N-Alkylation of 2-methoxy-10H-phenothiazine revisited. A facile entry to diversely N-substituted phenothiazine-coumarin hybrid dyes

Article abstract of DOI:10.1016/j.tetlet.2020.152582

N-Alkylation of 2-methoxy-10H-phenothiazine, a valuable building block for the synthesis of bioactive compounds and reaction-based fluorescent probes, has been revisited aimed at introducing a substituent easily convertible into cationic or zwitterionic side chains. We focused our attention on the 3-dimethylaminopropyl group since its derivatization through reactions with various alkyl halides or sultones is a well-established and effective way to enhance polarity of diverse hydrophobic molecular scaffolds. This two-step functionalization approach was applied to the synthesis of novel phenothiazine-coumarin hybrid dyes whose spectral features, especially their NIR-I emission, have been determined in aqueous media with the ultimate goal of identifying novel fluorescent markers for bioanalytical applications, including fluorogenic detection of reactive oxygen species (ROS) through selective S-oxidation reaction of phenothiazine scaffold.

Full text of DOI:10.1016/j.tetlet.2020.152582

Journal Pre-proofs
N-Alkylation of 2-methoxy-10H-phenothiazine revisited. A facile entry to di‐
versely N-substituted phenothiazine-coumarin hybrid dyes
Valentin Quesneau, Kévin Renault, Myriam Laly, Sébastien Jenni, Flavien
Ponsot, Anthony Romieu
PII:
S0040-4039(20)31081-9
DOI:
Reference:
https://doi.org/10.1016/j.tetlet.2020.152582
TETL 152582
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Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
24 August 2020
14 October 2020
17 October 2020
Please cite this article as: Quesneau, V., Renault, K., Laly, M., Jenni, S., Ponsot, F., Romieu, A., N-Alkylation of
2-methoxy-10H-phenothiazine revisited. A facile entry to diversely N-substituted phenothiazine-coumarin hybrid
dyes, Tetrahedron Letters (2020), doi: https://doi.org/10.1016/j.tetlet.2020.152582
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N-Alkylation
phenothiazine revisited. A facile entry to
diversely N-substituted phenothiazine-
coumarin hybrid dyes
Valentin Quesneau*, Kévin Renault, Myriam Laly, Sébastien Jenni, Flavien Ponsot, Anthony Romieu*
of
2-methoxy-10H-
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1
Tetrahedron Letters
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N-Alkylation of 2-methoxy-10H-phenothiazine revisited. A facile entry to diversely N-
substituted phenothiazine-coumarin hybrid dyes
Valentin Quesneaua,‡,*, Kévin Renaulta,‡, Myriam Lalya, Sébastien Jennia, Flavien Ponsota, Anthony
Romieua,*
aICMUB, UMR 6302, CNRS, Univ. Bourgogne Franche-Comté, 9, Avenue Alain Savary, 21078 Dijon cedex, France
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
N-Alkylation of 2-methoxy-10H-phenothiazine, a valuable building block for the synthesis of
bioactive compounds and reaction-based fluorescent probes, has been revisited aimed at
introducing a substituent easily convertible into cationic or zwitterionic side chains. We focused
our attention on the 3-dimethylaminopropyl group since its derivatization through reactions with
various alkyl halides or sultones is a well-established and effective way to enhance polarity of
diverse hydrophobic molecular scaffolds. This two-step functionalization approach was applied
to the synthesis of novel phenothiazine-coumarin hybrid dyes whose spectral features, especially
their NIR-I emission, have been determined in aqueous media with the ultimate goal of identifying
novel fluorescent markers for bioanalytical applications, including fluorogenic detection of
reactive oxygen species (ROS) through selective S-oxidation reaction of phenothiazine scaffold.
