Quest for novel fluorogenic xanthene dyes: Synthesis, spectral properties and stability of 3-imino-3H-xanthen-6-amine (pyronin) and its silicon analog

Article abstract of DOI:10.1016/j.tetlet.2018.11.031

To expand the range of primary aniline fluorophores available and suitable for the design of fluorogenic protease probes, the synthesis of 3-imino-3H-xanthen-6-amine (known as pyronin) and its silicon analog (Si-pyronin) was explored and presented here. A comprehensive photophysical study of these two fluorescent anilines, confirms the effectiveness of the heteroatom-substitution approach (O → SiMe₂) to yield dramatic red-shifts in absorption and fluorescence maxima of the xanthene scaffold (+85 nm). However, it also revealed its adverse effect on the hydrolytic stability of the Si-pyronin, especially at physiological pH. The pro-fluorescent character and utility of these two fluorogenic (hetero)xanthene dyes are also proved by the preparation and in vitro validation of activatable fluorescence “turn-on” probes for penicillin G acylase (PGA).

Full text of DOI:10.1016/j.tetlet.2018.11.031

Accepted Manuscript
Quest for novel fluorogenic xanthene dyes: synthesis, spectral properties and
stability of 3-imino-3H-xanthen-6-amine (pyronin) and its silicon analog
Anthony Romieu, Garance Dejouy, Ibai E. Valverde
PII:
S0040-4039(18)31359-5
https://doi.org/10.1016/j.tetlet.2018.11.031
TETL 50416
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 October 2018
7 November 2018
9 November 2018
Please cite this article as: Romieu, A., Dejouy, G., Valverde, I.E., Quest for novel fluorogenic xanthene dyes:
synthesis, spectral properties and stability of 3-imino-3H-xanthen-6-amine (pyronin) and its silicon analog,
Tetrahedron Letters (2018), doi: https://doi.org/10.1016/j.tetlet.2018.11.031
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Quest for novel fluorogenic xanthene dyes:
synthesis, spectral properties and stability of
3-imino-3H-xanthen-6-amine (pyronin) and
its silicon analog
Leave this area blank for abstract info.
Anthony Romieu*, Garance Dejouy, Ibai E. Valverde
Red-shifted Abs/Em maxima (+85 nm)
Enhanced reactivity of meso-position
Aqueous stability only pH < 5
Fluorogenic reactivity
Abs/Em maxima in the green region
Poor reactivity of meso-position
Aqueous stability
Fluorogenic reactivity

1
Tetrahedron Letters
Quest for novel fluorogenic xanthene dyes: synthesis, spectral properties and stability
of 3-imino-3H-xanthen-6-amine (pyronin) and its silicon analog
Anthony Romieua,b,, Garance Dejouya, Ibai E. Valverdea
aICMUB, UMR 6302, CNRS, Univ. Bourgogne Franche-Comté, 9, Avenue Alain Savary, 21078 Dijon cedex, France
bInstitut Universitaire de France, 1, Rue Descartes, Bâtiment MONGE, 75231 Paris, France
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
To expand the range of primary aniline fluorophores available and suitable for the design of
fluorogenic protease probes, the synthesis of 3-imino-3H-xanthen-6-amine (known as pyronin)
and its silicon analog (Si-pyronin) was explored and presented here. A comprehensive
photophysical study of these two fluorescent anilines, confirms the effectiveness of the
heteroatom-substitution approach (OSiMe2) to yield dramatic red-shifts in absorption and
fluorescence maxima of the xanthene scaffold (+85 nm). However, it also revealed its adverse
effect on the hydrolytic stability of the Si-pyronin, especially at physiological pH. The pro-
fluorescent character and utility of these two fluorogenic (hetero)xanthene dyes are also proved
by the preparation and in vitro validation of activatable fluorescence "turn-on" probes for
penicillin G acylase (PGA).
