHCl3 and in PBS + 5% BSA, see ESI† for Abs/Ex/Em spectra of other
compounds). A first general trend common to all triazole-based dyes
and alkyne 1 is the dramatic broadening of the main absorption band
assigned to S0-S1 electronic transition, compared to that of more
conventional DHX-hemicyanine fused fluorophore bearing a N,N-
dialkylamino group as C-6' substituent like 12, which explains the
notable lower molar extinction coefficient of dyes 2a-2o compared
to 12. A full-width half maximum (FWHM), Δλ1/2 max in the range of
3410-4370 cm-1 is observed depending primarily on the solvent used,
against only 695-990 nm for 12 and unlike the latter, well-defined
vibronic structures with two maxima and one pronounced blue-
shifted shoulder are observed in all absorption spectra. The lack of
an effective electron-donating group such as -NEt2, on C-6' position,
Scheme 3 A) Optimization of the CuAAC reaction for the synthesis of 6'-triazolyl-DHX- induces an expected hypsochromic shift of ca. 50 nm or 100 nm
hemicyanine fused dyes 2; B) Scope of the CuAAC reaction using aliphatic and aromatic
azides.
depending the local absorption maximum regarded for the triazole
derivatives, of the maximum absorption peak position for the DHX-
hemicyanine hybrid scaffold. This feature also contributes to the
dramatic decrease of molar extinction coefficients compared to
those of more conventional DHX-based fluorophores such as 12.
With one less carbon in its alkyl chain, (2-azidoethyl)benzene
11b behaved similarly to azide 11a and formed the
corresponding triazole 2b in 52% yield. 4-Chloro- and 4-
methoxybenzyl azide 11c and 11d were also successful (the
corresponding triazoles 2c and 2d were isolated in 74% and 36%
yields, respectively) and the two 2-(azidomethyl)pyridines 11e
and 11f developed by the Ting group to speed-up the CuAAC
reaction in cellulo gave triazole cycloadducts 2e and 2f in good
Red excitation at 620 nm produced the desired NIR emission in
the form of a vibronic structure with two well-defined maxima in the
range 690-760 nm. However, only a very weak fluorescence emission
intensity was observed, whatever the triazole derivative studied, the
solvent and concentration range (1-10 M) used. It prevented us to
This journal is © The Royal Society of Chemistry 20xx
New J. Chem., 2020, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 4 of 10
View Article Online
DOI: 10.1039/D0NJ01724H
ARTICLE
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fig. 1 Normalised absorption, emission (excitation at 620 nm for a-d and 650 nm for e-f) and excitation (emission at 760 nm for a-d, 830 nm for e or 800 nm for f) spectra of alkynyl-
based DHX-hemicyanine fused dye 1 (a in CHCl3 and b in PBS + 5% BSA), triazole-based DHX-hemicyanine fused dye 2o (c in CHCl3 and d in PBS + 5% BSA), and N,N-diethylamino-
DHX-hemicyanine fused dye 12 (e in CHCl3 and f in PBS + 5% BSA). For the excitation spectrum of 12 in PBS + 5% BSA, a peak at 400 nm (λex/2) assigned to Rayleigh scattering is
observed. See the Experimental section for details about these measurements.
accurately determine relative fluorescence quantum yields, roughly spectrally characterised in simulated physiological conditions (i.e.,
estimated at less than 1%. The very low values of these quantum PBS + 5% BSA). The capability of BSA protein to disrupt fluorophore
yields cannot be attributed to the formation of non-emissive aggregates in aq. media is well-documented but in the present case,
aggregates (i.e., H-type homodimers)26 because a good matching its surfactant behavior was not sufficiently effective to obtain a single
between the absorption and excitation spectra was observed except emissive species in solution.27 Alternatively, we attempted to assess
for some triazole derivatives (e.g., 2i, 2l and 2m, see ESI†) and 1 the relative fluorescence efficiency of dyes 2a-2o and 1 by calculating
4 |New J. Chem., 2020, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 5 of 10
View Article Online
DOI: 10.1039/D0NJ01724H
Journal Name
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
6-Ethynyl-2,3-dihydro-1H-xanthene-4-carbaldehyde (9). TMS-
protected terminal alkyne 8 (1.0 g, 3.3 mmol) was dissolved in dry
MeOH (30 mL) and treated with anhydrous K2CO3 (1.8 g, 13.2 mmol).