Available online
Keywords:
N-Alkylation
Coumarin
Fluorescent probe
Phenothiazine
Introduction
Since the discovery and first syntheses of methylene blue
(MB) [1] and 10H-dibenzo-1,4-thiazine (PTZ) [2] in the late 19th
century (Fig. 1), the interest shown by the scientific community
(especially chemists and biologists) for phenothiazine derivatives
has exploded [3]. This is notably reflected in the fact that there are
many thousands of different molecules belonging to this class of
N,S-heterocycles, and finding diverse applications in industry
(e.g., as dyestuffs, antioxidants in lubricants and fuels,
polymerization inhibitors, ...) and medicine (e.g., as antipsychotic
H2Sn), for analytical and diagnostics purposes respectively (see
Fig. 1 for selected examples) [8]. Curiously, the vast majority of
these reaction-based fluorescent probes and related fluorophores
display minor structural changes in their PTZ ring, which severely
limits opportunities for fine-tuning their spectral and
physicochemical properties (e.g., solubility in water) and/or
installation of a suitable handle for bioconjugation to perform
fluorescent biolabeling or to dramatically enhance their
performances as imaging agents (e.g., targeting properties). A
good illustration of this fact is N-substituent (i.e., the 10-position
of PTZ) found in the core structure of its photoactive PTZ-based
compounds. This is always a simple alkyl chain (i.e., ethyl or butyl
substituent) whereas this position may be better exploited to
introduce a post-synthetically derivatizable reactive group such as
amino, carboxylic acid, or hydroxyl. To the best of our knowledge,
few examples of such N-substituted derivatives of 2-methoxy-
10H-phenothiazine have been described in the literature even if
several modulations of its N-10 substituent were regarded in effort
to identify new drugs [9].
medications,
(photo)therapeutic
agents,
biological
staining/labeling agents, ...) [4]. Aside from these traditional
usages, PTZ-based molecular systems endowed with remarkable
electronic properties, have recently found further applications in
optoelectronics (e.g., bulk hetero-junction solar cells, dye
sensitized solar cells, organic light emitting diodes, ...) [5] and
fluorescent sensing and bioimaging [6]. Among the myriad PTZ
derivatives with different substitution patterns, some of them are
constructed from the structurally simple building block 2-
methoxy-10H-phenothiazine 1 (Fig. 1). They have been recently
popularized either as long-wavelength fluorescent dyes [7] or as
fluorogenic probes in the field of activity-based sensing. In this
latter context, they are effective tools for detecting reactive
analytes (e.g., phosgene, SO2, thiophenol) in different matrices or
tracking relevant species in biological systems (e.g., ROS/RNS,
In order both to fill this gap and to devise a novel approach towards
PTZ molecular diversity, we thought it would be interesting to
revisit N-alkylation of 2-methoxy-10H-phenothiazine to introduce
a substituent easily convertible into another group. Inspired by the
structure of levomepromazine (maleate salt, Fig. 1), a neuroleptic
———
* Corresponding authors. Tel.: +33-3-80-39-61-26 or +33-3-80-39-36-24; e-mail: valentin.quesneau@u-bourgogne.fr or anthony.romieu@u-
bourgogne.fr.
‡ These authors contributed equally to this work.

2
Tetrahedron Letters
drug used in palliative care and developed in the 1960s by a
dimethylaminopropyl substituent, would deserve to be explored to
meet demands in molecular diversity for such photoactive
compounds. Indeed, the current available methods are based on
formal exchange and/or post-functionalization of 3,6-(hetero)aryl
substituents, and are sometimes tricky and/or require de novo
synthesis [13c, 14].
Japanese company namely Yoshitomi Pharmaceutical Industries
[10], we have rapidly identified 3-dimethylaminopropyl moiety as
the appropriate substituent to achieve this goal. It is worth
mentioning that the use of this tertiary-amine-terminated alkyl
chain or related analogs was popularized for the water-
solubilization of various organic-based fluorophores, through their
site-specific introduction and post-synthetic quaternarization with
alkyl halides or sultones, thereby yielding positively-charged or
zwitterionic dyes [11],[12]. Similarly to PTZ uses,
diketopyrrolopyrroles (DPPs), a class of industrial pigments
known since the early 1970s, were recently applied as promising
fluorescent organic dyes owing to their valuable spectral
properties [13] but curiously, this N-functionalization strategy was
never applied to these bis-lactam-based molecules. Indeed, N-
alkylation of these cyclic amide moieties is often the preferred way
to convert DPP pigments into soluble dyes, and the practical
implementation of this reaction with versatile 3-
In this Letter, we report our findings related to optimization of
the N-alkylation of
1
with butyl bromide and 3-
dimethylaminopropyl chloride. A brief comparison with the
reactivity of selected DPP pigments was also established. The
availability of N-substituted 2-methoxyphenothiazine derivative 3
led us to consider the synthesis and photophysical characterization
of novel PTZ-coumarin hybrid dyes. Their ability to act as
ratiometric fluorescent chemodosimeters for hypochlorous
acid/hypochlorite (HClO/ClO-) detection, through oxidation of
their sulfur atom was finally assessed.