Available online
Keywords:
Amino group protection-deprotection
Fluorescent probe
Hetero-xanthene dye
Protease
Pyronin
———
 Corresponding author. Tel.: +33-3-80-39-36-24; e-mail: anthony.romieu@u-bourgogne.fr.

2
Tetrahedron Letters
1. Introduction
Fluorescent organic dyes bearing one or two primary aniline moieties are now recognized as being essential chemical tools for the
design of analyte-responsive fluorescent probes applied to sensing/imaging applications.1,2 Indeed, the protection-deprotection of an
optically tunable amino group is often an easy-to-implement method that produces optimal fluorescence "OFF-ON" response and thus
high sensitivity for the detection of a target analyte (i.e., enzyme) in complex media (Fig. 1)3. The vast majority of fluorogenic anilines
currently available are based on benzo[a]phenoxazine, coumarin, N-alkyl 1,8-naphthalimide and xanthene scaffolds. Well-known
examples are oxazine 9 (cresyl violet analog)4, coumarin 120 (i.e., 7-amino-4-methylcoumarin)5, N-butyl-4-amino-1,8-naphthalimide6
and rhodamine 1102 or its disulfonated analog Alexa Fluor 4887 (Fig. 1).
Recently, research efforts have been focused on the development and practical applications of alternative aniline-based
fluorophores8, with higher performances in terms of water solubility, cell permeability, (bio)conjugation ability (for further
functionalization) and/or spectral properties (especially, high brightness and optimum excitation/emission wavelengths to minimize
spectral interferences inherent to sample/medium to be analyzed). Among the various fluorogenic core structures explored, great
attention has been paid to rhodamine 110 analogs for which the size of -conjugated system was expanded or the bridging oxygen atom
was replaced by a group 14 element (i.e., C, Si or Ge)9,10-13. Less common hybrid structures having a dimethine bridge between an
aniline and a second chromophore unit, have also been explored to rapidly access to near-infrared (NIR) fluorogenic dyes for more
challenging biosensing/bioimaging applications, especially in vivo14-17 (Fig. 1).
Figure 1. (Top) Principle of the amino group protection-deprotection strategy applied to "OFF-ON" fluorogenic detection of enzyme activity; (middle) structures
of fluorescent anilines used in the design of conventional fluorogenic enzyme substrates (Rh110 = rhodamine 110, AF488 = Alexa Fluor 488); (bottom) aniline-
based fluorophores specifically developed for biosensing/bioimaging applications in living cells or in vivo (DHX = dihydroxanthene, DCM = dicyanomethylene-
4H-pyran chromophore).
Surprisingly, none of these studies concerned the most structurally simple xanthene dyes known: pyronins and succineins whereas
these diphenylmethane derivatives exhibit remarkable optical properties in the green-yellow spectral range.18 Despite the reactivity of
their meso-position towards nucleophiles, that may be problematic for some applications in complex biological media, there is a
renewed interest for this class of fluorescent anilines. Indeed, the amino-group protection-deprotection strategy can be easily applied to
give valuable activatable (or "smart") fluorescent probes. Furthermore, in situ formation of the electronic push-pull conjugated
backbone of pyronin dyes, from a non-fluorophore caged precursor and via a cascade reaction triggered by the analyte to be detected,
has been recently demonstrated by the Yang group and us. This enabled the emergence of "covalent-assembly" type probes whose
potential utility and superior performances in terms of detection sensitivity were highlighted through the sensing of several analytes
including Sarin nerve agent mimics19, Hg(II) cations20 and amidases/proteases (i.e., penicillin G acylase (PGA) and leucine
aminopeptidase (LAP))21.
