Massinissa Ayad

Philippe P Schollhammer

Yves Le Mest

Laurianne Wojcik

François Y Pétillon
email: francois-yves.petillon@univ-brest.fr

Nicolas Le Poul
email: nicolas.lepoul@univ-brest.fr

Dominique Mandon

Mononuclear copper(II) complexes containing a macrocyclic ditopic ligand: synthesis, structures and properties

Keywords: Copper complexes, Nitrogen ligands, Macrocylic ligands, copper-hydroperoxo species, hydrogen peroxide

come

Introduction

Iron and copper hydroperoxide complexes have been well investigated over the past 30 years, because these adducts are reactive intermediates for many biological and chemical oxidation processes [1][2][3]. In particular, [Cu-OOH] n+ (n = 1, 2) species have been proposed as being key intermediates involved in proton-coupled electron transfer (PCET) reactions promoted by monooxygenase enzymes, such as peptidylglycine α-hydroxylating monooxygenase (PHM) [4][5][6], lytic polysaccharide monooxygenase (LPMO) [7], or galactose oxidase (GO) [8]. Most of the reported biomimetic copper(II) hydroperoxide complexes have been shown to be unstable at room temperature [9][10][11][12][13][14][15][16][17][18][19][20][21]. These species are usually prepared upon reaction of a Cu(II) complex with H 2 O 2 in presence of a base (NEt 3 ). Reactivity of copper-hydroperoxo complexes was shown to be highly dependent on the mono-or dinucleating topology of the ligand, as well as its electronic and steric properties, which globally affect the O-O bond strength. For example, μ-1,1-hydroperoxo dicopper(II) complexes are electrophilic and typically allow Otransfer to nucleophilic substrates such as PPh 3 , but cannot perform H-atom abstraction [21]. In contrast, some mononuclear copper-hydroperoxo complexes were shown to be able to oxidize heterogeneous aromatic hydrocarbons, such as toluene or xylene [20]. Intramolecular aromatic hydroxylation and oxidative N-dealkylation of the supporting ligands have been also reported [15,16]. Hence, the full control of the ligand architecture is crucial to afford the characterization and studies of copper hydroperoxide complexes, as regards to their potential employment in catalytic systems.

We have recently developed a macrocyclic ligand L 1 (Scheme 1) which displays two coordinating sites (tris(methylpyridyl)amine (TPA), and pyridine-dicarboxamide (PydCA)) for metal ions attack [22]. This dissymmetrical ditopic ligand allows the generation of mono and dinuclear complexes. Our preliminary investigations were focused on mononuclear iron(II) complexes. Solid-state and solution studies demonstrated that the Fe(II) metal ion was coordinated in the TPA site. Reactivity of the complexes with H 2 O 2 in the absence of an exogenous substrate leads to an intramolecular aromatic hydroxylation. Notably, catalytic studies for the oxidation of cyclohexane into cyclohexanone and cyclohexanol show that the macrocyclic topology of the ligand and the nature of the counter-ion strongly impacted the turn-over number through different reaction mechanisms.

From these results, we present here the synthesis and characterization of three new mononuclear Cu II -(L n ) (n = 1, 2) complexes. Our aim was principally to rationalize the influence of the macrocyclic (rigid) structure of the ligands L 1 and L 2 (Scheme 1) on their structural, spectroscopic and electrochemical properties, in comparison to those of analogous copper complexes. The ligand L 2 bearing an isobutyl group was synthesized in order to enhance the solubility of the neutral copper complex [23]. Moreover, we have investigated the impact of the counter-ion (Cl -or OTf -), used in the synthesis on the spectroscopic and crystallographic features, as well as on their reactivity towards hydrogen peroxide to generate stable copper-hydroperoxo species. Scheme 1. Macrocylic and ditopic ligands L 1 and L 2 .

Experimental

General Procedures

All air sensitive organic reactions, as well as the handling and synthesis of copper complexes were routinely carried out under an argon atmosphere using standard Schlenk techniques. Further manipulations were performed in MBraun UNILab sp glovebox workstation under an argon atmosphere. All chemicals were purchased from Sigma-Aldrich and used without purification.

Solvents were either distilled immediately before use under nitrogen from appropriate drying agents or passed through MBraun MB SPS6800 solvent purification system. All dry solvents were degassed before use by bubbling N 2 through the liquid for 30 minutes or by freeze-thawing with nitrogen liquid under strict anaerobic conditions. CH 2 Cl 2 used for electrochemistry was freshly distilled from CaH 2 and kept under Ar in the glovebox. IR spectra were recorded on a Bruker-Vertex 70-Avatar spectrometer from solids. Chemical analyses were performed by the "Service de Microanalyse" ICSN-CNRS of Gif/Yvette (France). The UV-Vis measurements were carried out on a Jasco V-650 (190-10000 nm) spectrophotometer or a Varian Cary 05 E UV-Vis spectrophotometer equipped with an Oxford instrument DN 1704 cryostat in optically transparent Schlenk cells; HPLC-grade acetonitrile was degassed under argon and stored in a glove box. NMR spectra were recorded in CDCl 3 at ambient temperature on a Bruker AC 400 ( 1 H, 13 C) spectrometer. EPR spectra were obtained from a Bruker Elexsys E500 spectrometer, at a perpendicular mode X band (9.36 GHz); simulations were performed using the Bruker X-Sophe software. Electrochemical studies of the complexes were performed in a glove box (Jacomex) (O 2 1 ppm, H 2 O 1 ppm) with a home-designed 3-electrode-cell (WE: glassy carbon, RE: Pt wire in a Fc + /Fc solution, CE: Pt or a graphite rod). Ferrocene (Fc) was added at the end of the experiments to determine redox potential values. The potential of the cell was controlled by using an AUTOLAB PGSTAT 100 (Metrohm) potentiostat monitored by the NOVA software. The supporting salt NBu 4 PF 6 was synthesized from NBu 4 OH (Acros) and HPF 6 (Aldrich), then it was purified, dried under vacuum for 48 hours at 100°C, and finally kept under argon in a glovebox. Mass spectrometric measurements were performed on an Autoflex MALDI TOF III LRF 200 spectrometer by the "Service Commun de Spectrométrie de masse" of the Université de Bretagne Occidentale (Brest).

Syntheses of the ligands

The ligand L 1 was synthesized and characterized previously by us [22], according to a slight modified method of the literature [23]. The synthesis of L 2 was performed similarly to that of the cyclo [bis( (3-(pyridine-2-yl)-5-phenyl-2,6-dicarboxamide)amine] 2-pyridylmethyl (L 1 ) [24]. After the required purification steps the ligand was obtained as a pure, light-brown powder in valuable yields (62%).

Data for L 2 : IR (solid, cm -1 ): (NH) 3313 (w), (CO) 1681 (m). 1 H NMR (400 MHz, CDCl 3 ):  10.52 (s, 2H), 8.59 (d,J = 7.6 Hz,1H),8.33 (d,J = 4.4 Hz, 1H), 8.13 (s, 2H), 7.99 (s, 2H), 7.67 (t, J = 7.6 Hz, 4H), 7.50 (m, 8H), 7.31 (d, J = 7.0 Hz, 1H), 6.95 (t, J = 5.8 Hz, 1H), 4.12 (s, 4H), 4.07 (s, 2H), 3.95 (d, J = 6.4 Hz, 2H), 2.18 (m, 1H), 1.07 (d, J = 6.4 Hz, 6H). 13 C NMR (125.72 MHz, CDCl 3 ):  190.0 (2C, CH 3 , i OBut), 168.3, 161.7, 158.4, 151.0, 140.3, 138.4 (12C, C ipso ), 157.1 (2C, C=O), 137.3,132.2,132.1,131.9,129.5,128.6,128.5,123.0,122.8,121.8,120.9,119.8,119.0,112.0 (20 CH)

Syntheses of the Cu(II) complexes [CuCl 2 (L 1 )] (1)

To a THF (10 mL) solution of L 1 (50 mg, 0.08 mmol) in a Schlenk tube was added a blue solution of CuCl 2 (11.2 mg, 0.08 mmol) in THF (10 mL). Upon addition, the solution colored to green. The mixture was stirred for 8h at room temperature and then filtered. The resulting solution was concentrated by evaporation of the solvent, and Et 2 O (30 mL) was added to precipitate the product, that was washed with Et 2 O (2x5 mL) and dried in vacuum to yield the product 1 as a green solid.