S
+
S
S
Me2N
S
N
NMe2
Cl-
N
OMe
CO2H
N
H
N
H
OMe
Me2N
HO2C
Me
2-Methoxyphenothiazine 1
10H-Dibenzo-1,4-thiazine (PTZ)
Methylene blue (MB)
Levomepromazine (maleate salt)
O2N
O
H
reactive site
for SO2
CN
N
S
NO2
N
O
S
S
S
N
N
N
H
reactive site
for PhSH
H
BF2
reactive sites
for COCl2
N
O
N
O
N
OH
Bu
Bu
Et
"OFF-ON" probe responsive to phosgene
(dual N,O acylation reaction)
Ratiometric probe responsive to SO2
(Nu addition of HSO3- on C=N)
"OFF-ON" probe responsive to thiophenol
(deprotection of NH2 group by thiolysis of sulfonamide)
reactive site
for ClO-
N
N
S
S
S
S
CN
N
O
F
N
O
Bu
NO2
O
Bu
N
O
reactive site
for H2Sn
"Covalent-assembly" probe responsive to H2Sn
(phenol deprotection through a tandem SNAr/cyclization reaction,
followed by Pinner cylization)
Ratiometric probe responsive to hypochlorus acid
(sulfur atom oxidation mediated by ClO-)
Fig. 1. (Top) Structures of 10H-dibenzo-1,4-thiazine, 2-methoxy-10H-phenothiazine and methylene blue; (bottom) selected examples of reaction-based fluorescent
probes (intensiometric "OFF-ON" or ratiometric response) bearing a PTZ photoactive scaffold (Bu = n-butyl, COCl2 = phosgene, Et = ethyl, H2Sn = hydrogen
polysulfides, PhSH = thiophenol and SO2 = sulfur dioxide) [8m],[8i],[8b],[8h],[8e].
the conversion of commercial 2-methoxyphenothiazine 1 into the
Results and discussion
corresponding N-alkyl salicylaldehyde derivative (Scheme 1).
Thus, N-substitution of PTZ scaffold with n-butyl bromide or ethyl
iodide, was achieved by treatment of 1 with a large excess of this
alkylating reagent (10 equiv.), in the presence of NaOH and cat.
amount of KI in the case of 1-bromobutane, in dry DMSO heated
above 100 °C [8a], but experimental details for such reactions are
often omitted and only reference to former publications was given.
In our hands, these conditions led to disappointing results and
especially, the degradation of phenothiazine ring system was
Optimization of N-alkylation of 2-methoxyphenothiazine 1
As illustrated by the examples displayed in Fig. 1, N-alkyl 2-
hydroxyphenothiazine moiety is the structural unit common to a
wide range of analyte-responsive PTZ-based fluorescent probes.
The first stages of their multi-step preparation are aimed to achieve

3
observed.
A
literature survey on the preparation of
propanesultone [17] to install a zwitterionic side-chain onto PTZ
levomepromazine and related N-alkyl PTZ derivatives revealed
the use of other strong bases such as NaNH2 or NaH to perform
such N-alkylation reaction, often conducted in an aromatic
hydrocarbon solvent (toluene or xylene) possibly in mixture with
DMSO or DMF [6i, 10, 15]. By drawing on these previous works,
we have devised a new protocol based on the treatment of 1 with
2 equiv. of n-butyl bromide, in the presence of 2 equiv. of NaH in
dry DMF at room temperature, that provided the desired N-butyl
derivative 2 with a good isolated 85% yield (and deployable to
gram scale synthesis, Scheme 1). At this stage, it seemed relevant
for us to expand the scope of this optimized protocol to more
sophisticated alkyl pendant arms bearing a further reactive group
for post-synthetic functionalization of PTZ heterocycle. Thus, N-
alkylation of 1 with commercial 3-dimethylaminopropyl chloride
(HCl salt) conducted under the same conditions, was next
considered and the desired product 3 was obtained with a modest
23% yield. No substantial improvement was obtained with the
mesylate derivative freshly prepared from 3-dimethylamino-1-
propanol. These results are consistent with those previously
obtained by us with unsymmetrical DPP pigments 4-6 (Scheme 1
and Supplementary data) for which the N-alkylation of lactam
moieties is essential to impart to them both solubility and
functionality.