In this context, we strongly believe that the chemistry of 3-imino-3H-xanthen-6-amine 1 (Fig. 2), the only fluorogenic pyronin
known to date22, is worthwhile exploring to identify novel aniline-based fluorophores with valuable properties. We have thus revisited
the synthesis of 1 and here we report for the first time its spectroscopic and photophysical characterizations. The preparation and
spectral features of its silicon analog (Si-pyronin 2) are also described for the first time.23,24 The ability of these two anilines to act as
effective optical reporters in fluorogenic enzyme substrates has been finally assessed through the preparation and in vitro validation of
PGA-activated fluorescent probes.
N+
Cl-
H2N
X
+
N
X
NH2
-
CF3CO2
PY, pyronin Y, X = O
TMDHS, X = SiMe2
1, pyronin, X = O
2, Si-pyronin, X = SiMe2
Figure 2. Structures of already known (hetero)pyronins and fluorogenic analogs studied in this work.
Results and discussion

3
Synthesis of pyronin 1 and its silicon analog 2
To date, the synthesis of 3-imino-3H-xanthen-6-amine 1, through a conventional acid-catalyzed condensation between two
molecules of 3-aminophenol and formaldehyde followed by spontaneous aromatization, has been described in only one publication
written in German22 (Scheme 1). The work-up of the reaction mixture was not trivial, time-consuming and involved liquid-liquid
extractions with butanol, two size-exclusion chromatography over a Sephadex LH-20 column and a counter-ion exchange process to
obtain 1 as a perchlorate salt. Furthermore, the sole reported characterization data was curiously a UV-vis absorption spectrum recorded
in phosphate buffer (PB, 10 mM, pH 7.0).
In this context, we revisited the purification protocol of this polar fluorophore with a view both to simplify it and to rapidly obtain a
highly pure sample for spectroscopic characterization and photophysical studies. Butanol was replaced by the more conventional and
less expensive solvent mixture CHCl3-iPrOH (3:1, v/v)25, and the crude pyronin was easily purified by semi-preparative RP-HPLC
using a conventional liquid mobile phase (i.e., step gradient of CH3CN in aq. TFA 0.1%, pH 1.9). From 1.11 g of cheap starting
material 3-aminophenol, it was possible to recover 80 mg of 1 after only two independent HPLC purification runs (overall duration per
1
run = 60 min). This aniline-based fluorophore was fully characterized by IR, H, 13C and 19F NMR and ESI mass spectrometry (see
Figs. S4-S9) and all these spectroscopic data were in agreement with the structure assigned. Its high purity (>95%) was confirmed by
RP-HPLC analysis and the mass percentage of TFA (31%, ca. 1 TFA per molecule of fluorophore) in freeze-dried sample was
determined by ion chromatography.26
O
+
H2N
OH
H
H
H2N
O
NH2
1. conc. H2SO4, 100-145 °C
-
CF3CO2
2. H2O, NaOAc until pH 4-5
3. Liq.-liq. extractions
4. RP-HPLC purification
3-Aminophenol
1 (TFA salt)
yield 5%
Scheme 1. Synthesis of fluorogenic pyronin 1 (purification protocol revisited).
The synthetic route devised to obtain silicon analog of 3-imino-3H-xanthen-6-amine (Si-pyronin 2) through the formal substitution
OSiMe2 in the pyronin core structure is depicted in Scheme 2. The key step in the preparation of Si-xanthene dyes (e.g., TMDHS and
silicon analogs of some pyronin/rosamine derivatives) is the silylation/cyclization process involving the dilithium reagent generated
from 4 by halogen-metal exchange and SiMe2Cl2.12,24,27,28 Contrary to silicon-containing dyes previously published, the lack of alkyl
substituents in both amino groups necessarily implies the temporary protection of primary anilines. By analogy with the synthetic routes
towards silicon-fluorescein and sulfone-rosamine, respectively published by the Hanaoka group29 and us26, the allyl moiety was used as
protecting group (both to remove NH "acidic" protons and to enhance solubility of intermediates in organic solvents) that is stable under
a variety of conditions (especially in the presence of RLi/ArLi) and easily removed by Pd(0)-catalysis under mild and neutral
conditions.
