Sylvain Debieu

Anthony Romieu

Kinetics improvement of protease-mediated formation of pyronin dyes

Keywords:

A fluorescent probe for protease sensing and based on the "covalent-assembly" principle is reported. The basic rational for this unusual class of chemodosimeters proposed by the Anslyn and Yang groups entails the synthesis of non-fluorophore caged precursors full-stable and reactive towards the targeted analyte. Unlike the first generation of protease-sensitive "covalent-assembly" type probes recently published by ourselves (Org. Biomol. Chem. 2017, 15, 2575-2584), the availability of dicyanomethylidenyl and enzyme-labile phenylacetamide moieties within the core structure of mixed bis-aryl ether 2 enables its rapid conversion into a fluorescent pyronin dye at physiological pH and upon activation with penicillin G acylase (PGA). This is real progress towards the practical implementation of this ingenious activation mechanism to the detection of enzymes in their native environment (in cellulo or in vivo).

Introduction

Over the past two decades, activatable (or "smart") fluorescent probes have become established for bioanalytical landscape and more recently in the fields of molecular imaging and theranostics. 1,2,3 The vast majority are designed on the basis of well-known photophysical processes (e.g., self-quenching and exciton coupling, resonance energy transfer, PeT, ...) 2,4 and/or fluorogenic reactions that often involve transformation of a functional group acting as a fluorescence switch (typically, bond-cleavage reactions, organic addition and/or metal-ligand substitution reactions and cascade reactions). 1,5 These advanced chemical tools enable the detection of analytes-of-interest with good to high sensitivity and unrivaled spatiotemporal resolution, in the context of complex biological or environmental matrices. To overcome some limitations of these probes and to expand their scope of application, a new probe design principle namely the "covalent-assembly" approach was recently proposed by Anslyn and Yang. 6,[START_REF]For the two reviews on this topic, see : (a) Romieu[END_REF] The basic rational of the "covalent-assembly" type probes is the in situ formation of the electronic push-pull conjugated backbone of a fluorescent organic dye, from a nonfluorophore caged precursor and via a cascade reaction triggered by the analyte-of-interest. The main advantage of this approach is to produce reaction-based fluorescent probes (also known as fluorescent chemodosimeters or pro-fluorophores) with zero background signal and hence particularly useful for applications that require high detection sensitivity. Its practical utility in molecular sensing is highlighted by the large number of recent publications dealing with the detection of various analytes (i.e., biothiols, enzymes, metal cations, nerve agents and ROS/RNS) ---* Corresponding author. Tel.: +33-3-80-39-36-24; e-mail: anthony.romieu@u-bourgogne.fr.

through in situ formation of fluorescent 7-N,N-dialkylamino-or 7hydroxy-(2-imino)coumarin or pyronin/rosamine scaffolds. 6,8,9 Further extension of the "covalent-assembly" principle to other less popular fluorescent molecules (e.g., benzo[c]cinnoline 10 , benzotriazole 11 , coumarin-fused resorufin) 12 , diazachrysene) 13 , phenantridine 14 and pyrazino-benz [e]indole derivatives 15 ) was also reported but the cascade reaction triggered by the analyte-ofinterest is often related to the specific reactivity of the latter and therefore not suitable for developing a versatile molecular chemosensing approach. Finally, some pioneering works have demonstrated that fluorescence biosensing at longer wavelengths (in the orange-red, far-red or near-infrared (NIR) spectral range) can be readily achieved through internal construction of polymethine dyes. 16,17 Indeed, biocompatible addition-elimination reactions leading to the formation of azomethine or dimethine bridges found in (hemi)cyanine scaffolds, have already been used to illuminate biological structures in vitro or in living cells (e.g., DNA G-quadruplexes 16b and cellular retinoic acid binding protein II (CRABPII) 17 ).