scaffold (compound 14). Both reactions were conducted without
S
n-BuBr, KOH, KI,
DMSO, 100 °C
S
N
OMe
OMe
Yield 95% (ref. [8a])
but failure in our hands
N
H
OMe
Bu
2-Methoxyphenothiazine 1
2
S
RX, NaH, DMF,
RT, 90 min
S
N
R
X = Cl, Br or OMs
N
OMe
H
2-Methoxyphenothiazine 1
2, R = Bu, yield 85%
NMe2
3, R =
,
yield 23%
NMe2
H
N
N
Ar'
O
O
Ar'
O
O
Cl
NMe2 . HCl
NaH, TBAI, DMF,
80 °C, 24 h
Ar
Ar
N
N
H
Both
optimized
N-alkylation
methods
of
2-
4, Ar = p-Br-Ph, Ar' = p-MeO-Ph
5, Ar = p-Br-Ph, Ar' = p-MeO-Bn-OPh
6a, Ar = p-Br-Ph, Ar' = 4-Py
methoxyphenothiazine heterocycle can readily provide hundreds
of milligrams of 2 and 3. We wished to valorize these building
blocks in the synthesis of phenothiazine-coumarin hybrid dyes [7],
with the dual aim of accurately determining the spectral properties
of these NIR-I emitters under simulated physiological conditions,
and confirming the chemical inertness of N-alkyl pendant arm
during multi-step synthetic processes.
Me2N
7, Ar = p-Br-Ph, Ar' = p-MeO-Ph,
29%
N+
X-
NMe2
failure with DPP 5
failure with DPP 6
OMe
N
O
O
Synthesis of PTZ-coumarin hybrid dyes 10, 15 and 16
N
H
Br
8b
Coumarins fused with other (hetero)aromatic units are often
regarded as attractive fluorescent markers for performing
biosensing/bioimaging operations within the NIR-I spectral region
[16]. Among the myriad of -expanded coumarins currently
available and already studied in this context, 4H-
Scheme 1. (Top) Background information and revisited N-alkylation of 2-
methoxyphenothiazine with n-butyl bromide and 3-dimethylaminopropyl
chloride or mesylate (HCl salt); (bottom) N-Alkylation of DPP pigments of 3-
dimethylaminopropyl chloride (HCl salt, 10 equiv.) (OMs = mesylate, p-Br-Ph
= para-bromophenyl, p-MeO-Ph = para-methoxyphenyl, p-MeO-Bn-OPh =
para-methoxybenzyloxyphenyl, 4-Py = 4-pyridyl, RT = room temperature,
TBAI = tetrabutylammonium iodide, X- = Cl- or I-). aPlease note: for this DPP,
only 3 equiv. of alkylating agent was used and a mixture of N-alkyl pyridinium
and mono N-substituted lactam derivative was obtained. bPlease note: an
alternative structure for this side-product may be the N,O-dialkyl DPP
derivative.
benzo[1,4]thiazine-fused
compounds
(also
known
as
phenothiazine coumarin hybrids) have been used to produce a
fluorescence output upon the selective activation of "covalent-
assembly" type probes by targeted (bio)analytes (see Fig. 1 for
example of H2Sn-responsive probe) [8a, 8e, 8k, 8l].
A multi-step synthesis based on the preparation of the key
intermediate
N-alkyl
2-hydroxy-10H-phenothiazine-3-
carboxaldehyde and its subsequent Knoevenagel condensation
with a latent C-nucleophile (e.g., malononitrile, heteroaryl-
acetonitriles) complemented by an acid hydrolysis of iminolactone
to lactone, was devised to access to a set of PTZ-coumarin hybrid
dyes diversely substituted at the C-3 position. Both N-alkyl PTZ
derivatives 2 and 3 have been subjected to this reaction sequence
(Scheme 2). Vilsmeier-Haack formylation with POCl3/DMF
yielded the corresponding PTZ-based ortho-anisaldehydes in good
yields. Deprotection of aryl methyl ether was achieved with AlI3,
generated in situ from aluminum powder and iodine in dry MeCN.
From a practical point of view, we found that the use of a carousel-
type parallel reaction station is particularly needed for effective
gram scale synthesis of PTZ-based salicylaldehyde 12 because of
the low yield obtained when the reaction was performed at
hundreds of mg scale, in a single batch. A satisfying yield of 63%
was obtained for 12, compared to 92% for N-butyl derivative 9.
Next, chemoselective N-alkylation of 12 was performed either
with methyl iodide to obtain cationic derivative 13 or 1,3-
base to avoid undesired phenol substitution (10-20 equiv. of
alkylating reagent, in dry DCM at 40 °C for 17-24 h). Finally, the
three PTZ-coumarin hybrid dyes 10, 15 and 16 were synthesized
through Knoevenagel condensation reaction with malononitrile
under conventional conditions. All purifications were achieved by
semi-preparative RP-HPLC under acidic conditions (i.e., linear
gradient of MeCN in aq. TFA 0.1%, pH 1.9) that enable both
conversion of 2-iminocoumarins into coumarins 10, 15 and 16, and
their isolation in a pure form (>95%) required for photophysical
studies (vide infra). After recovery by freeze-drying, we noted that
the solid form of zwitterionic PTZ-coumarin hybrid dye 16, is
poorly soluble in water and polar organic solvent such as DMF,
DMSO, MeCN and MeOH. To overcome this solubility problem,
a further counter-ion exchange operation was performed by semi-
preparative RP-HPLC using aq. triethylammonium bicarbonate
buffer (TEAB, 50 mM, pH 7.5) and MeCN as eluents.
Unfortunately, the benefit was found to be negligible. All
spectroscopic data (see Supplementary data for the corresponding
spectra, Figs. S8-S10, S13, S33-S36, S39, S43-S44 and S47),

4
Tetrahedron Letters
especially NMR and mass spectrometry, were in agreement with
determined by ionic chromatography (see Supplementary data,
Figs. S11-S12, S37-S38 and S45-S46).
the structures assigned. The purity of each coumarin sample was
confirmed by RP-HPLC analysis with UV-vis detection at
different wavelengths, and mass percentage of TFA was
Scheme 2. (Top) Synthesis of PTZ-coumarin hybrid dye 10 from N-butyl 2-methoxyphenothiazine 1; (bottom) synthesis of cationic and zwitterionic PTZ-coumarin
hybrid dyes 15 and 16 from N-(3-dimethylaminopropyl) 2-methoxyphenothiazine 3: conditions A for 15 = piperidine, EtOH-MeCN (1:1, v/v), RT, 16 h; conditions
B for 16 = piperidine, anhydrous Na2SO4, EtOH-DMF (8:1, v/v), RT, 16 h. (O/N = overnight, FC (SiO2) = flash-column chromatography over silica gel, RT =
room temperature). aPlease note: compound 16 was isolated as triethylammonium salt.
determinations. Somewhat unexpectedly, the replacement of n-
Photophysical characterization of PTZ-coumarin hybrid dyes
butyl substituent by a polar pendant arm is not having a positive
effect on emissive effectiveness in aq. media. Indeed, in the
concentration range used for fluorescence measurements (10-7 M),
no formation of non-emissive H-type aggregates as evidenced by
the good matching between the absorption and excitation spectra
recorded in PBS, was observed whatever the N-substituent
introduced onto the PTZ scaffold (Fig. 2 and Figs S40-S42 and
Although several PTZ-coumarin hybrid dyes, especially the
derivative 17 bearing a 2-benzothiazolyl (BZT) moiety as C-3
substituent, have already been implemented in different biosensing
operations [8a, 8e], their spectral features have never been
determined in pure aq. buffers that mimic physiological
conditions. To fill this gap, we assessed the optical photophysical
properties of 10, 15 and 16 in phosphate buffered saline (PBS, pH
7.3) alone but also in the presence of a non-ionic surfactant
(Tween 80) to disrupt potential formation of aggregates. Further
spectral measurements in MeOH were also achieved. All results
S48-S50).
Further
molecular
motions
of
trimethylpropylammonium and sulfobetain moieties could
promote non-radiative deactivation and may explain the decrease
of quantum yields compared to those determined with N-butyl
derivative 10[11a].
are summarized in Table
1
(see Fig.
2
for the
absorption/fluorescence spectra of 10 and Supplementary data for
the absorption/fluorescence spectra of compounds 15 and 16, Figs.
S40-S42 and S48-S50). As already reported by Chen et al. for BZT
derivatives 17 [8a], all spectral features of PTZ-coumarin hybrids
10, 15 and 16 are characteristic of photoactive molecules where
strong internal charge transfer (ICT) process operates: (1) broad
structureless absorption and emission bands with full-width at half
maximum (FWHM) max in the range 80-110 nm and 130-165
nm respectively, (2) huge Stokes' shift values, and (3) NIR-I
emission with a very low fluorescence quantum yield in polar
solvents. Concerning this latter point, it is important to specify that
the standard used by Chen et al. (i.e., fluorescein in EtOH) for the
determination of relative fluorescence quantum yield of 17 was not
really the most suited because its absorption/emission curves do
not really display similarities with those of this class of ICT-based
compounds.