Yield: 46 mg, 78%. Blue crystals suitable for an X-ray analysis were obtained, at room temperature, by slow vapor diffusion of diethyl ether into a CH 3 CN solution of 1. IR (solid, cm -1 ): (NH) 3300 (w), (CO) 1679 (m). UV-Vis (MeCN)  max , nm (, M -1 cm -1 ): 261 (29990), 284 (28080), 646 (23) O: C,56.08;H,4.45;N,12.38. Found: C,55.06;H 4.03;N,10.80.

[CuCl 2 (L 2 )] (2)
Similarly, a blue solution of CuCl 2 (18.9 mg, 0.14 mmol) in dry THF (5 mL) was added to a brown solution of L 2 (100 mg, 0.14 mmol) in THF (5mL) at room temperature; upon addition the solution colored to blue. The reaction mixture was stirred overnight, after which time diethyl ether (50 mL) was added to precipitate the product. The supernatant was removed via cannula filtration and the product was washed with Et 2 O (3x15 mL), dried in vacuum, giving 2 as a blue-green powder. Yield 64 mg, 56%. In spite of several attempts no crystal of 2 suitable for X-ray analysis was obtained. IR (solid, cm -1 ): (NH) 3322 (w), (CO) 1675 (m). UV-Vis (MeCN)  max , nm (, M -1 cm -1 ): 258 (22460). 302 ( 18630), ESI-MS (CHCl 3 ) found (calcd) for [M-2Cl] + m/z: 738.38 (738.22). EPR (9.30 GHz;

CH 3 CN; 150 K): g// = 2.22, g = 2.05, A// = 164 G. [Cu(H 2 O) 2 (L 1 )](OTf) 2 , H 2 O (3)
To a yellow-brown solution of L 1 (60 mg, 0.09 mmol) in THF (3 mL) was added a blue solution of [Cu(OTf) 2 ] (36 mg, 0.09 mmol) at room temperature. Upon addition the solution colored to dark green. The mixture solution was stirred for 8h and after filtered, 20 mL of diethyl ether were then added to the filtrate to precipitate a green solid. The solvents were removed by filtration and the residue was washed with ether (3x5 mL) and dried in vacuum to yield product 3 as a blue-green powder. The formulation of 3 was deduced from elemental analysis as being [Cu(H 2 O) 2 (L 1 )](OTf) 2 , H 2 O. Yield: 50 mg, 56%. Crystals suitable for a X-ray diffraction study were obtained by slow vapor diffusion of Et 2 O into a CH 3 CN solution of 3 in a sealed tube. IR (solid, cm -1 ): (NH) 3334 (w), (CO) 1654 (w), (CF) 1027 (s). UV-Vis (MeCN)  max , nm (, M -1 cm -1 ): 257 (28110), 284 (26400), 666 (51), EPR (9.30 O: C,45.93;H,3.46;N,9.62. Found: C,45.72; H, 3.17; N, 9.23.

X-ray structural determination

Measurements for compounds 1 and 3 were made on an Oxford Diffraction X-Calibur-2CDD diffractometer equipped with a jet cooler device. Graphite-monochromated Mo K radiation ( = 0.71073 Å) was used in all experiments. The structures were solved and refined by standard procedures [START_REF] Sheldrix | SHELXS 97[END_REF][START_REF] Farrugia | [END_REF]. A nitrogen stream cryostat attached to the system enabled low-temperature measurements (170K). Intensity data were collected combining several runs (omega-scan, step 1°) in order to obtain a complete set of reflections (as far as possible down to d = 0.8 Å or less). Bond lengths, angles, data collection and processing parameters are given in Table 1 and in the SI.

Results and discussion

Syntheses and characterization of the ligands

The ligand L 1 has been synthesized previously [22]. The synthesis of L 2 was performed similarly to that of L 1 . L 2 was obtained as a light-brown powder in valuable yields (62%), by adding a tetrahydrofuran solution of 4-isobutylether-2,6-dicarbonyldichloride-pyridine, instead of 2,6-pyridine bicarbonylchloride to a tetrahydrofuran/acetonitrile solution of 2-aminophenyl-6-methylpyridine used for L 1 , as described in Scheme 2. L 2 was characterized by spectroscopy (see the Experimental part), and its geometry was confirmed by an X-ray diffraction study [24].

Scheme 2. Synthetic pathway for L 2

Syntheses and spectroscopic characterization of the Cu II complexes

The synthetic procedure to prepare the mononuclear complexes 1-3 is identical to that described for analogous iron(II) complexes [22] (Scheme 3). The metalation proceeds by addition of stoichiometric amounts of the metal salt, CuCl 2 or [Cu(OTf) 2 ], to a THF solution of the ligand L n (n = 1, 2), without the addition of a base. The complexes are obtained as green or blue-green powders in noticeable yields (see the Experimental part). Elemental analysis suggests that the complex 1 contains three solvate The formulation of 3 was deduced from elemental analysis as being the dicationic compound

[Cu(H 2 O) 2 (L 1 )](OTf) 2 .H 2 O
, which contains a solvate water molecule. This structure was confirmed by an X-ray diffraction study of crystals of the complex (see below; Figure 2), obtained by slow diffusion of diethyl ether into an acetonitrile solution of 3 with, however, the presence of a half molecule of diethyl ether, instead of 1 H 2 O.

The infrared spectra of complexes 1-3 display two main bands near 3300 and 1660 cm -1 , which can be assigned, respectively, to the vibrations of the NH and CO bonds of the uncoordinated imide moieties.

These frequency values are close to those observed for the free ligands L 1 and L 2 ( = 3313, and 1681 cm -1 ); these results are in agreement with the crystallographic data, which show complexation on the TPA site. The other spectroscopic data (UV-vis, EPR), ESI-MS and the molar conductivity accord also with the structures proposed for complexes 1-3 (see the Experimental part, and below). In particular, mass spectroscopy of complex 1 into a mixture of CHCl 3 /CH 3 CN (9:1) shows the presence of a peak at m/z = 666.86, which corresponds to the monocationic complex 1 deprived of the chloride ions, namely [Cu(L 1 )] + . Hence, it should be concluded to the decoordination of both chloride anions occurs in these experimental conditions.

X-ray diffraction study of 1 and 3

The crystal structure of 1 is shown in Figure 1. Selected bond lengths and angles are gathered in Table 2 (see SI for further details). The structural analysis reveals that crystals of 1 were formed with two solvate molecules of acetonitrile, and therefore this compound was formulated in the solid state as [CuCl 2 (L 1 )]. 2 CH 3 CN. The Cu(II) ion is tetra-coordinated by two chlorides and a bidentate N-donor ligand fragment, involving the TPA site; precisely, the nitrogen atoms are those of the tertiary amine (N( 1)) and the unsubstituted pyridine (N( 7)). The geometry can be considered as a distorted squareplane. For the nitrogen atoms N( 2) and N( 6) of the substituted pyridyl groups of the TPA ligand, the copper-nitrogen distances are long (Cu(1)-N ~ 2.91 Å) compared to the Cu(1)-N(1) and Cu(1)-N (7) ones (Cu(1)-N ~2.06 Å), suggesting at the most weak metal-ligand interaction. The dihedral angle  between the two plans defined by the Cl(1)-Cu(1)-N( 1) and Cl(2)-Cu(1)-N( 7) atoms equals 15.20°, which indicates that the square-plane is slightly distorted [27]. The chloride ion (Cl( 1)) in the proximity of the amide groups is at ~ 2.5 Å from the most proximal atoms (Table 3), which suggests only a light hydrogen-bonding stabilization. The solid-state structure of complex 1 is radically different from that of its analogous complex [CuCl(TPA)] + . Indeed, the latter displays a pentacoordinated copper ion in a bipyramid trigonal geometry, including four nitrogen atoms (Cu-N average distance: 2.06 Å) [28]. Such a discrepancy at solid state might be correlated to the rigidity of the ligand L 1 which does not allow coordination of all nitrogen atoms of the TPA core. Here, this lack of flexibility is compensated by the presence of a second chloride anion in the first coordination sphere.

Table 2. Selected bond lengths (Å) and angles (°) for complexes 1 and 3.