To implement the promising concept of "covalent assembly" to in vivo molecular imaging of disease-related enzymes [START_REF]For selected reviews about "smart" fluorescent probes suitable for NIR imaging of enzymes, see : (a) Tung[END_REF] , a first step was taken by our group with the clearly and indisputable demonstration of in situ formation of a yellow-orange emitting pyronin fluorophore 1, from the mixed bis-aryl ether 2 as caged precursor (Fig. 1), triggered by a protease (i.e., penicillin G acylase (PGA) or leucine aminopeptidase (LAP) enzyme). 19 However, the rate of cyclization/aromatization process was found to be slow and the use of an acidic buffer solution or additives known to activate

A R T I C L E I N F O A B S T R A C T
the carbonyl moiety of 2 (e.g., ammonium sulfate), was required to obtain its quantitative conversion into pyronin 1 (6-N,Ndiethylamino-3H-xanthen-3-imine) within 24 h of incubation with enzyme. To overcome this major shortcoming, particularly in view of implementing our small molecule detection platform for protease sensing in living biological systems (i.e., living cells and small animals), further structural optimization of this unusual type of fluorogenic enzyme substrates was considered. In this Letter, we report the valuable findings from this survey through the comprehensive description of synthesis, photophysical characterization, fluorogenic behavior and enzymatic activation of a novel PGA-sensitive caged precursor 3 rapidly converted to pyronin 1 under physiological conditions.

Results and discussion

The rationale behind a potentially profitable structural optimization of a pyronin caged precursor is to find the right balance between its reactivity (i.e., effectiveness of enzymetriggered cascade reaction) and its stability (i.e., inertness of the probe in the absence of the targeted enzyme). Starting from the core structure 2, the most obvious way to achieve this goal is to subtly enhance the electrophilic character of the aldehyde undergoing an intramolecular nucleophilic addition of the adjacent phenylogous amine unit unveiled by enzymatic hydrolysis. We thought to convert the formyl group into dicyanomethylidenyl moiety, whose electron-withdrawing properties are often exploited to design push-pull fluorophores with strong intramolecular charge transfer (ICT) character. 20 The presence of electrondonating N,N-diethylamino group in para position should confine the addition of water to this Michael acceptor leading to undesired hydrolysis of this benzylidenemalonitrile derivative. 21 Also, since the malonitrile anion is a better leaving group than the hydroxyde anion (pKa malonitrile = 11), a positive effect on the rate of 1,6elimination process contributing to xanthene aromatization, could also be expected.

Synthesis of dicyanomethylidene-based caged precursor 3

As previously reported by us 19 , the PGA-sensitive probe 2 was readily synthesized in two steps from commercial 3-iodoaniline (i.e., N-acylation with phenylacetyl chloride followed by Ullmantype coupling with 4-(N,N-diethylamino)salicaldehyde). Its conversion into dicyanomethylidene derivative 3 was achieved by reaction with malonitrile under mild conditions already optimized by our group for the synthesis of dual enzyme-responsive caged precursors of 7-hydroxy-(2-imono)coumarins: cat. piperidine, anhydrous Na 2 SO 4 , in EtOH and at room temperature (Scheme 1). Purification by conventional column chromatography over silica gel provided the dicyanomethylidene-based caged precursor 3 in a good 80% yield. All spectroscopic data (see Supplementary data), especially IR, NMR and mass spectrometry, were in agreement with the structure assigned. Its high level of purity was confirmed by RP-HPLC analysis and found to be in the range 96-99% depending the wavelength used for UV-visible detection.

Photophysical characterization of dicyanomethylene-based caged precursor 3

UV-visible and fluorescent measurements were conducted in phosphate buffer (PB, 100 mM, pH 7.6, simulated physiological conditions) containing less than 0.25% of DMSO (originating from dilution of 1.0 mg/mL stock solution in this latter solvent), and the corresponding spectral curves are given in Fig. 2. The PGA-sensitive probe 3 exhibits a strong electronic absorption in the violet-blue range (Abs l max = 467 nm and shoulder at l = 440 nm with e = 31 220 M -1 cm -1 and 27 770 M -1 cm -1 respectively) characteristic for an ICT transition from the N,N-diethylamino donor group to the dicyanomethylidenyl acceptor fragment. A further evidence of this is the large value of spectrum width (fullwidth half maximum , Dl 1/2 max = 90 nm). Contrary to the trend noted for aldehyde-based caged precursor 2, excitation at 440 nm (465 nm, or 485 nm), leads to a strongly Stokes-shifted ICT emission band centered at ca. 600 nm (fluorescence quantum yield F F = 6% determined using Ru(bpy) 3 Cl 2 as standard (F F = 4.2% in water) 22 ). Further investigation following up an unexpected result from HPLC-fluorescence analyses of enzymatic reaction mixtures (vide infra) has shown the complete loss of fluorescence properties in organic media (especially in CH 3 CN, see Fig. S1). This photophysical behavior is typical of molecules that exhibit aggregation-induced emission (AIE) behavior, and known as AIEgens. 23 Moreover, it is worthwhile mentioning that among the structural motifs found in AIEgens, the dicyanovinyl group is often used to decorate the popular tetraphenylethene (TPE) luminogen whose the AIE activity is ascribed to restricted intramolecular rotation (RIR) in aq. environment. The formation of such aggregates in PB is further supported by the recording of excitation spectrum of 3 that does not perfectly match with its absorption curve (Fig. 2). The main consequence of this lack of a zero background signal for the caged precursor 3 is that the detection of enzyme activity should be achieved through a ratiometric and not intensometric fluorogenic reponse as initially hoped. 2,4 Fortunately, the absorption ability of 3 at 525 nm is weak (Ex wavelength used for fluorescence-based in vitro assays) and its fluorescence emission at 545 nm (Em l max of pyronin 1 formed in situ) is almost negligible whatever the excitation wavelength used (440 nm, 465 nm or 485 nm) (Fig. 2 and4).