, K2CO3,
H2N
NH2
H2N
NH2
Br
N
N
Br2, Ag2SO4, conc. H2SO4
CH3CN, 60 °C, 16 h
40 °C, 90 min
52%
82%
Br
Br
Br
Br
4,4'-Diamino-
diphenylmethane
4
3
1. sec-BuLi, THF, -78 °C, 1 h
2. SiMe2Cl2, -78 °C, RT, 1 h
3. DDQ, CH2Cl2, RT, 15 min
Pd(PPh3)4, 1,3-DMBA, C2H4Cl2,
N+
+
N
Si
5
H2N
Si
NH2
CF3CO2
Ar, 40 °C, 90 min
-
Br-
overall yield (from 4)
after two RP-HPLC purifications 10%
2 (TFA salt)
Scheme 2. Synthesis of fluorogenic Si-pyronin 2 (DDQ = 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, 1,3-DMBA = 1,3-dimethylbarbituric acid).
Thus, the preparation of Si-pyronin 2 began with meta-bis-bromination of 4,4'-diaminodiphenylmethane. This SEAr reaction was
performed using bromine and Ag2SO4 in concentrated H2SO4 at 40 °C for 1 h30, and purification by automated flash-column
chromatography over 25 m silica gel was effective to perform challenging separation between 3 and its undesired regioisomers. N,N'-
Tetraalkylation of 3 was achieved with an excess of allyl bromide (5.1 equiv.) and anhydrous K2CO3 (4 equiv.) in dry CH3CN at 60 °C.
Purification by conventional column chromatography over silica gel provided the key synthetic intermediate 4 in a good 82% yield.
Next, halogen-metal exchange reaction with sec-BuLi (2.5 equiv.) in dry THF à -78 °C, generated the 2,2'-methylenebis(aryllithium)
species, on which the subsequent reaction with SiMe2Cl2 (1.8 equiv.) and finally DDQ-mediated oxidation in CH2Cl2, afforded the blue-
colored N,N,N',N'-tetraallyl-Si-pyronin 5. Our first attempts to isolate this dye in a pure form by column chromatography over silica gel
failed because a disproportionation reaction was occurred in the presence of air to give a mixture of 5 and its reduced and keto forms
(Figs. S24 and S25). Consequently, it became more relevant to do only washings with aq. NaHCO3 in order to remove reduced
hydroquinone form of DDQ and to subject directly the crude 5 to Tsuji-Trost deallylation conditions. Thus, this final deprotection step
was readily achieved by treatment with a cat. amount of Pd(PPh3)4 and a large excess of 1,3-dimethylbarbituric acid (4.4 equiv) in
degassed C2H4Cl2 at 40 °C. Purification by semi-preparative HPLC afforded pure Si-pyronin 2 as a TFA salt (mass percentage of TFA
in freeze-dried sample = 33.5%, determined by ion chromatography, ca. 1 TFA per molecule of fluorophore) and in a moderate yet not

4
Tetrahedron Letters
optimized overall yield (10%) from 4. The structure was proven by NMR (1H, 13C, 19F and 29Si) and ESI mass analyses (see
Supplementary data and Figs. S26-S33) and the high level of purity (>99% whatever the wavelength used for the UV-vis detection) was
confirmed by RP-HPLC-based analytical control (see Supplementary data and Figs. S33 and S34).
Stability and photophysical characterization of pyronin 1 and its silicon analog 2
Since the bioanalytical applications envisioned for these fluorogenic dyes generally involve to work under physiological conditions,
the photophysical properties of these two compounds were primarily determined in phosphate buffered saline (PBS, 100 mM + 150 mM
NaCl, pH 7.4). However, the poor stability of Si-pyronin 2 under these simulated physiological conditions (vide infra) pushed us to
consider acidic buffers as the aq. solvents for the photophysical characterization of this hetero-xanthene dye (see Table 1 for
absorption/emission properties and Fig. 3 for the corresponding spectra).