The mixture was stirred at room temperature for 5 h. The solvent
was removed, and the residue was taken up in 100 mL of DCM and
washed with 100 mL of deionised water. The organic layer was dried
over anhydrous Na2SO4 and concentrated under reduced pressure.
This compound was used in the next step without further
the ratio [(integration of emission curve)/absorption at 620 nm)] for
a single concentration (5 M) solution in CHCl3 of each compound (1
and 2a-2o). We observed a change in color of solution from sapphire
blue (for alkyne 1) to sky blue (for triazole derivatives 2a-2o) (see
ESI†) but could not obtain reliable values allowing an accurate and
relevant ranking of the fluorophores synthesized in this study, based
on their emissive capability.
1
purification, and the yield was assumed to be quantitative. H NMR
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Conclusions
(400 MHz, DMSO-d6): δ = 10.26 (s, 1 H), 7.36 (d, J = 7.9 Hz, 1 H), 7.34
(s, 1 H), 7.24 (dd, J = 7.8 Hz, J = 1.5 Hz, 1 H), 7.01 (s, 1 H), 4.37 (s, 1 H),
2.59 (ddd, J = 7.0 Hz, J = 5.3 Hz, J = 1.6 Hz, 2 H), 2.30 (t, J = 6.0 Hz, 2
H), 1.63 (m, 2 H) ppm; 13C NMR (101 MHz, CDCl3): δ = 188.1, 159.8,
151.7, 131.1, 127.7, 126.6, 125.9, 123.6, 122.0, 118.9, 113.9, 82.8,
79.3, 30.3, 21.6, 20.4 ppm; HRMS (ESI+): m/z 237.0910 [M + H]+, calcd
for C16H13O2+ 237.0910.
Quest for high-performance NIR-I chromophores/fluorophores
based on the attractive DHX-hemicyanine hybrid scaffold led us
to consider for the first time CuAAC "click" reaction as a simple
way of achieving high molecular diversity through the
straightforward synthesis of alkynyl-based DHX-hemicyanine
fused dye
1 and its "click" derivatisation with a wide range of
Alkynyl-based DHX-hemicyanine fused dye (1). To aldehyde 9 (236
mg, 1.0 mmol) in EtOH (2 mL) was added 1,2,3,3-tetramethyl-3H-
indol-1-ium iodide 10 (301 mg, 1.0 mmol) and the solution was
refluxed at 80 oC for 4 h. The reaction mixture was concentrated and
the crude product was purified by flash-column chromatography on
organic azides. In contrast to already published fluorogenic
alkynes photoactive in the blue-green spectral range, only a
weak fluorescence emission of triazole-based dyes was
observed within the NIR-I window. Thus, our efforts that
involved synthesis and photophysical characterization of a silica gel (eluent: 1% MeOH in DCM) to afford alkynyl-DHX 1 as a dark
library of 15 different compounds will require a further
structural optimization to obtain NIR-I fluorogenic "click-on"
dyes suitable for bioimaging. Interestingly, the specific
molecular absorption signature of both alkyne and triazole
derivatives (i.e., broad, structured and weakly solvent
dependent absorption band) might be used to design novel
broad spectrum dark quencher molecules suitable for the
construction of far-red or NIR-I fluorescence light-up probes
based on Förster resonance energy transfer (FRET)
mechanism.28
blue solid (345 mg, yield 88%). m.p >300 °C; 1H NMR (400 MHz,
DMSO-d6): δ = 8.56 (d, J = 15.3 Hz, 1 H), 7.80-7.73 (m, 2 H), 7.62 (s, 1
H), 7.58 (m, 1 H), 7.52 (m, 2 H), 7.39-7.33 (m, 2 H), 6.68 (d, J = 15.4
Hz, 1 H), 4.52 (s, 1 H), 3.95 (s, 3 H), 2.72 (t, J = 6.0 Hz, 2 H), 2.67 (t, J =
6.0 Hz, 2 H), 1.83 (m, 2 H), 1.77 (s, 6 H) ppm; 13C NMR (101 MHz,
DMSO-d6): δ = 179.0, 158.0, 151.8, 145.1, 142.4, 142.1, 131.1, 129.8,
128.8, 128.4, 127.8, 127.6, 123.8, 122.6, 122.2, 118.6, 114.3, 113.8,
107.2, 83.8, 82.6, 50.8, 33.2, 28.7, 26.9, 23.5, 19.7 ppm; HRMS (ESI+):
m/z 392.2019 [M]+●, calcd for C28H26NO+ 392.2009; HPLC (system A):
tR = 4.9 min (purity 94% at 260 nm and 98% at 600 nm); LRMS (ESI+,
●
recorded during RP-HPLC analysis): m/z 392.3 [M]+ (100), calcd for
C28H26NO+ 392.2; UV-vis (recorded during the HPLC analysis): max
=
559 and 592 nm (broad band).