Comparative enzymatic activation of "turn-on" fluorogenic probe 2 and ratiometric fluorogenic probe 3

Fluorescence-based in vitro assays were performed with commercial PGA (from Escherichia coli) through time-course measurements. The resulting kinetic curves are shown in Fig. 3. A rapid and gradual increase of fluorescence emission at 545 nm (Ex at 525 nm) was observed when dicyanomethylidene-based probe 3 was incubated with PGA at 37 °C and the plateau (meaning that 3 is quantitatively converted into pyronin 1) was reached within 1 h (for the corresponding time-dependant emission spectra, see Supplementary data). Conversely, when the parent aldehyde-based probe 2 was reacted with PGA under the same conditions, the level of fluorescence achieved is dramatically lower (225 AFU vs 9630 AFU, within 1 h) confirming the slow formation of the xanthenebased fluorophore. This valuable result confirms our hypothesis that the rate of cyclization/aromatization process can be readily promoted by enhancing (1) the electrophilic character of reactive position undergoing intramolecular nucleophilic addition of adjacent phenylogous amine, and (2) the rate of 1,6-elimination of C-9 substituent. Interestingly, a direct comparison of the fluorescence emission spectra of PGA-sensitive probe 3 recorded before and after incubation with enzyme (Ex at 440 nm and 485 nm, Fig. 4), highlighted a spectral blue-shift of 50 nm allowing a proper ratiometric detection. Further kinetics in two other aq. buffers at acidic and basic pH respectively (acetate buffer, 100 mM, pH 5.6 and borate buffer, 100 mM, pH 8.6) were performed (see Fig. S2). In contrast to that observed with aldehyde-based probe 2, acidic pH provided a significant lower fluorogenic response compared to that observed at physiological pH (6350 AFU vs 11240 AFU after 85 min of incubation). Indeed, dicyanomethylidenyl moiety cannot be further activated by protonation unlike the carbonyl group. Finally, blank experiments without PGA were performed and have clearly shown the full stability of both probes. Indeed, no fluorescence signal changes were observed in the absence of this amidase. The presence of pyronin dye 1 in these enzymatic reaction mixtures was unambiguously confirmed by RP-HPLC analyses (fluorescence detection, t R = 4.0 min) and compared with an authentic sample of synthetic pyronin 1 used as reference (Fig. 5). The unexpected lack of a peak for the starting probe 3 on the RP-HPLC elution profile with a fluorescence detection channel Ex/Em 440/660 nm, strongly supports the AIE character of this dye molecule. Indeed, its elution from the C 18 column was observed at ca. 100% CH 3 CN (t R = 6.0 min), a solvent in which the formation of fluorescent aggregates is not observed (vide supra).

Conclusion

In summary, we have found a clever and easily implementable solution to dramatically increase kinetic of a biocompatible reaction working under physiological conditions, triggered by a protease and leading to in situ construction of fluorescent pyronin dyes. This is a major achievement since it will help to apply the "covalent-assembly" probe design principle for interrogating various fundamental biological questions through biosensing/bioimaging operations. We are aware that the systemic toxicity of malonitrile (i.e., metabolic release of cyanide ions) might prevent the use of such dicyanomethylidene-based probes in living systems. Thus, other Michael acceptor adducts derived from easily enolizable Meldrum's acid or 1,3-dimethylbarbituric acid should be considered. More generally, a subtle balance between stability and reactivity of pyronin caged precursors depending on the matrix to be analyzed and targeted application, can be achieved by gradual increasing of the electrophilicity of carbon center undergoing intramolecular nucleophilic addition of adjacent phenylogous amine (i.e., formyl, a-trifluoromethyl carbonyl and dicyanomethylidenyl moiety). In addition to these valuable findings related to the rational design of assembly type probes, we have identified a novel red-emissive fluorophore scaffold with AIE characteristics. Further works aimed at converting this luminogen into useful biological probes are in progress and will be reported in due course. Lastly, the presented enzyme-triggered cyclization/aromatization reaction could be a useful source of inspiration to organic chemists working in the field of total synthesis of natural products bearing a xanthene scaffold, especially through the development of effective biomimetic cascades. [START_REF] Masters | For selected examples[END_REF]