Table 1. Photophysical properties of fluorophores and PGA-sensitive probes studied in this work, determined at 25 °C.

max Abs
(nm)a

max Em
(nm)
F
M-1 cm-1)
Cmpd
Solvent
(%)b,c
1
PBS
H2O
496
634
514
653
81 400
64 200
92
18
TMDHSd
aq. FA
0.1%e
NaOAc
PB
PB
NaOAc
2
580
597
42 200
89
2
7
9
9
580
238
267
265
597
47 900
23 000
22 650
28 600
89
f
f
-
-
f
f
-
-
f
f
-
-
aFurther bands with maxima within the UV spectral range are also observed in spectra of 1 (241, 260, 280 and 320 nm) and 2 (290 and 308 nm).
bDetermined using fluorescein as a standard (F = 95% in 0.1 M NaOH, Ex at 450 nm) for 1 and SR101 as a standard (F = 95% in EtOH, Ex at 545 nm) for 2.31
cAbsolute fluorescence quantum yield was also determined using an integration sphere (45% in PBS for 1 and 56% in CH3CN for 2, see Supplementary data for details).
dValues determined and reported by Fu et al.27
e0.1% aqueous formic acid (pH 2.5).
fNon-fluorescent.
Pyronin 1 exhibits spectral features in the green region, quite similar to popular fluorophores namely fluorescein and Alexa Fluor
488. The perfect matching between the absorption and excitation spectra (Fig. 3A) confirms the lack of non-fluorescent H-type
aggregates in neutral aqueous solutions. This may be credited to the small compact size and hydrophilic character of this xanthene dye.
The same photophysical study has been considered with the silicon analog 2 but we found that this compound is unstable in aq.
physiological conditions due to the marked electrophilicity of its meso-position (i.e., C-9 position of xanthene scaffold) and hence its
propensity to undergo nucleophilic attack by a water molecule or a hydroxyde anion. Indeed, despite lower electronegativity of Si atom
compared to O atom (1.9 against 3.5 in Linus Pauling's scale), the lack of electron-donating ability through +M effect seems to be
critical in this regard. The same poor hydrolytic stability was also evidenced in PB and ultrapure water, by the rapid discoloration of
initially pink solutions visible to the naked eye. To establish the pH range at which Si-pyronin 2 is fully stable and thus selecting the
appropriate aq. medium for photophysical measurements, its spectral behavior was studied over the pH range 3.5-9.5 through
incubation in various buffers (NaOAc, PB and borate, for 15 min at 23 °C) and subsequent absorbance measurement at 580 nm. By
drawing a pH-dependent absorption curve (Fig. 4), we were able to determine the pK value of the corresponding hydration equilibrium
between Si-pyronin 2 and Si-xanthydrol 6 (pK = 6.5). Furthermore, this curve clearly shows that the hydrolytic stability of 2 is optimal
at pH < 5. It is worth stating that pyronin 1 was found to be full-stable within the same pH range (see Supplementary data and Fig. S3),
supporting our hypothesis about the electrophilic character of meso-position weakened by +M effect of O-10 atom (vide supra).
We decided to investigate the photophysical properties of 2 in NaOAc buffer (100 mM, pH 4.8). 2 shows strong and sharp
absorption (full-width half maximum, FWHM, 1/2 max = 40 nm) and emission bands in the orange-red region of the spectrum (see Fig.
3B). The absorption maximum is centered at 580 nm, with a 85 nm bathochromic shift compared with pyronin 1. Molar extinction
coefficient ( = 47 900 M-1 cm-1) is of the same order of magnitude as that of silicon analog of pyronin Y (TMDHS,  = 64 200 M-1 cm-
1 in water)27. The emission band peaked at 595 nm is very narrow (25 nm FWHM) and the value of relative fluorescence quantum yield
(F = 89%) is really impressive and equivalent to that of larger fluorophores exhibiting the same spectral features (e.g., sulforhodamine
101, SR101, MW = 606.7, Abs/Em 575/595 nm, F = 95% in EtOH). As observed for pyronin 1, the absorption and excitation spectra
are perfectly superimposable (Fig. 3B), suggesting the presence of a single absorbing species in solution, and the lack of equilibrium
between Si-xanthene and Si-xanthydrol forms in NaOAc buffer.