Compound 1

Monocrystals of 3 have been obtained by slow diffusion of diethyl ether in an acetonitrile solution of the complex in a sealed tube. Crystals of 3 have been analyzed by X-ray diffraction. According to this analysis, complex 3 crystallizes as [Cu(H 2 O) 2 (L)][OTf] 2 .0.5 Et 2 O. X-ray structure of 3 (Figure 2) reveals the presence of two mononuclear complexes in the unit cell, but crystallographically distinct Cu(II) centers (Table 1 andSI As shown in Table 3, there is no interaction in complex 1 between the chlorine and ligand hydrogen

EPR spectroscopic studies

EPR spectroscopy of complexes 1 and 3 was recorded in frozen dichloromethane and acetonitrile (T = 155 K). All Cu(II) complexes display an anisotropic spectrum with similar features, except for complex 1 in CH 2 Cl 2 . Simulation of the spectra led to the determination of the g and A parameters.

(see Table 4 andSI). The values obtained for complex 1 in CH 3 CN, and complex 3 in both solvents (g // = 2.24-2.26; g⊥ = 2.06-2.08; A // = 166 10 -4 cm -1 ) are typical of a penta-coordinated copper(II) complex in a square-pyramidal geometry [29]. The complex 3 seems poorly affected by the change of solvent, indicating that substitution of H 2 O by CH 3 CN does not modify the coordination sphere of the copper(II) ion. This is in agreement with solid state data showing a nearly perfect square-pyramidal conformation in which water molecules can be easily substituted (vide supra). The effect of solvent was however more important for complex 1, since a rhombic signal (g 1 = 2.06 (A 1 = 30 10 -4 cm -1 ), g2 = 2.08 (A 2 = 35 10 -4 cm -1 ), g 3 = 2.24 (A 3 = 160 10 -4 cm -1 )) was obtained for the complex in dichloromethane, whereas an axial signature was detected in acetonitrile. Again, these results are consistent with X-ray data. Probably, the complex remains in the pseudo-axial conformation in CH 2 Cl 2 , as in solid state, but undergoes substitution of chloride ions by nitrilo ligands in acetonitrile. The UV-Vis studies showed that complexes 1 and 3 displayed similar spectroscopic signatures in acetonitrile (see Supplementary Information for spectra). Two intense bands located at 284-285 nm and 261 nm were detected, corresponding to π→π* transition in the pyridine ligand and phenyl groups, respectively (for comparison the ligand L 1 displayed two bands at 288 nm and 250 nm).

Complexes 1 and 3 also showed a low absorption band in the visible region ( max = 646 nm and 666 nm, respectively), which can be ascribed to a d-d electronic transition. These values are in the same range of those found for analogous penta-coordinated copper bis-pyridyl-amine complexes developed by Itoh et al [30].

Voltammetric studies.

Comparative voltammetric studies of complexes 1 and 3 were carried out in CH 2 Cl 2 / NBu 4 PF 6 0.1 M at a platinum working electrode. For both complexes, an irreversible peak was detected upon reduction on scanning negatively (Figure 4). The value of this reduction peak was found to be slightly more negative for 1 than for 3 (-0.72 V and -0.55 V vs. Fc, respectively), and is in the same range, for complex 1, as previously reported for [CuCl(TPA)] + in CH 2 Cl 2 (-0.75 V vs. Fc) [31]. This redox process is ascribed to the monoelectronic reduction of the Cu(II) into Cu(I). Cycling back led to the appearance of one or several oxidation peaks depending on the complex and the scan rate. The large peak separation between oxidation and reduction peaks is typical of copper complexes with strong rearrangement of the coordination sphere upon electron transfer [32]. This effect is associated with the different coordination and geometric properties between Cu(II) and Cu(I) redox states. Thus, the reduction at E pc (1) generates a Cu(I) unstable species that evolves into a copper complex by probable unbinding of one ligand. The reaction of complex 1 and 3 with H 2 O 2 in acetonitrile at room temperature was monitored by UV-Vis spectroscopy (see Figure 5). Both experiments clearly show that the complexes evolve towards a new species which displays an absorption band at  max = 387 nm. The wavelength value is close to that obtained for analogous copper-hydroperoxo complexes such as [Cu II (BPPA)(OOH -)] + (380 nm) [10] and [Cu II (TPA)(OOH -)] + (379 nm) [33]. The absorption bands are assigned to a Ligand to Metal Charge Tranfser (LMCT) from the hydroperoxide ligand to the Cu(II) ion. Hence, these results suggest here the formation of the complex [Cu II (L 1 )(OOH -)] + from both complexes 1 and 3 (Scheme 4). This indicates that the reaction with H 2 O 2 induced the unbinding of chloride and water ligand from the copper center. As shown in Figure 5, the formation of the hydroperoxo complex is slow at room temperature, the generated species being stable over several hours. Possibly, the Cu-OOH complex is stabilized through weak interactions (H-bonding) with the second coordinating core (PydCA).

Conclusions

In summary, we have reported here the synthesis and characterization of three new mononuclear copper(II) complexes 1-3 bearing ditopic macrocyclic ligands (L 1 or L 2 ). The solid state studies for 1 and 3 demonstrate that the Cu(II) ion is preferably coordinated in the TPA site, as previously found with the Fe(II)-L 1 analogous complex [22]. However, the coordination sphere of the copper centre is dictated by the Cu(II) salt used for the synthesis, since the tetracoordinated complex 1 prefers a distorted square-planar geometry at solid state while the pentacoordinated complex 3 displays an almost perfect square-pyramidal conformation. When dissolved in acetonitrile, both complexes adopt a square-pyramidal geometry, as emphasized by EPR studies. Electrochemical studies of complexes 1 and 3 show a strong rearrangement of the coordination sphere upon electron transfer. Remarkably, the reaction of 1 or 3 with H 2 O 2 yields a copper(II)-hydroperoxo species which is stable at room temperature. Future works will aim at studying the catalytic properties of the Cu II -OOH species towards organic substrates, and in particular the possible HAT of aliphatic hydrogenated substrates through monoelectronic oxidation.

, 75. 3 (

 3 CH 2 , i OBut), 61.5 (2C, N-CH 2 -Py), 59.4 (1C, N-CH 2 -Py). MS (CHCl 3 , m/z): Calcd for [M]: 675.29 (100%). Found: 674.4 for [M-H].

1 (Scheme 3 .

 13 Scheme 3. The synthetic pathways for the complexes 1 and 2 (path A), and 3 (path B).

Figure 1 .

 1 Figure 1. Molecular structure of 1 (thermal ellipsoids at 50% level). The hydrogen atoms are not displayed for clarity.

  ). Each copper(II) ion is pentacoordinated in the TPA site of the L 2 ligand, within a pseudo-square-based pyramidal geometry including the(O(1),N(1),N(6), N(7)) square and the apical O(2) atom. The value of Addison parameter  for this structure (=0.05) indicates an almost perfect square pyramidal geometry of the first coordination sphere of the metal ion. As shown in Table 2, the shorter Cu(1)-O(1) bond length (vs Cu(1)-O(2)) can be ascribed to a trans-effect of the nitrogen atom N(1) in the equatorial plan. The bond lengths for the solvate in 1 (CH 3 CN) and 3 (Et 2 O) are those expected for such a non-bonded molecule. In other respects, the structural analysis reveals that, by coordination to Cu(II) ion, the macrocycle L 1 is much more distorted in 3 than in 1.

Figure 2 .

 2 Figure 2. Molecular structure of 3 (thermal ellipsoids at 50% level). The hydrogen atoms are not displayed for clarity.

  (H-N(3/5)) atoms (H .. Cl~2.52 Å). Similarly, no obvious interaction is detected in complex 3 between oxygen atoms(O (5/8)) of the solvate and amine hydrogens of the TPA ligand (H-N(3/5)), with the average "O .. H" distance of 2.48 Å. However, a weak interaction can be observed in 3 between the hydrogens of the two bound water molecules (H-O(1/2)) and oxygen atoms of the two solvate molecules (d(O-H)~1.90 Å); a weak interaction is also detected between the TPA nitrogen atom, N(2), and the hydrogen of a water molecule (d(N-H(2v)~1.93 Å) (see SI).

Figure 3 .

 3 Figure 3. EPR spectra of complexes 1 (green: CH 2 Cl 2 ; red: CH 3 CN) and 3 (orange: CH 2 Cl 2 ; blue: CH 3 CN) at T=155 K in frozen solutions of solvents.

Figure 4 .

 4 Figure 4. Cyclic voltammograms of complexes 1 (A) and 3 (B) at a Pt working electrode in CH 2 Cl 2 /NBu 4 PF 6 0.1 M at different scan rates. E /V vs. Fc.

Figure 5 .Scheme 4 .

 54 Figure 5. UV-Visible spectroscopic monitoring of the reaction in CH 3 CN of A) complex 1 (3 mM) with H 2 O 2 (30 mM), and B) complex 3 (5 mM) with H 2 O 2 (15 mM) at room temperature. Inset: Plots of Abs(387 nm) vs. time.

  Elemental analysis calcd (%) for C 37 H 29 Cl 2 CuN 7 O 2 . 3H 2

, ESI-MS [CH 3 CN/CHCl 3 (1/9)] found (calcd) for [M-2Cl] + m/z: 666.86 (666.86). EPR (9.32 GHz; CH 2 Cl 2 ; 150 K): g// = 2.14, g = 2.08, A// = 171 G.

  GHz; CH 3 CN; 150 K): g// = 2.27, g = 2.05, A// = 166 G. Elemental analysis calcd (%) for C 39 H 29 CuF 6 N 7 O 8 S 2 . 1 H 2

Table 1 .

 1 Crystallographic data and refinement parameters of the complexes 1 and 3.

		1		3
	Empirical formula	C 41 H 35 Cl 2 CuN 9 O 2		C 82 H 76 Cu 2 F 12 N 14 O 21 S 4
	Formula weight	820.22		2076.89
	Temperature (°K)	170(2)		170(2)
	Wavelength (Å)	0.71073		0.71073
	Crystal system, space group	Monoclinic, P1 21/c1		Monoclinic, P1 21/n 1
	Unit cell dimensions : a (Å)	13.2568(6)		11.3061(3)
	b (Å)	21.6185(7)		18.4544(5)
	c (Å)	14.3023(6)		21.4444(5)
	(°)	114.1535(5)		97.720(2)
	Volume (Å 3 )	3740.1(3)		4433.8(2)
	Z	4		2
	D(calc) (Mg m -3 )	1.457		1.556
	Absorption coefficient (mm -1 )	0.778		0.679
	F(000)	1692		2128
	Crystal description	Flat spindle-shape needle		Prism, axis [ ] ?
	Crystal color	Clear light green		Light blue
	Crystal size (mm)	0.46x0.35x0.04		0.44x0.24x0.08
	Theta range for data collection (°) 3.44 to 26.37		3.38 to 26.37
	Limiting indices	-16 h 14, -26 k 27, -17 l	-13 h 14, -23 k 23, -25 l
		17		26
	Reflections collected/unique	23102/7627 [R(int) = 0.0545]	39084/9050 [R(int) = 0.0619]
	Completeness to theta = 26.37(%) 99.7		99.8
	Absorption correction	Semi-empirical	from	Analytical
		equivalents
	Max. and min. transmission	0.9696 and 0.7162		0.9477 and 0.7543
	Refinement method	Full-matrix least-squares on F 2	Full-matrix least-squares on F 2
	Data/restraints/parameters	11513/24/493		9050/60/654
	Goodness of fit on F 2	1.027		1.041
	Final R indices [I2(I)]	R 1 = 0.0452, wR 2 = 0.1031		R 1 = 0.0453, wR 2 = 0.1042
	R indices (all data) Largest diff. peak and hole (eÅ -3 ) 0.856 and -0.523 R 1 = 0.0694, wR 2 = 0.1155		R 1 = 0.0606, wR 2 = 0.1124 0.687 and -0.864

Table 3 .

 3 D-H..A distances (Å) and angles (°) in 1 and 3.

	D-H .. A	d(N-H)	D(H .. A)	d(N .. A)	D(D-H .. A)
	Compound 1
	N(3)-H(3N).Cl(1) 0.871(14)	2.571(15)	3.375(2)	154.0(15)
	N(5)-H(5N).Cl(1) 0.874(14)	2.476(16)	3.248(2)	147.7(16)
	Compound 3
	N(3)-H(3N).O(5) 0.850(17)	2.37(2)	3.135(3)	149(2)
	N(3)-H(3N).O(8) 0.850(17)	2.60(2)	3.123(3)	121(2)
	N(5)-H(5N).O(5) 0.866(17)	2.219(19)	3.033(3)	156(2)
	N(5)-H(5N).O(8) 0.866(17)	2.73(2)	3.158(3)	112.1(2)

Table 4 .

 4 EPR data for complexes 1 and 3 in CH 2 Cl 2 and CH 3 CN.

		Complex 1	Complex 3
	CH 2 Cl 2	g 1 = 2.06 (A 1 = 30 cm -1 )	g // = 2.26 (A // = 166 cm -1 )
		g2 = 2.08 (A 2 = 35 cm -1 )	g⊥ = 2.08
		g 3 = 2.24 (A 3 = 160 cm -1 )
	CH 3 CN	g // = 2.24 (A // = 166 cm -1 )	g // = 2.26 (A // = 166 cm -1 )
		g⊥ = 2.06	g⊥ = 2.08
	3.5 UV-Vis spectroscopic studies.

Acknowledgments

The authors acknowledge the Université de Bretagne Occidentale (UBO) for PhD grant (M. Ayad).

Dr. Francois Michaud is thanked for X-Ray diffraction analysis. The Agence Nationale de la Recherche (ANR-11-BS07-0024) is thanked for financial support.

Supplementary information

Experimental and simulated EPR spectra, UV-Vis spectra and crystallographic data for complexes 1 and 3 (CCDC 1921004-1921005). Crystallographic data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif,or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.


Laetitia Guerret-Legras

Jean-Frédéric Audibert

Indra M Gonzalez Ojeda

Galina V Dubacheva

Gilles Clavier

Fabien Miomandre

Time

Keywords: Time-resolved fluorescence, electrofluorochromism, SECM, tetrazine, quenching

come

Introduction

Fluorescence microscopy is a well-known imaging technique widely used to investigate chemical or biochemical events. Its advantages rely on its high sensitivity and its ability to localize events at very short scales using for example confocal [START_REF] Shuman | Confocal microscopy: an overview[END_REF] or TIRF 2 configuration. Its coupling with electrochemistry is now well documented in the literature [START_REF] Dias | Electrochemistry coupled to fluorescence spectroscopy: a new versatile approach[END_REF][START_REF] Loete | Application of total internal reflexion fluorescence microscopy for studying pH changes in an occluded electrochemical cell: Development of a waveguide sensor[END_REF][START_REF] Lei | Single molecule fluorescence spectroelectrochemistry of cresyl violet[END_REF][START_REF] Audebert | Electrofluorochromism: from molecular systems to set-up and display[END_REF][START_REF] Amatore | Coupling of electrochemistry and fluorescence microscopy at indium tin oxide microelectrodes for the analysis of single exocytotic events[END_REF] and even compact all-in-one devices now allow the simultaneous record of fluorescence and electrochemical signals through voltafluorograms or chronofluoamperograms [START_REF] Martin-Yerga | Time-Resolved Luminescence Spectroelectrochemistry at Screen-Printed Electrodes: Following the Redox-Dependent Fluorescence of Ru(bpy)3 2+[END_REF] . Electrochemistry can be coupled to fluorescence for various aims: as a trigger or as a complementary detection technique. The simplest configuration to do this is to use the substrate (microscope slide or coverslip) as the working electrode in a three electrode electrochemical cell [START_REF] Miomandre | Fluorescence microscopy coupled to electrochemistry: A powerful tool for the controlled electrochemical switch of fluorescent molecules[END_REF] . This requires the substrate to be both transparent and conductive enough with generally a trade-off between the optical and electrochemical performances. Another way of using an electrochemical input signal with fluorescence microscopy is through an ultramicroelectrode (UME) tip scanning the substrate surface, the typical configuration of Scanning ElectroChemical Microscopy (SECM) [START_REF] Bard | THEORY AND EXPERIMENT OF THE FEEDBACK MODE WITH[END_REF][START_REF] Liu | SCANNING ELECTROCHEMICAL AND TUNNELING ULTRAMICROELECTRODE MICROSCOPE FOR HIGH-RESOLUTION EXAMINATION OF ELECTRODE SURFACES IN SOLUTION[END_REF] . In that case, microscope glass coverslips can be used as substrates without any need of making them conductive because the electrochemical addressing can be made by the tip. This concept was already implemented by Bard et al. to combine SECM and Electrochemiluminescence (ECL) in a single experiment. [START_REF] Fan | Scanning Electrochemical Microscopy. 37. Light Emission by Electrogenerated Chemiluminescence at Secm Tips and Their Application to Scanning Optical Microscopy[END_REF][START_REF] Miao | Electrogenerated Chemiluminescence 69: The Tris(2,2 '-Bipyridine)Ruthenium(Ii), (Ru(Bpy)(3)(2+))/Tri-N-Propylamine (Tpra) System Revisited -a New Route Involving Tpra(Center Dot+) Cation Radicals[END_REF][START_REF] Choi | Electrogenerated Chemiluminescence (Ecl) 79. Reductive-Oxidation Ecl of Tris(2,2'-Bipyridine)Ruthenium(Ii) Using Hydrogen Peroxide as a Coreactant in Ph 7.5 Phosphate Buffer Solution[END_REF] Using fluorescence instead of ECL broadens the scope of the possible emitters to be used, with high luminescence yield and no need of coreactant.

A few examples combining SECM and fluorescence microscopy in a single experiment have been reported [START_REF] Boldt | Real-time pH microscopy down to the molecular level by combined scanning electrochemical microscopy/single-molecule fluorescence spectroscopy[END_REF][START_REF] Mukhopadhyay | SECM meets fluorescence microscopy[END_REF] and recently applied to the investigation of biochemical events like the detection of ROS in human cells [START_REF] Salamifar | Use of Combined Scanning Electrochemical and Fluorescence Microscopy for Detection of Reactive Oxygen Species in Prostate Cancer Cells[END_REF] or ion permeation through nuclear pore complex [START_REF] Kim | Ion Permeability of the Nuclear Pore Complex and Ion-Induced Macromolecular Permeation as Studied by Scanning Electrochemical and Fluorescence Microscopy[END_REF] but also to the analysis of nanometer-size defects in insulating layers [START_REF] Renault | Observation of Nanometer-Sized Electro-Active Defects in Insulating Layers by Fluorescence Microscopy and Electrochemistry[END_REF] . On our side we demonstrated the possibility to couple these two techniques using a single moiety as the redox fluorogenic probe either in organic [START_REF] Guerret-Legras | Combined scanning electrochemical and fluorescence microscopies using a tetrazine as a single redox and luminescent (electrofluorochromic) probe[END_REF] or in aqueous [START_REF] Guerret-Legras | SECMfluorescence microscopy using a water-soluble electrofluorochromic dye as the redox mediator[END_REF] medium. In these recent papers, we showed that various SECM modes (positive and negative feedback, generation-collection) could be coupled to epifluorescence microscopy in wide field to control the fluorescence state of the probe and obtain optical approach curves complementary to the electrochemical ones. The optical output signal was the luminescence intensity extracted from the CCD camera and the emission spectrum. Another valuable source of information can be obtained through luminescence lifetimes when pulse excitation is used. Fluorescence lifetime imaging microscopy (FLIM) is a widespread technique in the investigation of biological and biomedical issues [START_REF] Chen | Fluorescence Lifetime Imaging Microscopy for Quantitative Biological Imaging[END_REF][START_REF] Boreham | Time-Resolved Fluorescence Spectroscopy and Fluorescence Lifetime Imaging Microscopy for Characterization of Dendritic Polymer Nanoparticles and Applications in Nanomedicine[END_REF] . Performing FLIM under an electrochemical control is an innovative technique that might find valuable developments in all biological issues where redox states of cells need to be determined which is precisely done by fluorescence measurements [START_REF] Skala | In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia[END_REF][START_REF] Cao | Single-cell redox states analyzed by fluorescence lifetime metrics and tryptophan FRET interaction with NAD(P)[END_REF] .

We already reported before the possibility of using time-resolved fluorescence microscopy combined with electrochemistry [START_REF] Miomandre | Electrochemical Monitoring of the Fluorescence Emission of Tetrazine and Bodipy Dyes Using Total Internal Reflection Fluorescence Microscopy Coupled to Electrochemistry[END_REF][START_REF] Miomandre | Electrofluorochromism of a ruthenium complex investigated by time resolved TIRF microscopy coupled to an electrochemical cell[END_REF] and it was shown that the luminescence lifetime was electrochemically modulated as the emission intensity was, highlighting the role of the electrogenerated species in the quenching mechanism.

However, the electrochemical control of luminescence lifetime, although highlighted a long time ago in the case of ruthenium complexes [START_REF] Buttry | ELECTROCHEMICAL CONTROL OF THE LUMINESCENT LIFETIME OF RU(BPY)32+STAR INCORPORATED IN NAFION FILMS ON GRAPHITE-ELECTRODES[END_REF] has not been exploited so far as an analytical tool. We were interested in going further in this investigation and for that we implemented a pulsed laser source on the combined SECM-fluorescence microscope setup, allowing us to perform for the first time time-resolved fluorescence microscopy coupled to SECM. As the laser source we used delivers sub-nanosecond pulses, it is better suited to measure long luminescent lifetimes. Among electrofluorochromic probes available, tetrazines are organic molecules with outstanding long lifetimes in the range of 10 to more than 100 ns [START_REF] Clavier | s-Tetrazines as Building Blocks for New Functional Molecules and Molecular Materials[END_REF] . Therefore, we decided to focus our study on chloromethoxy-1,2,4,5-tetrazine (see scheme 1). This molecule is used as the redox fluorescent reporter and is particularly suited for this kind of measurement due its relatively high reduction potential (-0.99 V vs. Fc), electrochemical reversibility ensuring anion radical stability, high emission quantum yield (0.38) and very long lifetime (160 ns), [START_REF] Audebert | Synthesis and properties of new tetrazines substituted by heteroatoms: Towards the world's smallest organic fluorophores[END_REF] one of the highest among the tetrazine derivatives. The mechanism is investigated through the thorough analysis of lifetime evolution with the potential applied at the tip and when varying the tip-substrate distance. In the latter case, optical approach curves are obtained and analyzed through Comsol modelling. The results are analyzed successively for an insulating (glass) or a conductive (ITO) substrate.

Scheme 1 : Formula of chloromethoxy-1,2,4,5-tetrazine

Experimental

Chemicals. Chloromethoxytetrazine was synthesized according to a previous report [START_REF] Miomandre | Electrofluorochromism of a ruthenium complex investigated by time resolved TIRF microscopy coupled to an electrochemical cell[END_REF] and then dissolved in acetonitrile (Spectro. Grade, SDS) in presence of tetrabutylammonium hexafluorophosphate (puriss. from Fluka) 0.1 M. The approximate concentration of tetrazine is 1 mM. The solution is deaerated by argon bubbling.

DFT calculations. Three dimers were considered: two neutral tetrazines, one neutral and one reduced (radical anion) tetrazines and two reduced tetrazines (anion radicals). Their geometry was optimized at the APFD/6-31+g(d) level of theory with and without BSSE correction. The latest did not change the resulting geometry. A frequency calculation was done to confirm that a minimum was obtained. APFD was chosen because it includes empirical dispersion that is beneficial for modeling intermolecular interactions. Finally energy calculations were done at the APFD/6-311++g(d,p) and ωB97X-D/6-311++g(d,p) levels of theory with BSSE correction to estimate the complexation energy. The ωB97X-D hybrid long-range corrected functional was specifically designed to study non-bonded interactions. All calculations were done with Gaussian 16 (Revision B.01) 31 .

Electrochemical measurements. All electrochemical and SECM measurements were performed at room temperature using a homemade three-electrode cell on top of an inverted optical microscope (see below). A platinum wire and a Ag wire were used as counter and pseudo-reference electrode respectively. Glass (purchased from VWR, 1 mm thickness) and ITO (purchased from Solems, 25-35 ohm., 80 nm ITO thickness on glass) coverslips were employed as the substrates and working electrode in the case of ITO. A ultramicroelectrode (UME) made of a 25 µm diameter platinum disk (purchased from Ametek) was used as the tip and second working electrode and positioned above the substrate thanks to a mechanic arm connected to a piezo positioner (VersaSCAN motion controller) which enables to control the tip position under VersaSCAN Software. All electrochemical measurements are performed after 5 minutes of argon bubbling, so that dissolved oxygen has been removed.