Experimental†
See ESI† for the details about sections "General", "Instruments
and methods", and all experimental and spectral data
associated with synthesised compounds.
General procedure for the synthesis of tetrazole-based DHX-
hemicyanine fused dyes 2a-2o. To a mixture of alkynyl-based DHX-
hemicyanine dye 1 (784 mg, 2.0 mmol, 1.0 equiv.) and the
corresponding organic azide 11a-o (2.6 mmol, 1.3 equiv.) in
deionised water and CH2Cl2 (1:1 (v/v), 100 mL), sodium ascorbate
(79.2 mg, 0.4 mmol, 0.2 equiv.) was added, followed by the addition
of CuSO4 • 5 H2O (25 mg, 0.1 mmol, 0.05 equiv.). The heterogeneous
mixture was stirred vigorously at room temperature overnight.
Thereafter, the reaction mixture was concentrated under reduced
pressure and directly purified by flash-column chromatography on
silica gel (eluent: 1% MeOH in DCM) to afford the corresponding
triazole 2a-2o as a dark blue amorphous powder.
Synthesis
6-((Trimethylsilyl)ethynyl)-2,3-dihydro-1H-xanthene-4
carbaldehyde (8). Pd(PPh3)4 (67.5 mg, 8.3 mmol%) and CuI (23.6 mg,
0.1 mmol) were degassed in a flame-dried round bottom flask. NEt3
(1.2 mL, 8.3 mmol) in dry DMF (10 mL) was added with aryl bromide
6 (1.2 g, 4.1 mmol). The solution was degassed again then
trimethylsilylacetylene 7 was added (1.8 mL, 12.4 mmol). The
reaction mixture was heated to 85 oC for 24 h. After being cooled to
room temperature, the reaction mixture was concentrated under
vacuum and the resulting residue was purified by flash-column
chromatography on silica gel (eluent: 1% EtOAc in PE) affording pure
Photophysical characterisations
UV-visible spectra were obtained either on a Varian Cary 50
TMS-protected terminal alkyne 8 as a orange solid (1.0 g, yield 79%). scan (single-beam) or an Agilent technologies 60 (single-beam)
1H NMR (400 MHz, CDCl3): δ = 10.30 (s, 1 H), 7.17 (s, 1 H), 7.13 (dd, J
= 7.8 Hz, J = 1.2 Hz, 1 H), 7.05 (d, J = 7.8 Hz, 1 H), 6.62 (s, 1 H), 2.58
(ddd, J = 7.5 Hz, J = 5.8 Hz, J = 1.6 Hz, 2 H), 2.43 (t, J = 6.0 Hz, 2 H),
1.71 (m, 2 H), 0.25 (s, 9 H); 13C NMR (101 MHz, CDCl3): δ = 188.0,
159.9, 151.7, 130.8, 127.6, 126.5, 126.1, 124.6, 121.6, 118.6, 113.8,
104.0, 96.9, 30.3, 21.6, 20.4, 0.0 ppm; HRMS (ESI+): m/z 331.1141 [M
+ Na]+, calcd for C19H20O2SiNa+ 331.1125.
spectrophotometer (software Cary WinUV) by using rectangular
quartz cells (Hellma, 100-QS, 45 12.5 12.5 mm, pathlength:


10 mm, chamber volume: 3.5 mL), at 25 °C (using a temperature
control system combined with water circulation). Fluorescence
spectra (emission/excitation spectra) were recorded with an
HORIBA Jobin Yvon Fluorolog spectrofluorometer (FluorEssence
software) at 25 °C (using a temperature control system
combined with water circulation), with standard fluorometer
This journal is © The Royal Society of Chemistry 20xx
New J. Chem., 2020, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 6 of 10
View Article Online
DOI: 10.1039/D0NJ01724H
ARTICLE
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
10 D. Brunel and F. Dumur, New J. Chem., 2020, 44, 3546.
11 K. Sivakumar, F. Xie, B. M. Cash, S. Long, H. N. Barnhill and Q.
Wang, Org. Lett., 2004, 6, 4603.