We found that tris(2,2'-bipyridyl)ruthenium dichloride
(Ru(bpy3)Cl2) in air-saturated water is more suitable for such

5
The need for a comprehensive understanding of biological
functions of ROS linked to their positive effects and adverse
impacts on human health and the environment, has promoted the
development of luminescent probe-based strategies for detecting
such oxidative bioanalytes in vitro and in vivo [18]. This is true, in
particular for hypochlorous acid (HClO) that is produced by
neutrophils and monocytes in vivo upon the reaction between H2O2
and Cl- ions catalyzed by myeloperoxidase [19]. This reaction also
takes place in mitochondria and due to its pKa = 7.46, HClO is in
equilibrium with ClO- under physiological conditions. Among the
myriad of reaction-based fluorescent probes responsive to
HClO/ClO-, described in the literature [20], one of the preferred
strategies involves functionalizing a fluorescent scaffold with a
phenothiazine moiety that readily quenches its emission through
the photoinduced electron transfer (PeT) mechanism.
Hypochlorite-mediated oxidation of sulfur to sulfoxide prevents
this PeT process and thus triggered fluorescence unveiling [6b-d,
6f-i, 8f, 21]. Depending the chromophoric structure of the probe,
an intensiometric or a ratiometric response can be obtained. In this
context, we wished to examine the fluorogenic reactivity of PTZ-
coumarin hybrid dyes 10, 15 and 16 towards this ROS. Preliminary
experiments have revealed that reaction of these phenothiazine
derivatives with NaOCl (bleach) leads to a large hypsochromic
shift (-110 nm, -161 nm and -168 nm for 10, 15 and 16
respectively) of their emission maximum (data not shown). By
analogy with the recent work of the Hou group [8f] and to avoid
signal saturation, we selected the Ex/Em 400/520 nm pair for time-
dependent fluorescence analyses and to highlight the ratiometric
behavior of 10, 15 and 16. On treatment of the three probes with 1
equiv. of ClO- in PBS (pH 7.3) at 25 °C, an immediate and
dramatic fluorescence enhancement at 520 nm was observed in
each case. The most substantial increase in green fluorescence
emission intensity was obtained with cationic PTZ-based probe
15. Conversely, in vitro assays conducted with zwitterionic PTZ-
based probe 16 has led to the least spectacular fluorogenic
response. Furthermore, no significant fluorescence signal changes
were observed in the absence of ROS, confirming the stability of
the three ratiometric probes 10, 15 and 16 in PBS.
Fig. 2. Normalized absorption (blue), excitation (Em. 625 nm, slit 5 nm, green)
and emission (Ex. 450 nm, slit 5 nm, red) spectra of PTZ-coumarin hybrid dye
10 in MeOH (A), PBS (B) and PBS + Tween 80 (C) at 25 °C. Please note:
peak at 519 nm/530 nm is assigned to Raman scattering.
Likewise, addition of Tween 80 (300 M) in aq. buffer does not
have a positive impact on fluorescence efficiency of these ICT-
based fluorophores. Furthermore, in the present case, we were not
able to use a more biorelevant disaggregating agent such as bovine
serum albumin (BSA, typically added to PBS at a content of 5%
(w/v) to simulate body fluid) because emission spectrum of PBS +
5% BSA solution displays an intense broad band centered at 530
nm that partly overlap emission band of 10, 15 and 16. Despite
their poor fluorescence in aq. media, we finally examined the
ability of these phenothiazine derivatives to act as effective
fluorogenic chemodosimeters for detection of reactive oxygen
species (ROS).
Fig. 3. Fluorescence emission time course (Ex/Em 400/520 nm, slit 2 nm) of
PTZ-coumarin hybrid dyes 10, 15 and 16 (concentration: 2.0 M) in the
presence of hypochlorite ions (1 equiv.) in PBS (pH 7.3) at 25 °C.
N
In order both to have an accurate idea of the conversion rate of
such fluorogenic S-oxidation processes and to gain insights into
the sensing mechanism through addressing issues related to the
formation of a single or several oxidized species and the possible
existence of further reactions triggered by this oxidation event,
each reaction mixture was first subjected to RP-HPLC-
fluorescence analyses (see Supplementary data, Fig. S63-S75).
Under acidic conditions (i.e., linear gradient of MeCN in aq.
formic acid 0.1%, pH 2.5, as eluent) used for these analyses, the
S
S
N
O
O
Bu
17
Sensing response of PTZ-coumarin hybrid dyes to HClO/ClO-
analyte

6
Tetrahedron Letters
starting ICT-based probe 10 was detected on both Ex/Em
noted the formation of other minor products, one of which at tR =
3.4 min was possibly identified as the sulfinic acid derivative or
more probably the carboxamide derivative (MS(ESI+): m/z =
383.2 [M + H]+, calcd for C20H19N2O4S+ 383.1, see Fig. S90).