Optical set-up. Optical measurements were performed on an inverted microscope (Ti Eclipse Nikon) with a 40X NA 0.75 objective in a wide field epi-illumination. Chloromethoxytetrazine was excited by a Hg lamp (C-HGFI -Intensilight, Nikon) with an excitation filter (BP482 nm) or a pulsed laser diode at 470 nm (LDH-P-C-470, Picoquant) with pulse width of 30 ps and a repetition rate of 2.5 MHz. Emitted light is collected through a long pass emission filter (LP 520 nm). Acquisition time-lapses are carried out using a CCD Pixel Fly camera from PCO at 14.2 images/s under the μManager open source software for steady state measurements. Fluorescence intensity over time is reported as the mean intensity inside a square region of interest (ROI) of the total image centered on the tip (wide field configuration). Photon counting over time is done using a graded index multimode fiber APD (MPD-CCTC, Picoquant) with a mode field diameter of 60 µm in sample plane for time resolved measurement and analyzed with homemade script and broad numerical methods under Igor Pro Wavemetrics Inc. software.

Fluorescence lifetime and intensity measurements. The general principle is based on Time-Correlated Single Photon Counting (TCSPC), a technique which was already performed under voltage bias [START_REF] Tsushima | Time-resolved measurement system of electrofluorescence spectra[END_REF] . In our investigation photons are recorded in TTTR (Time-Tagged Time resolved) mode [START_REF] Wahl | Time-correlated single photon counting[END_REF] . According to the experimental parameters and the number of the collected photons over a same period, the expected accuracy for mono exponential lifetimes ranging from 25 ns to 160 ns estimated by weighted leastsquares method is 0.95% and 0.45% respectively [START_REF] Köllner | How many photons are necessary for fluorescence-lifetime measurements?[END_REF] .

The global setup can be represented in scheme 2. The substrate and UME tip can be polarized separately with the bipotentiostat. The two microscope outputs are the CCD camera for image and intensity recording on one hand and APD for lifetime measurement on the other hand. The first part allows us to investigate the mechanism of quenching by analyzing the modulation of the emission lifetimes induced by the potential signal at the tip (and possibly at the substrate if conducting). This can be used in the second part to reconstruct the optical approach curve (luminescence intensity vs. tip-substrate distance)

and explain its better sensitivity compared to the electrochemical approach curve. Finally Comsol simulation is performed to try to fit the experimental data with the best accuracy.

Results and discussion

SECM-time resolved fluorescence experiments on insulating substrate

The first experiment conducted was done on insulating substrate (glass) at a fixed position of the UME tip above the substrate. The tip-substrate distance is chosen equal to 30 µm, a value close to the tip diameter so that the tip is sensitive to what happens on the substrate. First, a cyclic voltammogram is recorded on the Pt tip (see figure 1) to determine the potential range where the reduction of chloromethoxytetrazine takes place. The classical waveform signal expected for a microelectrode scanned at low scan rate is actually visible. The halfwave potential can be estimated to -0.55 V vs. the pseudoreference used.

The substrate being insulating, the electrochemical monitoring signal was applied at the tip and consists (after a 10 s period under open circuit) of a double potential step from a value where the EFC probe is in its emissive form (typically 0 V) to progressively more negative values starting from -0.4 V to gradually induce luminescence quenching. The potential is finally stepped back to its initial value to check the reversibility. The results can be seen in figure 1 where the electrochemical current (1A), luminescence intensity (1B) and lifetime (1C) are simultaneously recorded and displayed as functions of time. When more and more negative potential values are applied at the tip, the fluorescence intensity drops more and more dramatically as expected, while the current gets larger in intensity. Interestingly the luminescence lifetime follows the same trend as the luminescence intensity, getting also smaller as the intensity decreases. This is indicative of a quenching mechanism, becoming more pronounced as the potential is more negative. Therefore, it can be confirmed that the fluorescence intensity drop is not only due to the disappearance of fluorophores induced by the electrochemical reaction, but also to a chemical reaction between the remaining fluorophores and a quencher. We can suppose that in absence of dissolved oxygen the main quencher is the electrogenerated species (tetrazine anion radical) but this must be quantitatively analyzed, especially by correlating the intensity and lifetime ratios (in absence vs. in presence of quencher) to the injected charge during the electrochemical process. These three parameters can be experimentally extracted for each potential value (figure 2A) and finally the two ratios plotted against the coulombic charge (figure 2B).

These two ratios increase with the injected charge as expected, meaning that quenching becomes more efficient as the electrochemical reaction goes forward. At first sight, the lifetime ratio seems to vary linearly with the coulombic charge, which fits the Stern-Volmer quenching model.

Indeed, according to this model the following equation (1a) should be valid: The probed volume can be estimated according to the tip position z by:

𝑉 = 𝜋 4 𝑤 2 𝑧 (1b)
where w is the Gaussian beam diameter measured at 1/e 2 (typically 40 µm in our case). Equation (1a), which combines Stern-Volmer relation and Faraday's law applies if only dynamic quenching is involved, that is if the quenching process involves the mutual diffusion of the emitter and the quencher, without any additional process.

The coulombic charge can be estimated by integrating the transient current on the time interval T necessary to establish the steady state diffusion of the reactant. It can be expressed as a function of electrode potential using Cottrell equation [START_REF] Bard | Electrochemical Methods : Fundamentals and Applications[END_REF] :

𝐶(𝐸) = ∫ 𝑖𝑑𝑡 𝑇 0 = ∫ 4𝜋𝑟 0 2 𝐷𝐹[𝑐 𝑏𝑢𝑙𝑘 -𝑐(𝐸)] ( 1 √𝜋𝐷𝑡 + 1 𝑟 0 ) 𝑑𝑡 𝑇 0 = 2𝑟 0 𝐼 𝑠𝑡𝑎𝑡 (𝐸) √𝜋𝐷 √𝑇 (2)
with

𝐼 𝑠𝑡𝑎𝑡 (𝐸) = 4𝜋𝑟 0 𝐷𝐹[𝑐 𝑏𝑢𝑙𝑘 -𝑐(𝐸)]
where r0 is the electrode radius, D is the diffusion coefficient, 𝑐 𝑏𝑢𝑙𝑘 is the bulk concentration of reactant and 𝑐 (𝐸)

is the concentration of reactant at the electrode polarized at potential E. Istat is the stationary current at the tip considered as a UME.

From the combination of ( 1) and ( 2), we can draw the equation for the variation of the charge, emission intensity and lifetime ratios with the potential. Then both ratios can be plotted vs. the coulombic charge. The results are shown in figure 2 (full lines) where they are compared with experimental data.

As expected, the charge, emission intensity and fluorescence lifetime ratios all increase as the potential is more negative. Moreover, a linear relationship for the lifetime ratio vs. charge is actually obtained, in agreement with the Stern-Volmer model. The linear fit leads to an estimation of the quenching rate constant kQ of 5.8 10 9 Lmol - 1 s -1 , (see table 1), which is very close to the value predicted by Smoluchowski equation [START_REF] Smoluchowski | Versuch einer mathematischen Theorie der Koagulationskinetik kolloider Lösungen[END_REF] for diffusion-limited bimolecular reactions assuming D equal to 10 -5 cm 2 s -1 and a distance between reactants of 1 nm.

However, in Stern-Volmer model the steady-state intensity ratio is supposed to vary linearly with the charge as the lifetime ratio, which is not the case according to figure 2. Therefore, a more complicated quenching mechanism must be envisioned. We may consider adding a static quenching, i.e. the possibility that a quencher may quench all the fluorophores in a given volume around it without necessity of mutual diffusion.

A combined dynamic and static quenching model leads to the following equation ( 3):

𝐼 0 𝐼 = (1 + 𝐾 𝑆𝑉 [𝑄])(1 + 𝐾 𝑆 [𝑄]) = 𝜏 0 𝜏 (1 + 𝐾 𝑆 𝑉𝐹 𝐶) ( 3
)
where Ks is the equilibrium constant for the formation of the complex between the emitter (in the fundamental state) and the quencher.

In that model, the lifetime ratio still varies linearly with the quencher concentration as previously, because only the intensity ratio is affected. If that model is valid, a quadratic relationship should appear between the intensity ratio and the injected charge C:

𝐼 0 𝐼 = 1 + (𝐾 𝑆𝑉 + 𝐾 𝑠 )[𝑄] + 𝐾 𝑆𝑉 𝐾 𝑠 [𝑄] 2 = 1 + 𝐾 𝑆𝑉 +𝐾 𝑠 𝑉𝐹 𝐶 + 𝐾 𝑆𝑉 𝐾 𝑠 (𝑉𝐹) 2 𝐶 2 (4)
Therefore, we proposed a quadratic function to fit the experimental data (figure 2, bottom panel). A good fit is observed demonstrating that the model is relevant. Numerical values for KSV and Ks can be deduced from the combination of a linear fit for lifetime ratio (giving KSV through equation ( 1)) and a quadratic fit for intensity ratio (giving Ks knowing KSV using equation ( 4)).

The results are listed in table 1. The values have the order of magnitude expected for a very efficient quenching process [START_REF] Lunardi | Stern-Volmer quenching and binding constants of 10-alkyl-9(10H)-acridone probes in SDS and BSA[END_REF] .

Table 1: Quenching rate constant, Stern-Volmer and static quenching constants for the various substrates extracted from experimental data.

To go further, the authenticity of this additional static quenching assumption has been checked by computational chemistry. DFT calculations have been implemented to assess the interaction between a neutral s-tetrazine and its anion radical by evaluating the stability of a complex between these two moieties.

Three possible tetrazine-tetrazine complexes were studied: the two neutral, one neutral and one reduced (anion radical) and the two anion radicals. In the case of two anion radicals, no calculation method and relative orientations of the two aromatics did converge toward a stable geometry. It was thus concluded that the interaction between two tetrazine anion radicals is unfavorable. Conversely, stable complexes could be obtained for the two other cases. Table 2 shows the complexation energy values obtained by applying the basis set superposition error (BSSE) correction method. Whatever the calculation method, the value is negative, demonstrating that this interaction is favorable. Moreover, the values are more negative for the mixed Tz,Tz  complex than in the case of the supramolecular interaction between two neutral Tz.

Table 2 : Energy (kcal mol -1 ) calculated for the complexes formed by the interaction between two neutral tetrazines (Tz,Tz) or one neutral tetrazine and its anion radical (Tz,Tz  ). Molecular orbitals (MOs) and spin density for the supramolecular system (Tz,Tz  ) are represented in figure 3.

Method (Tz,Tz) (Tz,Tz

One can notice that MOs lost their molecular character with a contribution spread on the two units. In particular, the SOMO is highly delocalized and the spin density is especially high in the interspace. These two factors demonstrate that a close interaction is possible between Tz and Tz  , making the static quenching of Tz * by Tz  likely to occur.

Another experiment is performed by moving the tip polarized at a fixed potential (allowing the tetrazine reduction) in the vertical direction z. This consists in the typical approach curve which is systematically used to position the tip at the closest distance of the substrate surface before scanning it for analysis. The results obtained at various tip positions are shown in figure 4. The electrochemical current (top panel) varies consistently with a negative feedback mode, that is the current drops as the tip approaches the surface, as expected for an insulating substrate. As the tip gets closer to the surface, the emission intensity displays a gradual decrease in the 'on' state because the probed volume gets smaller as well as a modulation in the 'off' state, which has been analyzed in detail in a previous publication [START_REF] Guerret-Legras | Combined scanning electrochemical and fluorescence microscopies using a tetrazine as a single redox and luminescent (electrofluorochromic) probe[END_REF] . The normalized difference between these 'on' and 'off' intensities (IonIoff)/Ion, which can be considered as the normalized optical contrast, can be plotted as a function of the tipsubstrate distance and shows a gradual increase when the gap shortens. This constitutes the optical approach curve (green curve, top panel in figure 4) as already defined in a previous paper [START_REF] Guerret-Legras | Combined scanning electrochemical and fluorescence microscopies using a tetrazine as a single redox and luminescent (electrofluorochromic) probe[END_REF] . Finally, a similar behavior is also observed for the normalized difference of emission lifetimes (onoff)/on when this factor is plotted vs. the tip-substrate distance 1 (blue curve), which is a new result.

1 the point at closest distance should be considered with care as lifetime is extracted from a very low intensity.

This variation of the lifetime modulation amplitude with the tip position is an indication that quenching plays a role in the optical approach curve based on the luminescence intensity. This will be analyzed in more details below.

Interestingly the lifetime and intensity modulation amplitudes vary more significantly at large tip-substrate distances than the electrochemical current does. This is because the electrochemical current is sensitive to diffusion processes and the presence of a boundary (substrate) does not change the diffusion rate until very short distances are reached. The optical signal is not only influenced by diffusion but also by the quenching mechanism as suggested above and demonstrated further. Globally the variation of fluorescence intensity and lifetime vs. distance shows a variation spreading over 200 µm, while the electrochemical current starts to vary only below 100 µm.

SECM-time resolved fluorescence measurements on conductive substrates

Similar measurements have been performed on a conductive substrate (ITO). When ITO is left under open circuit conditions, a positive feedback is expected like already demonstrated in our previous work [START_REF] Guerret-Legras | Combined scanning electrochemical and fluorescence microscopies using a tetrazine as a single redox and luminescent (electrofluorochromic) probe[END_REF] . When polarized at a potential where no reoxidation of the anion radical is possible (0.23 V), this positive feedback is not possible anymore. Figure 5 shows the results obtained for these two situations. For both, when the tip potential is stepped to more and more negative values, the electrochemical current increases (in absolute value) while the fluorescence intensity and fluorescence lifetimes drop. When comparing more closely figures 5A and 5B, it can be seen that the lifetime modulation is deeper in the case of polarized ITO than for ITO left at open circuit. Indeed, in the first case the anion radical can not be reoxidized on the substrate, so that the amount of quencher is higher and therefore the lifetime is shorter, the other parameters (tip potential, tip-substrate distance) being kept constant.

As in the previous section for insulating substrate, the variation of fluorescence lifetime and fluorescence intensity ratios are plotted against the coulombic charge (see figure 6). Linear fitting works well for the lifetime ratio as it did previously for the insulating substrate, but leads to lower slopes, which corresponds to smaller values for the Stern-Volmer constant. This is consistent with the fact that the quenching is less efficient, because the anion radical quencher has been partly reoxidized on the substrate. Polarizing ITO to a value inhibiting the reoxidation of the anion radical leads to a situation close the one of the glass substrate, meaning that a negative feedback occurs on the substrate under this polarization value. All these results tend to demonstrate that the anion radical produced actually acts as a quencher of the luminescence emission and its amount can be tuned depending on the substrate nature and polarization.

Optical approach curves modelling

The tip-substrate space and the region of interest (ROI) where the fluorescence is collected are represented in We previously reported a modelling of the SECM -fluorescence coupling by finite element method enabling to calculate the concentration fields of neutral and electrogenerated radical anion of tetrazine, respectively [Tz](r,z)

and [Tz  ](r,z) for each tip-substrate distance [START_REF] Guerret-Legras | Combined scanning electrochemical and fluorescence microscopies using a tetrazine as a single redox and luminescent (electrofluorochromic) probe[END_REF] . This modelling considered the fluorescence intensity collected in a fictive ROI as directly proportional to the number of emitting species in this ROI. Nevertheless, such an assumption was unable to explain the better sensitivity of the optical approach curves compared to the electrochemical ones. So we decided to refine the model by taking into account the quenching mechanism mentioned above.

For this purpose, we use the local expression of the fluorescence intensity derived from Beer-Lambert law:

𝑑𝐼 𝑘𝑞 (𝑟 = 0, 𝑧) = 𝜖 𝜑 𝑘𝑞 (𝑟 = 0, 𝑧)𝐼 0 [𝑇𝑧](𝑟 = 0, 𝑧)ln(10)

Where 𝜖 is the extinction coefficient and 𝜑 𝑘𝑞 (𝑟 = 0, 𝑧) is a local quantum yield :

𝜑 𝑘𝑞 (𝑟 = 0, 𝑧) = 𝑘 𝑟 𝑘 𝑟 +𝑘 𝑛𝑟 +𝑘 𝑞 [𝑇𝑧 -](𝑟=0,𝑧) (6)
In equation ( 6) 𝑘 𝑟 and 𝑘 𝑛𝑟 are the radiative and non-radiative constants respectively. It states that the excite state may evolve by either radiative pathways (luminescence), or non-radiative pathways among which the quenching by electron transfer with the electrogenerated anion radical produced at the tip (r = 0).

For each tip-substrate distance d, the fluorescence intensity collected in the ROI is obtained by integration of the local one between z=0 and z=d. This leads to the following expressions for the intensities with and without electrochemical reaction at the tip, Imin and Imax respectively: [𝑇𝑧] 0 𝑑𝑧 = 𝜖 𝐼 0 ln (10) 𝑘 𝑟 𝑘 𝑟 +𝑘 𝑛𝑟

[𝑇𝑧] 0 𝑑 (8) Equation ( 7) clearly shows that 𝐼 𝑚𝑖𝑛 (𝑑) is not directly proportional to the number of emitters in the ROI when considering an inhomogeneous quantum yield.

The normalized fluorescence intensity modulation is then obtained for each tip-substrate distance by calculating the following expression:

𝐼. 𝑀. 𝑘𝑞 (𝑑) = 𝐼 𝑚𝑎𝑥 (𝑑)-𝐼 𝑚𝑖𝑛 (𝑑) 𝐼 𝑚𝑎𝑥 (𝑑) (9)
Comsol is used to evaluate the value of the integral in equation ( 7) thanks to experimental values of kr and knr, and then to calculate a theoretical optical approach curve using equation ( 9).

Such a curve can be simulated for various values of the kq parameter, from 0 in the absence of quenching to about 10 10 for a diffusion-limited quenching reaction.

The modelling results are reported in Figure 7A. It can be seen that the variation of the modulation spreads over a larger distance as the quenching rate constant is higher: as expected, the more efficient the quenching is, the better the sensitivity of the simulated approach curves. Nevertheless, the behavior of the modelling curves differs from the experimental ones for the long tip-substrate distances. This is probably due to optical effects in the experiment, as differences were observed according to the excitation source (laser or white lamp). Indeed, the tip looks more and more tiny in the ROI of constant size when the tip-substrate distance increases. Probably, modelling results could also be improved by considering a local quenching constant kq(r,z) depending on the location instead of a global one.

Beyond the fluorescence intensity, it is interesting to model the lifetime as well. For this purpose the Stern-Volmer relation was used in which emission lifetime is related to the quencher concentration [Tz  ]. A mean lifetime is thus calculated for each tip-substrate distance d after calculation of the mean anion radical concentration in the ROI :

[𝑇𝑧 -] 𝑅𝑂𝐼,𝑑 = 1 𝑑 ∫ [𝑇𝑧 - 𝑑 0 ](𝑟 = 0, 𝑧)𝑑𝑧 (10)
The lifetime based optical approach curve is finally obtained by calculating the normalized amplitude modulation:

𝜏 𝑀,𝑘𝑞 (𝑑) = 𝜏 0 (𝑑)-𝜏 𝑘𝑞 (𝑑) 𝜏 0 (𝑑) (11)
As for fluorescence intensity, kq is used as a parameter and an optical approach curve is calculated for each value (figure 7B).

As expected, a strong dependence of the kq value is observed on the curve shape. For short tip-substrate distances, the model matches pretty well with experimental data considering the highest quenching rate constant values, corresponding to a diffusion-limited reaction, up to 100 µm. At longer tip-substrate distances, a gap between the modelling and the experimental curves appears, because the model is probably limited by the fact that mean values are considered and the same optical effects as previously mentioned should also explain the differences observed.

Conclusion

SECM was successfully combined with time-resolved fluorescence microscopy in a single experiment for the first time, with chloromethoxytetrazine as the redox-active fluorescent probe. Compared to our previous works, we

show that luminescence lifetime, as luminescence intensity, is modulated according to the tip potential and tipsubstrate distance. This shows that the luminescence intensity drop is not only due to the disappearance of the emitter due to the electrochemical reaction, but also to a quenching reaction, involving the electrogenerated anion radical. The quenching mechanism is a mix of dynamic and static quenching since the variation of intensity ratio (in presence vs. in absence of quencher) vs. coulombic charge cannot be fitted by a linear variation. A similar behavior is observed whatever the substrate nature (insulating or conducting), but when a positive feedback occurs on the substrate, the lifetime drop is deeper. Polarizing the ITO substrate at value inhibits the positive feedback and this can be seen directly on the variation of both fluorescence intensity and fluorescence lifetime.

Finally, approach curves based on the variation of fluorescence lifetime with the tip-substrate distance can be constructed with a variation spreading over much larger distance values than the electrochemical current. This

  photons are recorded in TTTR (Time-Tagged Time resolved) mode 33 at 2.5MHz with a TCSPC 260Nano PCIe card from Picoquant with a global IRF resolution of 550 ps FWHM. The discrete intensity average lifetime for a sampling period 𝑗 over the total acquisition time is estimated from the number 𝑛 𝑖 of photons emitted after a 𝑑𝑡 𝑖 delay-time respect to the pulse excitation for each channel 𝑖, according to the following equation:

Scheme 2 .

 2 Scheme 2. Time-resolved fluorescence microscope (bottom, orange part) combined with Scanning Electrochemical microscope (top, blue part). DM = dichroic mirror. EM = emission filter. APD= Avalanche PhotoDiode. RE = Reference electrode. CE = counter electrode. WE = working electrode. UME = Ultramicroelectrode. TCSPC = Time Correlated Single Photon Counting.

  + 𝐾 𝑆𝑉 [𝑄] = 1 + 𝐾 𝑆𝑉 𝐶 𝐹𝑉 (1a) With: 𝐼 0 , 𝐼 : steady-state emission intensities resp. in absence and in presence of quencher 𝜏 0 , 𝜏: emission lifetimes resp. in absence and in presence of quencher 𝐾 𝑆𝑉 = 𝑘 𝑄 𝜏 0 : Stern-Volmer constant (𝑘 𝑄 : quenching rate constant). C = coulombic charge injected during the electrochemical reaction F = Faraday constant (F = 96500 C mol -1 ) V= volume of solution probed.

scheme 3 : 3 :

 33 scheme 3:

  𝐼 𝑚𝑖𝑛,𝑘𝑞 (𝑑) = 𝜖 𝐼 0 ln (10) ∫ 𝑘 𝑟 𝑘 𝑟 +𝑘 𝑛𝑟 +𝑘 𝑞 [𝑇𝑧 -](𝑟=0,𝑧)

Figure 1 :

 1 Figure 1: Current (A), fluorescence intensity (B) and fluorescence lifetime (C) vs. time at various potentials (same color codes for all curves) for a fixed position of the Scanning Electrochemical Microscope tip (30 µm above the substrate). The potential varies from 0 V (0-30 s) to the indicated value (30-60 s) and back to 0 V (60-120 s) and refer to the CV recorded at the microelectrode tip at 50 mV/s (D).

Figure 2 :

 2 Figure 2: Coulombic charge measured by integrating current from 30 to 60 s (red), fluorescence intensity I (blue) and fluorescence lifetime T (green) ratios vs. potential (top panel) on glass substrate. Ratios are measured between the oxidized state and the given potential. Same fluorescence intensity and lifetime ratios vs. charge (bottom panel). Full line shows the fitting of experimental data.

Figure 3 :

 3 Figure 3: Frontier molecular orbitals (top) and spin density (bottom) for the complex formed by neutral chloromethoxytetrazine and its anion radical (Tz,Tz  ) calculated with APFD theory.

Figure 4 :

 4 Figure 4: Current (top panel), fluorescence intensity (medium panel) and fluorescence lifetime (bottom panel) vs. time at various tip positions (same color codes for all curves) for a potential signal of: 0 V (0-30 s); -0.7V (30-60 s); 0 V (60-100 s).

Figure 5 :

 5 Figure 5: Electrochemical current (top panel), fluorescence intensity (medium panel) and fluorescence lifetime (bottom panel) vs. time at various potentials (same color codes for all curves) for a fixed position of the tip (30 µm) above the ITO substrate under open circuit (A) or polarized at -0.23 V (B).The potential varies from 0 V (0-30 s) to the indicated value x V (30-60 s) and back to 0 V (60-120 s).

Figure 6 :

 6 Figure 6: Luminescence intensity and luminescence lifetime ratios vs. charge on glass (pink), ITO at open circuit (green), ITO polarized at -0.23 V (blue). Full lines are the fitting curves, dots are the experimental points.

Figure 7 :

 7 Figure 7: Simulation of the modulation amplitude of luminescence intensity (A) and lifetime (B) vs. tip position for various values of the quenching rate constant (full lines). Experimental data are shown by dots.

Acknowledgments

CHARMMMAT Labex (ANR-11-LABX-0039) and Region Ile-de-France (C'Nano network) are acknowledged for financial support.

The University of Michigan, promotor of the program 'Optics in the city of lights' is acknowledged for funding the internship of I.M. Gonzalez Ojeda.

This work was granted access to the HPC resources of CINES under the allocation 2018-A0050810547 made by GENCI and HPC resources from the "Mésocentre" computing center of CentraleSupélec and École Normale Supérieure Paris-Saclay supported by CNRS and Région Île-de-France (http://mesocentre.centralesupelec.fr/) for DFT calculations.