Figure 1 .

 1 Figure 1. Proposed detection mechanism of proteases (PGA chosen as model enzyme, also known as penicillin amidase, EC 3.5.1.11) based on the "covalent-assembly" principle and the use of caged precursors 2 and 3 convertible into fluorescent pyronin 1.

Scheme 1 .

 1 Scheme 1. Synthesis of PGA-sensitive fluorogenic probe 3.

Figure 2 .

 2 Figure 2. Normalized absorption (blue), excitation (Em. 580 or 620 nm, slit 5 nm, green) and emission (Ex. 440 or 470 nm, slit 5 nm, red) spectra of pyronin 1 and dicyanomethylidenyl-based caged precursor 3 in PBS at 25 °C. Please note: for excitation spectrum, intense peak at 290 or 310 nm (lEx/2) is assigned to Rayleigh scattering.

Figure 3 .

 3 Figure 3. Time-dependant changes in the green-yellow fluorescence intensity (Ex/Em 525/545 nm, slit 5 nm) of fluorogenic probes 2 and 3 (concentration: 1.0 µM) in the presence of PGA (1 U) in PB (100 mM, pH 7.6) at 37 °C. Please note: PGA was added after 5 min of incubation of probe in PB alone.

Figure 4 .

 4 Figure 4. Fluorescence emission spectra of fluorogenic probe 3 before and after incubation with PGA (1 U, 240 min of incubation in PB at 37 °C). (A) Ex at 440 nm (slit 5 nm) and (B) Ex at 485 nm (slit 5 nm).

Figure 5 .

 5 Figure 5. RP-HPLC elution profiles (fluorescence detection, system B) of fluorogenic probe 3 before incubation with PGA (top) and after incubation with PGA (1 U, 250 min of incubation in PB at 37 °C, bottom left), and authentic sample of pyronin 1 (bottom right). Please note: for RP-HPLC elution profile of blank (fluorogenic probe 3 incubated in PB alone) see Fig. S3.

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. Financial support from Institut Universitaire de France (IUF), the Burgundy region ("FABER" programme, PARI Action 6, SSTIC 6 "Imagerie, instrumentation, chimie et applications biomédicales"), especially for the Ph. D. grant of S. D., and GDR CNRS "Agents d'Imagerie Moléculaire" (AIM) 2037 are also greatly acknowledged. The authors 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. 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. Ibai Valverde and Garance Dejouy for a critical reading of the manuscript before publication, Dr. Jean-Alexandre Richard (ICES, A*STAR, Singapore) for helpful discussion and Marcel Soustelle (University of Burgundy, ICMUB, UMR CNRS 6302) for elemental analysis.

Supplementary data

Supplementary data (synthetic procedure and spectroscopic characterizations of compound 3, in vitro fluorescence assays and HPLC-fluorescence analyses described in this work) associated with this article can be found, in the online version:


Anthony Romieu
email: anthony.romieu@u-bourgogne.fr.

Garance Dejouy

Ibai E Valverde

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

Keywords:

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 profluorescent 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).

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. [START_REF] Chen | [END_REF]2 Indeed, the protectiondeprotection of an optically tunable amino group is often an easyto-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-naphthalimide 6 and rhodamine 110 2 or its disulfonated analog Alexa Fluor â 488 7 (Fig. 1).