Indeed, the nitrile moiety introduced onto the C-3 position of
coumarins, is prone to facile hydrolysis [23]. The same valuable
information has been gathered with the cationic PTZ derivatives
(see Supplementary data, Figs. S83-S85 and S91, tR = 3.0 min, m/z
= 426.2 [M]+, and tR = 3.2 min, m/z = 408.2 [M]+ for the sulfoxide
derivatives bearing -CONH2 and -CN as the C-3 substituent). We
have also confirmed the presence of a small amount of sulfoxide
derivative in the blank experiment (i.e., incubation of probe in PBS
alone) whose formation may arise from a photooxidation process
(i.e., sulfide oxidation mediated by singlet oxygen, formed by
triplet energy transfer to molecular oxygen since PTZ scaffold is
known to be an effective photosensitizer) [6a, 24]. Regarding the
PTZ-coumarin hybrid 16 bearing a sulfobetain pendant arm, its
poor reactivity towards ClO- was further confirmed by these RP-
HPLC-MS analyses (see Supplementary data, Figs. S87-S89).
Therefore, thanks to these complementary analytical tools, we
have shown for the first time that fluorogenic reactions between
PTZ-based probes and stoichiometric amount of ClO- is not a
quantitative and unequivocal process. Furthermore, depending on
the nature of N-substituent (length and net electrostatic charge) of
PTZ scaffold, the ability of sulfide moiety to be readily oxidized
and thus both quality and intensity of the fluorogenic ratiometric
response are dramatically impacted.
channels (400/520 nm and 450/700 nm, tR = 5.7 min). After
treatment with 1 equiv. of ClO-, a new major peak at tR = 4.6 min
was detected, characterized by an intense emission at 520 nm and
a negligible emission at 700 nm, that was assigned to the sulfoxide
derivative. When the S-oxidation reaction was conducted with 10
equiv. or 100 equiv. of ClO-, a slight decrease of peak intensity or
its complete disappearance was observed (see Supplementary data,
Figs. S66-S67). That would suggest that the sulfoxide derivative
may undergone over-oxidation (formation of sulfone derivative?
[22]) and subsequent degradation of PTZ heterocycle. RP-HPLC-
fluorescence results obtained with cationic PTZ derivative 15 are
quite similar (see Supplementary data, Figs. S68-S70, tR = 3.3 min
and 3.9 min for sulfoxide and starting PTZ derivatives
respectively). Conversely, a less substantial result was revealed
with zwitterionic PTZ derivative 16 even a new peak (tR = 3.4 min)
assigned to sulfoxide product was detected. The large remaining
amount of 16 observed on the elution profile (see Supplementary
data, Figs. S71-S73, tR = 3.9 min) highlighted the poor conversion
rate for this S-oxidation reaction. To confirm that the reaction
between the probes 10, 15 and 16 and ClO- led to the formation of
a sulfoxide derivative, the same mixtures were next analyzed by
RP-HPLC-MS (see Supplementary data, Figs. S76-S91). The
partial disappearance of the probe 10 peak (tR = 5.6 min) and the
formation of sulfoxide derivative (tR = 4.6 min) was clearly
observed and the structure of this latter green-emissive
fluorophore was supported by MS-ESI+ data (both in "full scan"
and single ion monitoring (SIM) modes). Interestingly, we also
Table 1 Photophysical properties of PTZ-coumarin hybrid dyes studied in this work, determined at 25 °C.
Stokes' shift
(nm / cm-1)b
Cmpda
Solvent

max Abs (nm)
 (M-1 cm-1)

max Em (nm)
F (%)c
10
10
MeOH
PBS
309, 451
310, 478
314, 462
309, 433
310, 443
309, 443
307, 432
307, 440
308, 440
10 800, 11 950
10 300, 9 850
11 550, 11 250
21 550, 20 700
19 650, 21 810
19 425, 22 050
9 400, 8 850
678
664
645
678
700
700
672
706
702
227 / 7 423
186 / 5 860
183 / 6 141
245 / 8 345
257 / 8 287
257 / 8 287
240 / 8 267
266 / 8 563
262 / 8 482
1.6
2.8
PBS + Tween 80
10
4.8
15
MeOH
1.6
15
PBS
0.2d
0.4d
1.6
0.3d
0.3d
PBS + Tween 80
15
16e
16e
16e
MeOH
PBS
8 150, 9 275
PBS + Tween 80
8 750, 9 125
PB-MeCN
(7:3, v/v)
17f
467
-
662
195 / 6 307
7
a
Stock solutions (1.0 mg/mL) of fluorophores were prepared in peptide synthesis grade DMF. Please note: do not use DMSO to achieve this because this solvent accelerates the
photooxidative degradation of these PTZ-based fluorophores.