5
A
B
Figure 3. Normalized absorption (blue), excitation (Em 550 or 660 nm, slit 5 nm, green) and emission (Ex 450 or 545 nm, slit 5 nm, red) spectra of pyronin 1 in
PBS (pH 7.4) (A) and Si-pyronin 2 in NaOAc buffer (pH 4.8) (B) at 25 °C.
In order to complement our study devoted to these two unusual fluorescent anilines, it was essential to demonstrate their fluorogenic
behavior through the reversible masking of their primary amino groups, in the context of fluorescence "turn-on" detection of analytes.
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Figure 4. pH-Dependant maximum absorbance (580 nm) curve for Si-pyronin 2 (concentration: 4.5 M in the corresponding buffer, 15 min of incubation before
absorbance measurement).
Fluorogenic reactivity of pyronin 1 and its silicon analog 2. Synthesis and in vitro validation of PGA-sensitive probes 7 and 9
To achieve this goal, we explored the synthesis of two unusual PGA substrates in which the green/orange-red fluorescence of
pyronin/Si-pyronin reporter is unveiled through two distinct and sequential events: enzymatic hydrolysis of phenylacetamide moieties
and 1,6-elimination process promoted by the released phenylogous amine. PGA (also known as penicillin amidase, EC 3.5.1.11) was
chosen as model protease because this hydrolytic enzyme has two clear advantages: (1) a structurally simple substrate
(phenylacetamide) than can be easily installed on a wide range of fluorescent anilines through amidification of their primary amino
group(s) and (2) a commercial availability at low cost32.
Bis-amidation of 1 and 2 was achieved by treatment with an excess of phenylacetyl chloride (PhAcCl, 17 equiv.) and DIEA (4-4.5
equiv.) in dry DMF (Schemes 3 and 4). We intentionally used a large excess of acyl chloride in order to promote the introduction of
phenylacetoxy (PhAcO-) moiety onto the meso-position. Indeed, once complete, the reaction was quenched by adding aq. TFA 1% to
produce a significant amount of phenylacetic acid (PhAcOH) that may able to displace chloride or other leaving groups onto the C-9
position of the (hetero)xanthene scaffold. In the case of pyronin 1, these conditions led to the desired fluorogenic PGA substrate 7 as the
major product. After isolation by semi-preparative RP-HPLC, the structure of 7 was confirmed by NMR and ESI mass analyses (see
Supplementary data, Figs. S35-S37).
PGA-sensitive
trigger
PGA
O
O
O
O
- PhAcOH
Ph
Ph
H2N
O
NH
N
O
7
N
H
H
1. PhAcCl, DIEA, DMF,
4 °C to RT, 105 min
1
2. 1% aq. TFA, RT, 15 min
then RP-HPLC purification
Fluorescent !!!
Non-fluorescent
yield 13%
Scheme 3. Synthesis of PGA-sensitive probe 7 and its enzymatic activation.

6
Tetrahedron Letters
Conversely, when Si-pyronin 2 was subjected to the same acylation conditions, the expected PGA-sensitive probe 8 was only formed
as a minor product. Surprisingly, the major compound isolated by semi-preparative RP-HPLC was identified as the -phenyl 9-Si-
pyronin-acetaldehyde derivative 9 (see Supplementary data for NMR and ESI mass analyses, Figs. S41-S43). The proposed mechanism
to explain the conversion of 8 into 9, via a Vilsmeier-type reaction is shown in Scheme 4. Once again, the unexpected reactivity of
phenylacetoxy ester 8 in line with its poor stability in aq. buffers (see Supplementary data), can be explained by the fact that the
electropositivity of the intracyclic Si atom cannot fully compensate the electrophilicity of the C-9 position.
O
PGA-sensitive
trigger
PGA
H
O
O
- PhAcOH, - PhCH2CHO
Ph
Ph
H2N
Si
NH
N
Si
9
N
H
H
1. PhAcCl, DIEA, DMF,
4 °C to RT, 90 min
2
2. 1% aq. TFA, RT, 15 min
then RP-HPLC purification
Fluorescent !!!
Non-fluorescent
yield 12%
O
O
O
O
DIEA
O
O
O
O
Ph
Ph
Ph
N
Ph
N
Si
8
N
Si
N
H
H
H
H
poorly stable compound ?
- CO2
Cl
O
H
N
H
H+, H2O
O
O
O
O
- NH(CH3)2
Ph
Ph
Ph
Ph
N
Si
9
N
N
Si
N
H
H
H
H
full-stable compound
O
Cl
O
+
Cl
H
N
H
N
-O
O
Chloroiminium salt
(Vilsmeier-type reagent)
Scheme 4. (Top) Synthesis of PGA-sensitive probe 9 and its enzymatic activation; (bottom) proposed mechanism to explain the conversion of 8 into 9.
As expected, the two probes 7 and 9 exhibit absorption only in the UV range (220-370 nm, Figs. S40 and S46) and are non-
fluorescent in both acetate and phosphate buffers (pH 4.8 and 7.4 respectively). Their sensing response to commercial PGA (from
Escherichia coli) was studied by time-dependent fluorescence analyses. The resulting kinetic curves are shown in Fig. 5 for 9 (see Fig.
S51 for the curves obtained with 7). When the enzymatic activation was performed at pH 4.8 (pH where the released Si-pyronin is full-
stable but not optimal for stability and activity of PGA), a significant and gradual increase of orange-red fluorescence of Si-pyronin
(Ex/Em 580/595 nm) was observed (Fig. 5). A plateau indicating the complete hydrolysis of 9 by PGA and subsequent aromatization of
the intermediate through 1,6-elimination process (the rate-limiting step of probe activation) was reached only after a lengthy incubation
time (more than 6 h).
However, after 15 h (overnight incubation), a dramatic 2600-fold increase in fluorescence was obtained (Fig. S53). Furthermore, no
fluorescence signal changes were observed in the absence of amidase, confirming the full stability of the probe 9 in acetate buffer.
Interestingly, the same fluorescence-based in vitro assay conducted in PB (pH 7.4) provided a lower but significant "OFF-ON" response
(a 9-fold decrease compared to enzyme activation in NaOAc buffer, Fig. S54) whereas Si-pyronin 2 is assumed to be unstable under
these simulated physiological conditions. A stabilization/protective effect of the local environment of enzyme in contact with 2 could be
advanced to explain this unexpected but positive result. Further experiments are currently in progress to clarify the behavior of
fluorescent Si-pyronin 2 in complex biological media. Finally, the presence of Si-pyronin dye 2 in the enzymatic reaction mixtures was
unambiguously confirmed by RP-HPLC analyses (fluorescence detection, tR = 3.5 min) and compared with an authentic sample of
synthetic Si-pyronin 2 used as reference (Figs. S56 and S57). The same methodology was used to identify the fluorescent species
(pyronin 1) found in enzymatic reaction mixtures related to activation of probe 7 with PGA (Fig. S55). All these results demonstrate the
potential utility of O-/Si-pyronin dyes bearing two primary anilines, as fluorogenic labels in enzyme-activatable molecular probes.

7
Figure 5. Time-dependent changes in the orange-red fluorescence intensity (Ex/Em 580/595 nm, slit = 5 nm) of fluorogenic probe 9 (concentration: 1 M) in the
presence of PGA (1 U) at 37 °C. Please note: PGA was added after 5 min of incubation of probe in buffer alone.
Conclusion
To the best of our knowledge, the present work is the first comprehensive survey devoted to the synthesis and characterizations of
the most structurally simple fluorogenic xanthene dyes known to date. These fluorophores namely 3-imino-3H-xanthene-6-amine 1 and
its silicon analog Si-pyronin 2 exhibit outstanding spectral properties in aq. solution, and dramatic bathochromic shift as a result of the
substitution of the pyronin oxygen bridge atom by silicon enables to cover a wide spectral range from green to red (ca. 450-650 nm)
while keeping a small compact size and a marked hydrophilic character. A study of the hydrolytic stability of Si-pyronin 2 has outlined
the pH range (pH < 5) of aq. media wherein this orange-red emitter is actually usable. The availability of two primary anilines within
the (hetero)xanthene core structure of these dyes gives them a unique fluorogenic reactivity and to place them high in the list of
fluorophores applicable to the design of reaction-based small-molecule probes for analyte sensing/bioimaging. Since the reactivity of
the meso-position can be finely tuned by the heteroatom-substitution approach (OSiMe2), the nucleophilic displacement of the C-9
substituent of fluorogenic enzyme substrates related to 8, may be an entrance gate towards "smart" theranostic drug delivery agents that
provide a cytotoxic chemotherapeutic response while facilitating fluorescence imaging of cancer cells33.
Acknowledgments
This work is supported by 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. G. D. gratefully
acknowledges the Burgundy Franche-Comté region for his Ph. D. grant and bioMerieux company (R&D Microbiology) as industrial
sponsor. Financial support from Institut Universitaire de France (IUF, 2013-2018), the Burgundy region ("FABER" programme, PARI
Action 6, SSTIC 6 "Imagerie, instrumentation, chimi ") and GDR CNRS "Agents d'Imagerie Moléculaire"
(AIM) 2037 are also greatly acknowledged. The authors thank the "Plateforme d'
Bourgogne" (PACSMUB, http://www.wpcm.fr) for access to spectroscopy instrumentation. COBRA lab (UMR CNRS
6014) and Iris Biotech company are warmly thanked for the generous gift of some chemical reagents used in this work. The authors also
thank Dr. Sylvain Debieu for preliminary research about fluorogenic pyronin dyes, Dr. Myriam Laly (University of Burgundy,
PACSMUB) for the determination of TFA content in samples purified by RP-HPLC, Dr. Ewen Bodio (University of Burgundy,
ICMUB, UMR CNRS 6302, OCS team) for access to SAFAS Xenius XC spectrofluorimeter (equipped with an integrating sphere) and
Dr. Jean-Franck Bussotti / M. Olivier Chaudon (SAFAS Monaco company) for their precious advice regarding the determination of
absolute fluorescence quantum yields.
Supplementary data
Supplementary data (all synthetic procedures, spectroscopic and photophysical characterizations of fluorophores and PGA-sensitive
probes, stability studies, in vitro fluorescence assays and HPLC-fluorescence analyses described in this work) associated with this
article can be found, in the online version:
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9
Graphical Abstract
Red-shifted Abs/Em maxima (+85 nm)
Enhanced reactivity of meso-position
Aqueous stability only pH < 5
Fluorogenic reactivity
H2N
Si
NH
Abs/Em maxima in the green region
Poor reactivity of meso-position
Aqueous stability
Fluorogenic reactivity
H2N
O
NH
Highlights:
The synthesis of 3-imino-3H-xanthene-6-amine dye (pyronin) was revisited.
The orange-red emitting silicon analog (Si-pyronin) was prepared for the first time.
Both compounds exhibit outstanding fluorescence properties in aqueous solution.
Aqueous stability of fluorogenic Si-pyronin dye only at pH < 5.
Novel fluorogenic (Si-)pyronin substrates for detection of penicillin G acylase.