Recently, research efforts have been focused on the development and practical applications of alternative anilinebased fluorophores [START_REF] Chevalier | For unsymmetrical analogs of sulforhodamine 101 bearing a primary aniline[END_REF] , 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 p-conjugated system was expanded or the bridging oxygen atom was replaced by a group 14 element (i.e., C, Si or Ge) [START_REF] Grimm | For the first example of fluorogenic carbopyronin[END_REF][10][START_REF]For reviews about Si-xanthene dyes and related probes[END_REF][12][START_REF] Kushida | For selected examples of Si-rosamines[END_REF] . 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 vivo [START_REF] Kong | For examples of probes based on amino-hemicyanine dye[END_REF][START_REF] Xie | For examples of probes based on DHX-hemicyanine hybrids[END_REF][START_REF] Sun | For examples of probes based on DCM scaffold[END_REF][START_REF] Liu | For examples of probes based on isophorone scaffold[END_REF] (Fig. 1).

NH 2 N + R NH 2 X - N + R X - O NH 2 X CN NC NH 2 R 1 R 2 DHX-hemicyanine Amino-hemicyanine DCM, X = O, R 1 , R 2 = fused Ph ring & R 3 = ⦰ Isophorone derivative, X = CH 2 , R 1 = H & R 2 = R 3 = Me R 3 A R T I C L E I N F O A B S T R A C T
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 mimics 19 , Hg(II) cations 20 and amidases/proteases (i.e., penicillin G acylase (PGA) and leucine aminopeptidase (LAP)) 21 .

In this context, we strongly believe that the chemistry of 3imino-3H-xanthen-6-amine 1 (Fig. 2), the only fluorogenic pyronin known to date 22 , 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. [START_REF] Urano | Si-pyronin 2 is claimed in a patent but never synthesized[END_REF][START_REF] Hanaoka | unsymmetrical Si-rosamines bearing a primary aniline[END_REF] 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.

Results and discussion

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 German 22 (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 counterion 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 CHCl 3 -iPrOH (3:1, v/v) 25 , and the crude pyronin was easily purified by semipreparative RP-HPLC using a conventional liquid mobile phase (i.e., step gradient of CH 3 CN 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 run = 60 min). This aniline-based fluorophore was fully characterized by IR, 1 H, [START_REF] Kushida | For selected examples of Si-rosamines[END_REF] C and 19 F 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 Scheme 1. Synthesis of fluorogenic pyronin 1 (purification protocol revisited).

The synthetic route devised to obtain silicon analog of 3imino-3H-xanthen-6-amine (Si-pyronin 2) through the formal substitution O®SiMe 2 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 SiMe 2 Cl 2 . 12,[START_REF] Hanaoka | unsymmetrical Si-rosamines bearing a primary aniline[END_REF]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 siliconfluorescein and sulfone-rosamine, respectively published by the Hanaoka group 29 and us 26 , 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. Thus, the preparation of Si-pyronin 2 began with meta-bisbromination of 4,4'-diaminodiphenylmethane. This S E Ar reaction was performed using bromine and Ag 2 SO 4 in concentrated H 2 SO 4 at 40 °C for 1 h 30 , and purification by automated flash-column chromatography over 25 µm 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 K 2 CO 3 (4 equiv.) in dry CH 3 CN 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 SiMe 2 Cl 2 (1.8 equiv.) and finally DDQmediated oxidation in CH 2 Cl 2 , 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 andS25). Consequently, it became more relevant to do only washings with aq. NaHCO 3 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(PPh 3 ) 4 and a large excess of 1,3-dimethylbarbituric acid (4.4 equiv) in degassed C 2 H 4 Cl 2 at 40 °C. Purification by semipreparative 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 optimized overall yield (10%) from 4. The structure was proven by NMR ( 1 H, [START_REF] Kushida | For selected examples of Si-rosamines[END_REF] C, 19 F and 29 Si) and ESI mass analyses (see Supplementary data and Figs. S26-S33) and the high level of purity (>99% whatever the wavelength used for the UVvis detection) was confirmed by RP-HPLC-based analytical control (see Supplementary data and Figs. S33 andS34).

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). X NH 2 + H 2 N 1, pyronin, X = O 2, Si-pyronin, X = SiMe 2 X N + N PY, pyronin Y, X = O TMDHS, X =

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 Sipyronin 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). a Further 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).

b Determined using fluorescein as a standard (FF = 95% in 0.1 M NaOH, Ex at 450 nm) for 1 and SR101 as a standard (FF = 95% in EtOH, Ex at 545 nm) for 2. 31 c Absolute 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). d Values determined and reported by Fu et al. 27 e 0.1% aqueous formic acid (pH 2.5).

f Non-fluorescent.