b Only the maximum absorption wavelength corresponding to S0-S1 transition was used for this calculation.
c Determined using Ru(bpy)3Cl2 as a standard (F = 2.8% in water, Ex. at 450 nm) [25].
d Fluorescence efficiency was too low to establish a linear relationship between Abs at 450 nm and Em 470-880 nm with four distinct points. Only one measurement was possible (and
additional zero) to calculate these values below 1%.
e Difficulties were encountered to completely solubilize this compound in DMF (1.0 mg/mL) despite prolonged sonication. That explains lower values of molar extinction coefficients
obtained for this dye.
f Values determined and reported by Chen et al. [8a] Relative fluorescence quantum yield was determined using fluorescein as a standard (F = 79% in EtOH).
example, three PTZ-coumarin hybrid dyes with a marked ICT
character, have been synthesized and photophysically
characterized. Despite their modest fluorescence quantum yields
in neutral aq. buffer, N-butyl and cationic PTZ derivatives 10 and
15 have been identified as practical fluorogenic probes for
ratiometric detection of highly oxidative ROS, namely
hypochlorite ions, through selective S-oxidation of their
phenothiazine heterocycle. For the first time, the accurate analysis
of such reaction by means of RP-HPLC coupled to fluorescence
and mass detections, has enabled to show that this activity-based
Conclusion
In summary, we have revisited and extended N-alkylation
reaction of 2-methoxyphenothiazine 1 to the convertible 3-
dimethylaminopropyl substituent. Selective quaternarization of its
tertiary amine moiety has been achieved and this N-
functionalization approach has opened the way for preparing novel
hydrophilic PTZ-based pharmacophores and fluorophores. As an

7
sensing strategy did not necessarily result in the quantitative
formation of a single oxidized species (i.e., sulfoxide derivative).
We are convinced that the implementation of such analytical
methodology by (bio)chemists working in the field of reaction-
based fluorescent probes would be helpful for deciphering
precisely their activation mechanism and thus improving their
performances.
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Declaration of Competing Interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
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J. Sheng, W. Shen, X. Yue, C. Huang CN Pat.
108250220A, 2018.
This work is part of the project "Pharmacoimagerie et Agents
Theranostiques", supported by the Université de Bourgogne and
Conseil Régional de Bourgogne through the Plan d'Actions
Régional pour l'Innovation (PARI) and the European Union
through the PO FEDER-FSE Bourgogne 2014/2020 programs.
Financial support from Agence Nationale de la Recherche (ANR,
AAPG 2018, DetectOP_BChE, ANR-18-CE39-0014 and
LuminoManufac-Oligo, ANR-18-CE07-0045), especially for the
post-doc fellowships of Drs. Valentin Quesneau and Kévin
Renault is also greatly acknowledged. Part of this work devoted to
chemistry of DPP dyes was supported by the French
"Investissements d'Avenir" program, project ISITE BFC (contract
ANR-15-IDEX-0003), especially for the post-doc fellowship of
Dr. Sébastien Jenni. Flavien Ponsot gratefully acknowledges the
French Ministry of National Education, Higher Education and
Research for his Ph. D. grant (2017-2020). The authors thank the
"Plateforme d'Analyse Chimique et de Synthꢀse Molꢁculaire de
l'Universitꢁ de Bourgogne" (PACSMUB, http://www.wpcm.fr)
for access to analytical instrumentation, especially Dr. Quentin
Bonnin (CNRS, PACSMUB) for his help with variable
temperature NMR experiments, COBRA lab (UMR CNRS 6014)
and Iris Biotech company for the generous gift of some chemical
reagents used in this work.
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Supplementary data
Supplementary data (all synthetic procedures, spectroscopic
and photophysical characterizations of PTZ-coumarin hybrid dyes
described in this work) associated with this article can be found, in
the online version:
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9
Highlights:
N-Alkylation of 2-methoxy-10H-phenothiazine was optimized
Three novel NIR-emissive phenothiazine-coumarin hybrid dyes were synthesized
Phenothiazine-coumarin hybrid dyes act as ratiometric fluorescent probes for HClO
Fluorogenic hypochlorite-mediated oxidation was deciphered by RP-HPLC-fluorescence/MS