12 (a) P. Shieh, M. J. Hangauer and C. R. Bertozzi, J. Am. Chem. Soc.,
2012, 134, 17428; (b) P. Shieh, V. T. Dien, B. J. Beahm, J. M.
Castellano, T. Wyss-Coray and C. R. Bertozzi, J. Am. Chem. Soc.,
2015, 137, 7145.
13 P. Shieh, M. S. Siegrist, A. J. Cullen and C. R. Bertozzi, Proc. Natl.
Acad. Sci. U S A, 2014, 111, 5456.
14 (a) A. Herner, I. Nikić, M. Kállay, E. A. Lemke and P. Kele, Org.
Biomol. Chem., 2013, 11, 3297; (b) G. B. Cserép, A. Herner and P.
Kele, Methods Appl. Fluoresc. , 2015, 3, 042001; (c) Z. Pünkösti, P.
Kele and A. Herner, J. Het. Chem., 2018, 55, 1183.
15 G. T. Hermanson, in Bioconjugate Techniques (Third Edition),
Academic Press, Boston, 2013, ch. 3, pp. 253.
16 E. Pǎunescu, L. Louise, L. Jean, A. Romieu and P.-Y. Renard, Dyes
Pigm., 2011, 91, 427.
17 (a) J. Qi and C.-H. Tung, Bioorg. Med. Chem. Lett., 2011, 21, 320;
(b) J. Qi, M.-S. Han, Y.-C. Chang and C.-H. Tung, Bioconjugate
Chem., 2011, 22, 1758.
18 J. Li, M. Hu and S. Q. Yao, Org. Lett., 2009, 11, 3008.
19 (a) R. Weissleder and U. Mahmood, Radiology, 2001, 219, 316; (b)
J. V. Frangioni, Curr. Opin. Chem. Biol., 2003, 7, 626; (c) I. Martinić,
S. V. Eliseeva and S. Petoud, J. Lumin., 2017, 189, 19.
20 (a) J.-A. Richard, Org. Biomol. Chem., 2015, 13, 8169; (b) A.
Romieu and J.-A. Richard, Tetrahedron Lett., 2016, 57, 317; (c) M.
J. H. Ong, S. Debieu, M. Moreau, A. Romieu and J.-A. Richard, Chem.
Asian J., 2017, 12, 936; (d) S. Zheng, G. Lingyue, M. J. H. Ong, D.
Jacquemin, A. Romieu, J.-A. Richard and R. Srinivasan, Org. Biomol.
Chem., 2019, 17, 4291.
cells (Labbox, LB Q, light path: 10 mm, width: 10 mm, chamber
volume: 3.5 mL). The absorption and fluorescence emission
spectra were recorded with dye solutions of concentrations in
the range of 10-5-10-6 M. The emission spectra were recorded in
the range of 635-850 nm after excitation at 620 nm (shutter:
Auto Open, integration time = 0.1 s, 1 nm step, HV(S1) = 950 V,
excitation slit = 5 nm and emission slit = 5 nm). The excitation
spectra were recorded in the range of 400-750 nm after
emission at 760 nm (excitation slit = 5 nm for spectra recorded
in CHCl3 and 12 nm for spectra recorded in EtOH or PBS + 5%
BSA and emission slit = 5 nm). All excitation/emission spectra
are corrected.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
This work is supported by SPST, Tianjin University (P.R. China), ICES,
A*STAR (Singapore), and the CNRS, Université de Bourgogne and
Conseil Régional de Bourgogne through the "Plan d'Actions Régional
pour l'Innovation (PARI) and the "Fonds Européen de
Développement Régional (FEDER)" programs. Financial supports
from Agence Nationale de la Recherche (ANR, AAPG 2018, PRCI
LuminoManufacOligo, ANR-18-CE07-0045-01), especially for the
post-doc fellowship of K. R., the National Research Foundation
Singapore (NRF, LuminoManufacOligo, NRF2018-NRF-ANR035) and
GDR CNRS "Agents d'Imagerie Moléculaire" (AIM) 2037 are also
greatly acknowledged. The authors thank Ms Doris Tan (ICES,
Singapore) and Dr. Quentin Bonnin (CNRS, PACSMUB) for the
recording of HRMS spectra. A. R. and K. R thank the "Plateforme
d'Analyse Chimique et de Synthèse Moléculaire de l'Université de
Bourgogne" (PACSMUB, http://www.wpcm.fr) for access to
spectroscopy instrumentation.
21 H. Chen, B. Dong, Y. Tang and W. Lin, Acc. Chem. Res., 2017, 50,
1410.
22 M. J. H. Ong, R. Srinivasan, A. Romieu and J.-A. Richard, Org. Lett.,
2016, 18, 5122.
23 (a) N. U. Hofsløkken and L. Skattebøl, Acta Chem. Scand., 1999,
53, 258; (b) T. V. Hansen and L. Skattebøl, Org. Synth., 2005, 82,
64; (c) T. V. Hansen and L. Skattebøl, Org. Synth., 2012, 89, 220.
24 C. Uttamapinant, A. Tangpeerachaikul, S. Grecian, S. Clarke, U.
Singh, P. Slade, K. R. Gee and A. Y. Ting, Angew. Chem. Int. Ed.,
2012, 51, 5852.
25 (a) J. Dong, L. Krasnova, M. G. Finn and K. B. Sharpless, Angew.
Chem. Int. Ed., 2014, 53, 9430; (b) A. S. Barrow, C. J. Smedley, Q.
Zheng, S. Li, J. Dong and J. E. Moses, Chem. Soc. Rev., 2019, 48,
4731; (c) A. Marra, C. Nativi and A. Dondoni, New J. Chem., 2020,
44, 4678.
26 M. Ogawa, N. Kosaka, P. L. Choyke and H. Kobayashi, ACS Chem.
Biol., 2009, 4, 535.
27 Pauli, M. Grabolle, R. Brehm, M. Spieles, F. M. Hamann, M.
Wenzel, I. Hilger and U. Resch-Genger, Bioconjugate Chem.,
2011, 22, 1298.
Notes and references
1 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem. Int. Ed.,
2001, 40, 2004.
2 (a) J.-F. Lutz, Angew. Chem. Int. Ed., 2007, 46, 1018; (b) H.
Nandivada, X. Jiang and J. Lahann, Adv. Mater., 2007, 19, 2197.
3 H. C. Kolb and K. B. Sharpless, Drug Discov. Today, 2003, 8, 1128.
4 (a) J. A. Prescher and C. R. Bertozzi, Nat. Chem. Biol., 2005, 1, 13;
(b) M. Boyce and C. R. Bertozzi, Nat. Methods, 2011, 8, 638; (c) C.
R. Bertozzi and P. Wu, Curr. Opin. Chem. Biol., 2013, 17, 717; (d) J.
G. Rebelein and T. R. Ward, Curr. Opin. Biotechnol., 2018, 53, 106.
5 (a) J. M. Baskin and C. R. Bertozzi, QSAR & Comb. Sci., 2007, 26,
1211; (b) E. M. Sletten and C. R. Bertozzi, Angew. Chem. Int. Ed., 28 (a) T. Myochin, K. Hanaoka, S. Iwaki, T. Ueno, T. Komatsu, T. Terai,
2009, 48, 6974; (c) E. M. Sletten and C. R. Bertozzi, Acc. Chem. Res.,
2011, 44, 666.
6 (a) C. Le Droumaguet, C. Wang and Q. Wang, Chem. Soc. Rev., 2010,
39, 1233; (b) P. Shieh and C. R. Bertozzi, Org. Biomol. Chem., 2014,
12, 9307.
7 (a) C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem., 2002,
67, 3057; (b) M. Meldal and C. W. Tornøe, Chem. Rev., 2008, 108,
2952.
8 V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless, Angew.
Chem. Int. Ed., 2002, 41, 2596.
T. Nagano and Y. Urano, J. Am. Chem. Soc., 2015, 137, 4759; (b)
J. Demuth, R. Kucera, K. Kopecky, Z. Havlínová, A. Libra, V.
Novakova, M. Miletin and P. Zimcik, Chem. Eur. J., 2018, 24, 9658.
.
9 R. Huisgen, Proc. Chem. Soc., 1961, 357.
6 |New J. Chem., 2020, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 10
New Journal of Chemistry
Please do not adjust margins
1
2
3
4
5
6
7
8
9
ARTICLE
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Table 1 Photophysical properties of alkynyl-based DHX-hemicyanine fused dye 1, triazole derivatives 2a-2o and reference dye 1222 at 25 °C. For structures, see ESI†.
Abs maxa [nm]
Em maxc,d [nm]
EtOH
Stokes' shifte [cm-1]
 [M-1 cm-1]
Entry
Dye
CHCl3
EtOH
PBSb
CHCl3
PBS
CHCl3
EtOH
PBS
CHCl3
EtOH
1935
PBS
569
607
654
579
617
670
579
619
671
579
618
670
579
618
670
580
620
671
580
618
669
580
562
597
640
572
608
656
573
608
656
571
607
656
571
608
656
572
607
656
571
607
656
571
566
598
645
578
614
662
575
612
661
573
612
662
573
611
663
570
607
661
576
613
662
572
25 700
45 200
38 300
34 200
52 050
40 600
41 800
63 500
48 600
38 100
58 900
45 800
40 400
59 050
43 500
42 100
62 200
46 100
37 200
55 300
41 500
37 800
38 750
42 200
22 850
35 200
44 200
28 200
42 100
53 100
33 700
45 100
56 450
35 500
44 250
55 850
35 600
43 050
54 150
34 200
35 750
44 800
28 250
39 350
31 150
31 000
16 700
28 600
34 550
22 500
35 700
41 250
26 450
36 700
39 300
25 300
38 500
41 000
27 050
38 950
35 700
23 350
30 500
38 700
27 500
33 700
685
746
675
728
681
741
1
2
3
4
5
6
1
1876
1777
1683
1730
1751
1720
2038
693
755
686
741
688
751
2a
2b
2c
2d
2e
1870
1849
1854
1849
1876
1752
1784
1805
1789
1939
691
755
685
742
687
748
692
752
684
744
688
747
693
754
685
749
686
748
694
751
685
745
688
741
694
758
687
743
686
749
7
8
2f
1772
1720
1918
1897
1736
1816
2g
694
686
686
Please do not adjust margins

New Journal of Chemistry
Page 8 of 10
Please do not adjust margins
1
2
3
ARTICLE
New Journal of Chemistry
4
5
6
7
8
9
620
672
583
621
672
581
619
670
580
620
671
581
619
671
581
619
671
579
619
672
580
618
670
585
624
677
724
607
657
574
609
658
572
608
656
572
608
657
573
609
658
571
606
655
571
607
655
571
607
655
573
610
658
717
610
661
583
616
667
570
605
674
577
612
667
573
612
656
573
610
665
573
607
662
574
611
655
577
613
665
724
758
746
751
58 400
45 500
23 250
37 400
30 900
37 800
56 700
43 700
38 700
58 600
45 350
37 650
57 100
44 150
39 100
60 100
47 300
34 550
54 400
43 250
36 400
55 100
42 750
15 800
27 050
25 500
194 100
49 600
31 400
27 000
33 700
21 450
41 500
51 150
31 650
44 700
56 000
35 350
40 600
51 000
32 500
43 350
53 000
32 300
39 100
47 550
28 900
44 600
54 050
32 700
20 900
27 000
17 950
35 700
23 750
22 800
25 950
17 100
33 100
25 650
14 100
35 300
38 700
25 800
19 250
20 800
17 400
37 050
35 250
21 950
37 900
35 250
21 600
36 400
37 000
22 900
18 500
23 000
15 900
693
755
684
746
688
751
9
2h
2i
1673
1766
1782
1725
1746
1662
1751
1800
1849
1891
1864
1903
1833
1790
1700
2015
1868
1868
1900
2003
1916
695
758
685
748
689
751
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
10
11
12
13
14
15
697
758
687
749
691
746
2j
693
757
687
747
691
749
2k
2l
694
753
685
742
690
748
690
756
683
745
691
748
2m
2n
693
760
681
745
692
747
697
761
684
747
688
749
16
17
2o
12
1678
262
1773
360
1778
262
738
736
738
163 550 131 850
8 |New J. Chem., 2020, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 9 of 10
New Journal of Chemistry
Please do not adjust margins
1
2
3
Journal Name
New Journal of Chemistry
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
This journal is © The Royal Society of Chemistry 20xx
New J. Chem., 2020, 00, 1-3 | 9
Please do not adjust margins

New Journal of Chemistry
Page 10 of 10
1
2
3
4
View Article Online
DOI: 10.1039/D0NJ01724H
Table of contents:
5
6
7
A copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) to explore the fluorogenic potential of near-
infrared (NIR) dihydroxanthene (DHX) triazole dyes
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60