Pyronin 1 exhibits spectral features in the green region, quite similar to popular fluorophores namely fluorescein and Alexa Fluor â 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, Dl 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 (e = 47 900 M -1 cm -1 ) is of the same order of magnitude as that of silicon analog of pyronin Y (TMDHS, e = 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 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 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 Sixanthydrol forms in NaOAc buffer. 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.

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 cost [START_REF]For instance, only 100 euros for 1000 units[END_REF] .

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). 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 aphenyl 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. As expected, the two probes 7 and 9 exhibit absorption only in the UV range (220-370 nm, Figs. S40 and S46) and are nonfluorescent 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 Sipyronin 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 1,6elimination 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 2600fold 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. experiments are currently in progress to clarify the behavior of fluorescent Sipyronin 2 in complex biological media. Finally, the presence of Sipyronin dye 2 in the enzymatic reaction mixtures was unambiguously confirmed by RP-HPLC analyses (fluorescence detection, t R = 3.5 min) and compared with an authentic sample of synthetic Si-pyronin 2 used as reference (Figs. S56 andS57). 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.

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 smallmolecule probes for analyte sensing/bioimaging. Since the reactivity of the meso-position can be finely tuned by the heteroatom-substitution approach (O®SiMe 2 ), 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 cells 33 .

Figure 1 .

 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 â 488); (bottom) aniline-based fluorophores specifically developed for biosensing/bioimaging applications in living cells or in vivo (DHX = dihydroxanthene, DCM = dicyanomethylene-4H-pyran chromophore).

Figure 2 .

 2 Figure 2. Structures of already known (hetero)pyronins and fluorogenic analogs studied in this work.

Figure 3 .

 3 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.

Figure 4 .

 4 Figure 4. pH-Dependant maximum absorbance (580 nm) curve for Si-pyronin 2 (concentration: 4.5 µM in the corresponding buffer, 15 min of incubation before absorbance measurement).

Scheme 3 .

 3 Scheme 3. Synthesis of PGA-sensitive probe 7 and its enzymatic activation.

Scheme 4 .

 4 Scheme 4. (Top) Synthesis of PGA-sensitive probe 9 and its enzymatic activation; (bottom) proposed mechanism to explain the conversion of 8 into 9.

Figure 5 .

 5 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 µM) 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.

  SiMe 2

									O
									H 2 N	OH	H	H	H 2 N	O	NH 2 +
									1. conc. H 2 SO 4 , 100-145 °C	CF 3 CO 2 -
									3-Aminophenol	2. H 2 O, NaOAc until pH 4-5 3. Liq.-liq. extractions 4. RP-HPLC purification	1 (TFA salt)
									yield 5%
	Cl -				CF 3 CO 2	-
	H 2 N	NH 2	40 °C, 90 min Br2, Ag2SO4, conc. H2SO4	H 2 N				NH 2	CH3CN, 60 °C, 16 h Br , K2CO3,	N	N
	4,4'-Diamino-		52%			Br	Br	82%	Br	Br
	diphenylmethane							3	4
									1. sec-BuLi, THF, -78 °C, 1 h
									2. SiMe2Cl2, -78 °C, RT, 1 h
									3. DDQ, CH2Cl2, RT, 15 min
			H 2 N	Si	NH 2	+	Pd(PPh3)4, 1,3-DMBA, C2H4Cl2, Ar, 40 °C, 90 min	N	Si	N +
					CF 3 CO 2	-	overall yield (from 4)	Br -
								after two RP-HPLC purifications 10%
				2 (TFA salt)				5

Table 1 .

 1 Photophysical properties of fluorophores and PGA-sensitive probes studied in this work, determined at 25 °C.

	Cmpd	Solvent	lmax Abs (nm) a	lmax Em (nm)	e (M -1 cm -1 )	FF (%) b,c
	1	PBS	496	514	81 400	92
	TMDHS d	H2O	634	653	64 200	18
	2	aq. FA 0.1% e	580	597	42 200	89
	2	NaOAc	580	597	47 900	89
	7	PB	238	-f	23 000	-f
	9	PB	267	-f	22 650	-f
	9	NaOAc	265	-f	28 600	-f

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(IUF, -2018)), the Burgundy region ("FABER" programme, PARI Action 6, SSTIC 6 "Imagerie, instrumentation, chimie et applications biomédicales") and GDR CNRS "Agents d'Imagerie Moléculaire" (AIM) 2037 are also greatly acknowledged. The authors 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. 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

Supplementary data

Supplementary data (all synthetic procedures, spectroscopic and photophysical characterizations of fluorophores and PGAsensitive 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: