Rayan Zaiter
email: rayan.zaiter@icmcb.cnrs.fr

Marc Dussauze

Marcelo Nalin

Evelyne Fargin

Frédéric Adamietz

Sylvain Danto

Olivier Toulemonde

Thierry Cardinal

Thermal and structural modification in transparent and magnetic gallogermanate glasses induced by Gd 2 O 3

Keywords: magneto-optical materials, gallogermanate glasses, vibrational Raman and infrared spectroscopies, magnetic susceptibility

A series of new transparent and magnetic barium gallogermanate glasses in the system (x) Gd 2 O 3 -

with x = 0, 8, 14, 18, 22 and 25 mol% were synthesized. Their thermal, structural and magnetic properties were characterized. Based on the differential scanning calorimetry results, one determined the composition domain exhibiting the highest thermal stability toward crystallization and provided a detailed experimental fabrication method for the production of optical fibers. The local glass structure investigated using combined Raman and Infrared vibrational spectroscopies shows a progressive depolymerization of the 3D glass germanate network accompanied with an increase of non-bridging oxygens when increasing the Gd 2 O 3 content. The incorporation of gadolinium ions tends to extend the IR transmission window. Thanks to the magnetic susceptibility measurements, the paramagnetic behavior was evidenced, and increases with the Gd 3+ content. The combination of the optical, thermal and magnetic properties of Gd 2 O 3 gallogermanate glasses as well as their ability to be shaped into optical fibers make them promising materials for their integration in MIR functional optical components.

Introduction

Magneto-optical materials that can be used as magneto-optical current transducers or Faraday isolators are of interest for the development of mid-infrared (MIR) lasers, lab-on-chip photonic devices as well as in biological imaging and drug targeting [START_REF] Fernandes | T f p g g g induced by Gd 2 O 3[END_REF]- [9]. While most of the research effort has been dedicated for the exploration of magneto-active materials for the visible and near-infrared (NIR) regions, the investigation on such materials transparent in the MIR domain are still very limited [4].

Although the magneto-optical phenomena seem to be more pronounced in crystals compared to glasses, the latter ones possess unique assets, among which their ability to be shaped into optical fibers. In the meantime, the synthesis of vitreous materials with high concentrations of rare earth oxides is challenging due to their tendency to devitrify during the cooling process [10], making it difficult to develop optical components. Besides, several glass systems possessing high concentrations of rare earth ions (even up to 37.5 mol% [11]) have been developed and reported in the literature such as germanates [12], [13], germanoborates [START_REF] Fernandes | T f p g g g induced by Gd 2 O 3[END_REF], [START_REF] Franco | g -optical borogermanate glasses and fibers containing Tb 3+[END_REF], [14], borates [15], aluminoborates [16],

borosilicates [11], [17] aluminosilicates [18] and fluorophosphates [19]. However, none of these common oxide glass systems exhibits a transmission window extending from the ultraviolet region (UV) up to the MIR as the one represented by gallogermanate glasses. Such glasses also possess superior chemical, mechanical, and thermal resistance [START_REF] Bayya | O-Ga 2 O 3 -GeO 2 glasses w p p[END_REF]. Among them, barium gallogermanate glass (so-called BGG), developed by the US Naval Research laboratory, were successfully employed as optical components either as low-loss optical fibers or bulk optical components [START_REF] Bayya | O-Ga 2 O 3 -GeO 2 glasses w p p[END_REF]- [23]. Glass formation, structure, and properties of BGG glasses have been discussed earlier in the literature [START_REF] Bayya | O-Ga 2 O 3 -GeO 2 glasses w p p[END_REF], [START_REF] Bayya | G g w w f g gy[END_REF], [START_REF] . K W | R p p f O-Ga 2 O 3 -GeO 2 g[END_REF], [25].

In the present work, we report on the thermal, structural and magnetic evolution of a novel series of transparent glass in the system Gd 2 O 3 -O-G 2 O 3 -GeO 2 . The characteristic temperatures and the glass thermal stability against crystallization were studied using Differential Scanning Calorimetry (DSC). The fiber drawing ability of these new glasses was also assessed. The glass structure was elucidated using the complementary vibrational spectroscopy techniques Raman and Infrared. Finally, the magnetic susceptibility measurements of the paramagnetic Gd 3+ ions were characterized as a function of temperature.

Material and methods

Synthesis of glasses and preforms

Glasses were prepared by the traditional melt-quenching technique. The following raw materials : barium carbonate BaCO 3 (Fox Chemicals, 99.99%), gallium oxide Ga 2 O 3 (Fox Chemicals, 99.999%), germanium oxide GeO 2 (Fox Chemicals, 99.999%) and gadolinium oxide Gd 2 O 3 (Alfa Aesar, 99.999%) were weighed in the required proportions to prepare a series of batches of 7 grams. Then the powders were mixed in a platinum crucible and heated up to 1550 °C for 1 to 2 h depending on the composition. The melted glasses were formed by quenching in ambient air. Glasses were then annealed at T g -40°C for 4 h and slowly cooled down to room temperature to reduce residual mechanical stress. All the resulting glasses were colorless, transparent and bubble free. Finally, glasses were cut and polished on both parallel faces for further characterizations.

Elaboration of the preforms for fiber drawing experiments was performed by melting and quenching 25 grams of glass into a stainless steel mold. Then preforms with dimensions of 1 cm in diameter and 7 cm in length were annealed (T g -40°C) and drawn following a protocol explained further below.

Physical and thermal characterizations

Characteristic temperatures which include the glass transition temperature (T g ), the onset of crystallization (T x ), and the maximum of the exothermic peak corresponding to the crystallization (T C ) were measured by diff erential scanning calorimetry using the Netzsch DSC Pegasus 404PC apparatus, on glass chunks in a Pt pan at a heating rate of 10 °C/min up to 1000 °C with a precision of ±2 °C. The thermal stability (ΔT= T x -T g ) was also calculated. The vitreous state of the samples studied was evaluated by XRD technique PAN y X'p PRO PD ff ractometer in gg-θ-θ g y q pp w y X' strip detector. Each measurement was made within an angular range of 2θ = 8 -80°. The Cu Kα radiation was generated at 45 kV and 40 mA (λ = 0.15418 nm).

The density, ρ, was obtained from the average of four measurements per sample using Archimedes' method by immerging a glass chunk in diethyl phthalate at room temperature on a Precisa XT 220A

weighing scale with an estimated error of 0.005 g/cm 3 .

The refractive index was determined by measuring the Brewster angle at 532 nm with an estimated error of ±0.005.

The transmission spectra in the UV-Visible-NIR and the IR ranges were respectively obtained from Agilent Cary 5000 (UV-Visible-NIR) and Bruker Equinox 55 (FTIR) spectrometers. The short wavelength cut-off (λ UV ) and the multiphonon cut-off (λ IR ) were determined from the recorded spectra for an absorption coefficient of 10 cm -1 .

Structural characterizations

Unpolarized Raman spectra were recorded with a confocal micro-Raman spectrometer LabRAM HR Evolution (Horiba Jobin Yvon) equipped with a Synapse CCD detector using a 532 nm radiation from a diode pumped solid state laser (output power = 20 mW). The incident laser beam was focused onto the sample through a microscope with a 10x objective (NA = 0.27, Olympus). Scattered light was dispersed by 1200 grooves/mm holographic grating system. Raman spectra have been corrected by the Bose-Einstein factor.

Reflectance infrared spectra have been recorded from 150 to 1500 cm -1 with a spectral resolution of 4 cm -1 using a Fourier Transform Spectrometer Vertex 70 V. A MIR source has been used (globar type).

All measurements were performed in vacuum. The spectra have also been recorded from an average of 200 scans. Kramers-Kronig transforms have been performed in order to extract the real (n 0 ) and imaginary (k) parts of the refractive index n according to the relation n = n 0 +ik. Then the absorption coefficient was calculated using the relation α = 4πνk, where ν is the frequency.

Magnetic susceptibility measurement

Magnetic susceptibility was collected using a Quantum Design MPMS-7XL magnetometer in the temperature range of 200 -400 K under 1 T in a zero-field cooled mode.

Results and discussion

Physico-chemical properties

The investigated glass materials were selected in the system (x)

Gd 2 O 3 -(100-x) [20 O-G 2 O 3 -65GeO 2 ]
, with x = 0, 8, 14, 18 and 22 and 25 mol%. Their acronyms, nominal compositions in molar and cationic percent are summarized in Table 1. The glass transition temperature (T g ), onset of the crystallization event (T x ) and the crystallization peak (T C ) for each composition were determined by DSC and are summarized in Table 2. The difference ΔT between the two characteristic temperatures T g and T x is often considered as a reliable criterion for evaluating the thermal stability vs crystallization of a glass. It makes it important in particular for its shaping by thermal process like glass fiber drawing (with targeted values of ΔT ≥ 100 °C). One should note that the 8Gd 2 O 3 glass sample shows the highest thermal stability against crystallization with a value around 138 °C, which progressively decreased when increasing concentration of Gd 2 O 3 , to attain 76 °C for the 25Gd 2 O 3 glass. The measured density of the glasses is given in Table 2. Likewise the T g evolution, density increases linearly with the addition of Gd 2 O 3 from 4.568 g.cm -3 up to 6.156 g.cm -3 between the 0Gd 2 O 3 and 25Gd 2 O 3 samples respectively. This result can be readily explained by a higher density of the Gd 2 O 3 component (7.407 g.cm -3 ) compared to the other constituents of the glass. One can also note that the refractive index increases with increasing the Gd 2 O 3 content, from 1.715±0.005 (0Gd 2 O 3 ) to 1.867±0.005 (25Gd 2 O 3 ). The absorption coefficient spectra of the gadolinium-containing gallogermanate glasses are displayed in Fig. 3. Spectra have been corrected for Fresnel reflections. A large transmission window is observed from the UV up to ~5500 nm for all the samples. The cut-off wavelengths have been determined for a given linear absorption coefficient of 10 cm -1 in the UV and the MIR.

Regarding the infrared multiphonon cut-off, the insertion of gadolinium in the glasses leads to an extending of the transparency window from 5810 nm (0Gd 2 O 3 ) to 6000 nm (25Gd 2 O 3 ). The infrared cut-off is dependent on the presence of an absorption band around 6.2 µm observed as a shoulder and the multi-phonon edge above 6.5 µm. The shift to the IR while increasing the Gd 2 O 3 content corresponds to a shift toward high wavelengths of both the multi-phonon edge and the 6.2 µm absorption feature. Regarding the short wavelength cut-off, as shown in Fig. 3 and from the values presented in

Fabrication of magneto-optical glass fiber

One now turn our effort to produce optical fibers by the standard preform-to-fiber method. Owing to its highest thermal stability parameter against devitrification, two macroscopic mono-index glass preforms were selected for drawing, the 8Gd 2 O 3 (ΔT = 138 °C) and the 14Gd 2 O 3 (ΔT = 120 °C).

Preform with the highest amount of gadolinium (14 mol% of Gd 2 O 3 ) is shown in Fig. 4a. The preforms were drawn using a dedicated optical fiber drawing towers equipped with an annular resistive furnace having a sharp temperature profile. The heating chamber was kept under continuous oxygen gas flow (3 L.min -1 ). Preforms were fed into the furnace and the temperature was gradually increased up to ~600 °C using a heating rate of 20 °C/min, then to ~920 °C for 8Gd 2 O 3 and ~950 °C for 14Gd 2 O 3 with a heating rate of 10 °C/min to soften the bottom-sections of the preforms. Following this procedure, ~20-meter long fibers with diameters ranging from ϕ = 200 µm down to 100 µm (Fig. 4b) were fabricated.

Shaping gallogermanates into fibers is a difficult task due to the formation of zeolite crystal phase at temperatures above the glass transition temperature [START_REF] Agg W | I f T p Germanate Glass[END_REF]. We have shown a proof of concept regarding this system because gadolinium addition allows to improve the thermal stability towards crystallization which is in agreement with findings of reference [28] stating that introduction of rare earth ions in germanogallate systems can contribute in mitigating the detrimental glass surface crystallization. In addition, it induces new magnetic properties as will be detailed below.

In order to qualitatively demonstrate the magnetic behavior of the 14Gd 2 O 3 glass fiber, one approach a neodymium magnet in its vicinity (Fig. 4c). It shows a small deviation of the fiber due to the magnetic attraction of the magnet. While magnetic property of the mono-index fiber is highlighted, the optical losses recorded at this stage (in the range 10-20 dB/m at 1 µm) remain too high for practical use. Such losses relate to the high viscosity of the melt, which leads to strong density fluctuation within the preform. Work is currently in progress to improve the preforms optical homogeneity, involving both the optimization of the glass composition and of the elaboration protocol. Unpolarized Raman and Infrared spectra for the investigated glass series are presented in Fig. 5. First, we focused on the vibrational spectra of the gallogermante matrix (0Gd 2 0 3 ) which can be decomposed into low (200 -400 cm -1 ), intermediate (400 -640 cm -1 ) and high frequency regions (640 -000 cm -1 ). In the high-frequency region, vibrations can be assigned to stretching features of tetrahedral units of germanium or gallium [29]. The low and intermediate frequency envelopes can be attributed mainly to highly coupled bending δ (T [4] -O -T [4] ) modes of an oxygen surrounded by two cornersharing tetrahedral units with T being either gallium or germanium; the brackets refer to the coordination number of the atoms [29]-[31]. One should remind that for the stretching (high frequency) spectral domain, the IR modes are always active. However, in Raman they are almost inactive for a GeO 2 glass and they are observable with a non-uniform charge distribution on the oxygen anions of the glass network by two reasons: (1) with mixed gallogermanate glass matrices, and the formation of Ga [4] -O --Ge [4] bridges [29] or (2) by the presence of non-bridging oxygens [START_REF] Kamitsos | R an and Infrared Structural Investigation of xRb 2 O•( -)G O 2 G[END_REF], [START_REF] S Y H. . W G | Q p k g g[END_REF]. In our case, for the gallogermanate matrix (0Gd 2 0 3 ), we have calculated Q = [2BaO + 3GdO 3/2 ] / [GaO 3/2 ] > 1 as can been seen in Table 1, thus we expect to see both latter modes.

Vibrational spectroscopy

To observe the evolutions with the Gd 2 O 3 content, we normalized the Raman spectra to the band centered at 515 cm -1 because it is linked to the 3D network of germanium and gallium [START_REF] . K W | R p p f O-Ga 2 O 3 -GeO 2 g[END_REF], and importantly it does not change too much in shape and position (~9 cm -1 ) with the increase of Gd 2 O 3 content. However, for the IR spectra, this band is located at 550 cm -1 and shifts down to 530 cm -1 for 25 mol% of Gd 2 O 3 , as well as it merges with the low frequency bands. That is why it was easier to normalize the IR spectra to the most intense broad symmetrical band located around 790 cm -1 .

In the low frequency domain, a broad band rises in the same range (360-390 cm -1 ) in both Raman and IR, and which increases with the Gd 2 O 3 concentration. It can be related to the vibration of the Gd -O -Gd bonds attributed in cubic Gd 2 O 3 as the F g mode [START_REF] Luyer | W g R spectroscopy of sol-gel Gd 2 O 3 f[END_REF], [START_REF]g q[END_REF]. The high-frequency region increases largely in intensity with the addition of gadolinium in Raman, and shifts from 793 cm -1 to 748 cm -1 with the disappearance of the right tail band in IR. This proves that the germanate network is affected either by the content of NBOs or the nature of charge compensation of these NBOs. This evolution can be compared to the red shift observed in the absorption spectra in Fig. 3. One can propose that the band observed around 6.2 µm corresponds to first overtone of the IR main mode around 800 cm -1 .

In order to enable a clear visualization of the spectral evolution of the gallogermanate network in the high-frequency region, deconvolution of the Raman spectra were conducted w 600 -000 cm -1 range as shown in Fig. 6a-f. We have used the model from reference [31] for deconvolution because it works well with five Gaussian peak functions: at 690 cm -1 (mode 1), 758 cm -1 (mode 2), 785 cm -1 (mode 3), 838 cm -1 (mode 4) and 890 cm -1 (mode 5). The variation of the peak position was maintained within ~5% while the width was kept fixed. The intermediate frequency region has been also fitted with a single Gaussian to enable a proper fitting of the high ones. The vibrational band assignments are presented in Table 3. According to the literature [START_REF] S Y H. . W G | Q p k g g[END_REF], modes 2 and 4 can be assigned to NBO on germanium tetrahedral moieties, respectively to Q 2 (Ge 2 O 2 ) 2-and Q 3 (Ge 3 O) -. Raman modes 3 and 5 can be attributed to localized asymmetric stretching vibrational modes involved in Ge [4] -O --Ga [4] bridges [29]. The band at 700 cm -1 was necessary for the deconvolution and its origin will be discussed later. The key points observed in the structural evolution of the different entities are that Q 2 (Ge 2 O 2 ) 2-and Q 3 (Ge 3 O) -increase largely and become the dominant ones with the addition of gadolinium, while the Ge -O --Ga species are quite stable as shown in Fig. 6g. This model works well if taking into account the shift of the mode # 4, which shifts from ~854 cm -1 to ~810 cm -1 . This ~44 cm -1 shift is in accordance with the shift observed in IR. We can explain it by the change of the charge compensation of the NBOs. The 700 cm -1 band increases in the same manner as the NBO units, so it is certainly related to structural entities which are negatively charged, but at this stage we are not sure to distinguish between its attribution to modes in gallate units [START_REF] I H. Y T. K W | w phonon energies and wideband optical windows of La 2 O 3 -Ga 2 O 3 glasses prepared using an y q[END_REF] or change in germanate or gallate coordination number [START_REF] Kamitsos | R an and Infrared Structural Investigation of xRb 2 O•( -)G O 2 G[END_REF], [START_REF] S Y H. . W G | Q p k g g[END_REF]. Complementary structural studies need to be performed by nuclear magnetic resonance (NMR) spectroscopy for instance, to have more detailed vibrational band assignments especially regarding the origin of the vibration around 700 cm -1 .

Magnetic properties

In order to study the magnetic behavior of the samples and to quantify the Gd 2 O 3 content with respect to the targeted concentration, a magnetic susceptibility study was carried out over the temperature range 220 -400 K. 4. Even if p g f θ would suggest the occurrence of ferromagnetic interactions between the rare earth ions, we believe that these high values are overestimated due to the temperature range that was considered during the data collection.

For x cationic mol% of GdO 3/2 , one expects a targeted Gd 3+ ion content of 2x cationic mol%. Fig. 7(b) shows that our analysis presents a good agreement between the targeted and the probed Gd 3+ ion concentrations. The deviation of the highest probed concentration (41.93 cationic mol% of GdO 3/2 ) from linearity might be probably due to GeO 2 evaporation from the melt because of the very high temperature of the synthesis (~1550 °C) which could lead to germanium oxide loss and then over estimation of the gadolinium content. The results ascertain the paramagnetic behavior of the Gd 3+ ions, which were expected to be welldispersed throughout the glass matrix. As can be seen in Figure 1, the glass is attracted by a magnet.

Such observation supports the fact that Gd 3+ ions can align themselves along the magnetic field. Once the magnetic field is not present anymore, no magnetic behavior is observed.

Based on their unique magnetic properties, but also on their extended optical transmission and the successful demonstration of their drawing into fibers, one considered the newly developed Gd 2 O 3containing glasses exposed here as materials of interest for further effective integration into MIR functional optical components.

Conclusions

The introduction of high loading of Gd 3+ ions in barium gallogermanate glass matrix provides a paramagnetic character of the materials. In the glass system Gd 2 O 3 -O-G 2 O 3 -GeO 2 in which up to 25 mol% of Gd 2 O 3 have been hosted, we have clearly confirmed that the Gd 3+ ions are the only paramagnetic species. Raman and infrared analysis showed that the Gd 2 O 3 incorporation in the gallogermanate glass matrix induced depolymerization of the tetrahedral germanium oxide network forming non-bridging oxygens on the germanate moieties. Besides exhibiting good optical properties such as a transparency window up to 5.5 µm, glasses up to 18 mol% of Gd 2 O 3 content showed a good thermal stability against crystallization with T x -T g above 100 °C. This merit combined with their magnetic behavior make such glasses promising materials for the manufacture of optical fibers which has been effectively demonstrated.

  Fig. 1a shows a photograph of the synthesized glasses in this work. The samples with 0 up to 25 mol% of Gd 2 O 3 are well transparent, colorless, homogeneous and chemically stable. Moreover, after synthesis, the glassy state was confirmed by the X-ray diffraction technique (results not shown). Fig.1bshows the attraction of the sample with 22 mol% of Gd 2 O 3 to a magnet. The same behavior was observed for all the other samples. However, based only on qualitative (visual) inspection at this stage, one should note that the magnetic force associated with the samples appears to be dependent on the concentration of paramagnetic ions as confirmed later below by the magnetic measurements.

Fig. 1

 1 Fig. 1. a) A photograph of glasses from 0 up to 25 mol% of Gd 2 O 3 after cutting and polishing; b) sample with 25 mol% of Gd 2 O 3 attracted by the magnet.

Fig. 2

 2 shows the evolution of T g and ΔT as a function of Gd 2 O 3 concentration. The introduction of Gd 2 O 3 is characterized by a linear increase of T g from 668 °C for 0Gd 2 O 3 to 815 °C for 25Gd 2 O 3 . Therefore, the presence of Gd 2 O 3 in the glass increases the average strength of the chemical bonds. Later in the paper, based on the Raman and IR spectroscopy techniques, the structural evolution is described to better understand the connectivity evolution of these systems versus the amount of Gd 2 O 3 introduced. For the 0Gd 2 O 3 sample, a crystallization peak appears around 794 °C. With the introduction of 8 mol% of Gd 2 O 3 , the peak shifts to 882 °C. For samples with concentrations greater than 8 mol% of Gd 2 O 3 , a secondary crystallization peak appears indicating the crystallization of different phases.

Fig. 2 .

 2 Fig. 2. The glass transition temperature (left) and the glass stability parameter against crystallization (right) as a function of the Gd 2 O 3 concentration.

Fig. 3 .

 3 Fig. 3. Absorption coefficient of the glasses in the Gd 2 O 3 -containing gallogermanate glasses.

Fig. 4 .

 4 Fig. 4. Preform-based optical fiber manufacturing for 14Gd 2 O 3 glass (a) Photograph of macroscopic 14Gd 2 O 3 glass preform prior to drawing (b) glass fiber on drum and (c) fiber attracted to a Nd magnet.

Fig. 5 .

 5 Fig. 5. Normalized (a) Raman and (b) Infrared spectra of samples with Gd 2 O 3 concentrations from 0 to 25 mol%.

2 (

 2 Fig. 6. Raman spectra deconvolutions with an error of 5%: (a) 0Gd 2 O 3 , (b) 8Gd 2 O 3 , (c) 14Gd 2 O 3 , (d) 18Gd 2 O 3 , (e) 22Gd 2 O 3 , (f) 25Gd 2 O 3 . (g) The integrated Gaussian area of the modes TO n , Q 2 (GeO 4 ) 2-, T -O -T, Q 3 (GeO 4 ) -and T -O -T as a function of the Gd 2 O 3 concentration for a normalization at the 515 cm -1 band.

Fig. 7

 7 (a) shows the temperature dependence of magnetic susceptibilities for all glasses. When the temperature tends to zero, a paramagnetic behavior with an asymptotic trend is expected. In the inset of Fig.7fitted according to the Curie-Weiss law, , which allows to estimate the Curie constant (C) from the slope and the paramagnetic Curie temperature from the intercept. The values together with the coefficient of determination (R 2 ) to describe the quality of the fit are presented in Table

Fig. 7 .

 7 Fig. 7. (a) Magnetic molar susceptibility (χ m ) vs temperature measurements for a series of glasses according to the Gd 2 O 3 concentration. The inset shows the 1/χ vs T plot demonstrating the validity of the Curie-Weiss law in the temperature range 220 -400 K. (b) Plot of the targeted Gd 3+ content versus the probed one (error bars are plotted ranging from ± 0.01 to ± 0.05, however they are masked by the size of the red dots).

Table 1 .

 1 Investigated glasses (acronyms, nominal compositions in molar and cationic percent) and their theoretical cationic ratios, i.e. (3GdO 3/2 + 2BaO)/GaO 3/2 .

										Cationic
		Nominal compositions (mol %)	Nominal compositions (cat mol %)	ratios
										(cat mol %)
	Sample name	Gd 2 O 3	BaO	Ga 2 O 3	GeO 2	GdO 3/2	BaO	GaO 3/2	GeO 2	Q = (3GdO3/2 + 2BaO) / GaO3/2
	0Gd 2 O 3	-	20	15	65	-	17.4	26.1	56.5	1.33
	8Gd 2 O 3	8.0	18.4	13.8	59.8	13.1	15.1	22.7	49.1	3.06
	14Gd 2 O 3	14.0	17.2	12.9	55.9	22.1	13.6	20.3	44.1	4.61
	18Gd 2 O 3	18.0	16.4	12.3	53.3	27.6	12.6	18.9	40.9	5.71
	22Gd 2 O 3	22.0	15.6	11.7	50.7	32.9	11.7	17.5	37.9	6.98
	25Gd 2 O 3	25.0	15	11.3	48.8	36.7	11.0	16.5	35.8	8.00

Table 2 .

 2 Thermal and physical properties of the investigated glass samples (T

	Sample name	T g (±3 °C)	T x (±3 °C)	T C1 (±3 °C)	T C2 (±3 °C)	ΔT (±4 °C)	ρ (g.cm -3 )	 IR (nm)	 UV (nm)	n 532nm (±0.005)
	0Gd 2 O 3	668	776	794	-	108	4.568	5810	332	1.715
	8Gd 2 O 3	719	857	882	-	138	5.169	5840	316	1.810
	14Gd 2 O 3	752	872	904	925	120	5.566	5860	314	1.847
	18Gd 2 O 3	775	892	923	938	117	5.793	5900	308	1.841
	22Gd 2 O 3	802	910	933	958	108	6.023	5950	284	1.858
	25Gd 2 O 3	815	891	910	943	76	6.156	6000	284	1.867

g : glass transition temperature, T x : onset of the crystallization temperature T C : crystallization temperature and ΔT: thermal stability criterion (T x -T g )) (ρ: density,  UV : short wavelength cut-off,  IR : multiphonon cut-off and n 532nm : refractive index at 532 nm).

Table 2 ,

 2 one observe a small decrease from 332 (0Gd 2 O 3 ) to 284 nm (25Gd 2 O 3 ) with increasing the gadolinium concentration. This is expected because Gd 2 O 3 has the largest band gap

compared to all the other oxides used. One can notice an absorption band around 3 µm and a weak one around 4.2 µm related to the presence of OH groups in the glasses [26]. These absorption features are related to the absence of dehydration steps during the glass synthesis, similarly to previous observations made in tellurites [27].

Table 3 .

 3 Raman band assignments of the high frequency region in the Gd 2 O 3 -O-G 2 O 3 -GeO 2 glasses.

	Band number	ν (cm -1 ) Band assignments

Table 4 .

 4 Parameters obtained from the Curie-Weiss fits of the magnetic susceptibility data for all the studied glasses, where C is the Curie constant, θ W p , χ diam is the diamagnetic susceptibility and R 2 is the coefficient of determination.

	mol% of Gd 2 O 3	C (emu.K/mol)	θ (K)	(*10 -5 emu/mol) [38]	R 2
	8%	1.011 ± 0.001	40.64 ± 0.29	-1.69	0.99961
	14%	1.841 ± 0.001	39.11 ± 0.22	-1.86	0.99981
	18%	2.275 ± 0.003	38.15 ± 0.39	-1.98	0.99938
	22%	2.728 ± 0.003	41.06 ± 0.31	-2.09	0.99884
	25%	3.302 ± 0.004	35.93 ± 0.36	-2.18	0.99944

Acknowledgements

This research was supported by Agence Nationale de la Recherche (ANR) (ANR Grant ANR-18-CE08-0004-02), the CPER Campus B, and the New Aquitaine region (Grant 2016-1R10107). M.

Nalin acknowledge the

São Paulo Research foundation FAPESP (grant numbers 2013/07793-6 and 2019/01223-0). This project has received f g f E p ' H z 2020 and innovation program under the Marie-Skł w k -Curie grant agreement N°823941 (FUNGLASS).


Léa Vilcocq

Ana Paez

Victoria D S Freitas

Laurent Veyre

Pascal Fongarland

Régis Philippe

Unexpected reactivity related to support effects during xylose hydrogenation over ruthenium catalysts

Xylose is a major component of hemicelluloses. In this paper, its hydrogenation to xylitol in aqueous medium was investigated with two Ru/TiO2 catalysts prepared with two commercial TiO2 supports. A strong impact of support on catalytic performances was evidenced. Ru/TiO2-R led to fast and selective conversion of xylose (100 % conversion in 2 h at 120°C with 99 % selectivity) whereas Ru/TiO2-A gave a slower and much less selective transformation (58 % conversion in 4 h at 120°C with 17 % selectivity) with the formation of several by-products. Detailed characterization of catalysts with ICP, XRD, FTIR, TEM, H2 chemisorption, N2 porosimetry, TPR and acid-base titration were performed to elucidate the role of each support.

TiO2-R has a small specific surface area with large ruthenium nanoparticles in weak interaction with TiO2 support and no acidity, whereas TiO2-A is a mesoporous material with a large specific surface area, mildly acidic, and bears small ruthenium particles in strong interaction with TiO2 support. The former was very active and selective for xylose hydrogenation to xylitol whereas the latter was less active and poorly selective. Moreover, careful analysis of reaction products also revealed that TiO2 anatase can catalyze undesired side-reactions such as xylose isomerisation to various pentoses, and therefore the corresponding unexpected polyols (arabitol, ribitol) were produced during xylose conversion by hydrogenation. In a first approach of the kinetics, a simplified kinetic model was built to compare quantitatively intrinsic reaction rates of both catalysts. The kinetic constant for hydrogenation was 20 times higher for Ru/TiO2-R at 120°C.

Introduction

Biomass is a unique source of renewable carbon on our planet. In the current context of fossil resources depletion, increase in the world demand for fuels and chemicals, and global climate change due to CO2 emissions from fossil sources, the development of biobased chemicals appears critical for a sustainable future. Today, biobased chemistry represents 177 M€/year and 7.9 Mt/year in Europe, i.e. 35 % of the chemical industry market and 10 % of all carbon-based materials, without including biofuels nor food manufacture. As 85 % of chemical compounds could be technically biosourced, European Union's target is 25 wt.% of biosourced carbon-based materials in 2030. [START_REF] Panchaksharam | Roadmap for the Chemical Industry in Europe towards a Bioeconomy[END_REF] Lignocellulose, as the main constituent of fibrous plants (e.g. herbs, straws, trees), is a major source of biomass. It is constituted by three components: cellulose, a crystalline glucose homopolysaccharide, lignin, a phenolic macropolymer, and hemicellulose, an amorphous heteropolysaccharide. The latter represents 20 to 40 wt.% of lignocellulose. [START_REF] Vassilev | [END_REF] Hemicellulose is a polymer of different sugars bearing six carbon atoms (hexoses, e.g. glucose, mannose, galactose) or five carbon atoms (pentoses, e.g. xylose, arabinose), arranged in linear and branched structures, in which acetyl groups can also be found. [START_REF] Ebringerová | Polysaccharides I[END_REF] The composition of hemicelluloses varies with the type of biomass. For example, xylans are predominant in hardwood hemicelluloses. [START_REF] Scheller | [END_REF] Xylose is the main ex-hemicellulose sugar from xylan. It is a starting material for various processes, and as such was identified as one of the Top 10 and then Top 12 platform biomolecules. [START_REF] Werpy | Top Value Added Chemicals from Biomass[END_REF][START_REF] Bozell | [END_REF] It is notably used to produce furfural, furan dicarboxylic acid (FDCA), γ-valerolactone (GVL), glycols, etc. However, the main industrial xylose-based process is the production of xylitol. This polyol is used in food industry and pharmaceutical industry as an additive and as a low calorie sweetener. Its market is evaluated ca. 300 kton/year. [START_REF]Research and Markets, Xylitol Market: Global Industry Trends, Share, Size, Growth, Opportunity and Forecast[END_REF] It is industrially produced by selective catalytic hydrogenation. Other polyols can be produced from pentoses, such as arabitol, from arabinose, lyxose or ribulose, and ribitol (also known as adonitol) from ribose or ribulose. Both arabitol and ribitol are used as pharmaceutical ingredients. [START_REF] Moreni | [END_REF] Historically, Raney nickel was used as heterogeneous catalyst for the hydrogenation of glucose and xylose. [START_REF] Wisniak | [END_REF][12][13] Nickel is advantageous because of its low price, but can leach easily in aqueous phase and leads to toxicity issues. Thus, some authors tried to reduce Ni leaching from nickel catalysts. [14][15][16][17] Ni catalyst supported on nitrogen-doped carbon was used for 40 h presenting high catalytic activity. Although, high temperatures (ca. 150°C) were necessary to obtain high xylitol yields. 16 Bimetallic catalysts were also used in literature. [17][18][19] The use of Sn associated to Pt was beneficial to the conversion and selectivity of the catalyst to xylitol synthesis. Thus, the increase of Sn amount in catalyst favoured by-products synthesis. 18 Aiming to reduce the catalyst deactivation, Ni was used associated with Re. Both monometallic and bimetallic catalysts presented Ni leaching in reaction media, but the presence of Re decreased this deactivation, helping to increase catalyst stability. 17 However, few catalysts presented high catalytic activity associated to high selectivity towards sugar alcohols. Ru/C is today the main catalyst for xylose hydrogenation at laboratory scale. 13 Indeed, ruthenium is an oxophilic metal particularly active for aqueous phase hydrogenation of carbonyl groups. 20 It presents a catalytic activity superior to other metals in the xylose hydrogenation reaction, in the following the order: Ru > Ni ≈ Co > Pt > Rh ≈ Pd. [START_REF] Wisniak | [END_REF]21 Its price is also interesting for industrial applications, as ruthenium is much less expensive than palladium or platinum. 22 The choice of supports is critical for such an aqueous phase reaction, as water can induce several deactivation phenomena such as leaching, phase changes through hydration, collapsing of porous structures, etc. Few candidates have been identified as stable supports in water: carbon materials, titanium dioxide (TiO2), zirconium dioxide (ZrO2). 23 Among them, TiO2 has the property to assist H2 activation via a spillover phenomenon 24 and is thus a promising support for ruthenium in aqueous phase hydrogenation reactions. TiO2 can exist in several crystalline phases but the most common for catalysis applications are anatase and rutile. Ru/TiO2 was already studied for xylose and glucose hydrogenation reactions 14,25,26 but the impact of support properties, such as crystalline phase, was reported in only one article so far. Hernandez-Mejia et al compared the activities of Ru/TiO2 catalysts with different TiO2 supports from 100 % anatase to 100 % rutile and observed a higher yield of xylitol when rutile was present. 27 In their experimental conditions, TiO2 rutile lead to smaller ruthenium nanoparticles during catalyst synthesis. Therefore, they attributed the higher activity in xylose hydrogenation to the higher number of surface ruthenium atoms, i.e. the catalytic sites, on the catalyst surface. However, they did not explain the decrease of selectivity observed with TiO2 anatase as a support nor identify reaction by-products. The decrease of activity for TiO2 anatase was interpreted only as a consequence of low ruthenium dispersion on the support. The impact of TiO2 support on ruthenium particle size was also described for other catalytic applications, but contradictory results were obtained. For example, Zhang et al observed smaller ruthenium nanoparticles on TiO2 anatase than on TiO2 rutile, 24 whereas other authors observed smaller ruthenium nanoparticles and a more homogeneous repartition of nanoparticles on TiO2 rutile. [27][28][29][30] In this paper, the impact of TiO2 support on the reactivity of ruthenium catalysts in xylose hydrogenation was investigated with a focus on the differences in reactivity observed when different supports are used. Two catalysts with two different commercial TiO2, anatase and rutile, were prepared, characterized and tested for xylose hydrogenation. Xylose conversion and the production of xylitol and other by-products were studied to understand the role of each support on the behaviour of Ru/TiO2 catalysts.

Experimental Materials

Ruthenium (III) chloride (RuCl3•xH2O) and xylose were purchased from Sigma-Aldrich and xylitol was purchased from Acros Organics with purity higher than 98%. Two TiO2 supports were used: TiO2 anatase with high specific surface area from Alfa Aesar (CAS 1317-70-0, reference 44429) and TiO2 rutile from Sigma Aldrich (CAS 1317-80-2, reference 224227). All the materials were used without further purification.

Catalyst preparation

The catalysts were prepared by incipient wetness impregnation. TiO2 was crushed in powder, sieved below 90 µm, and dried 2 h at 120°C. The precursor solution was prepared using a volume of ethanol necessary to wet the support and the appropriate amount of RuCl3•xH2O. The solution was added drop by drop to the dry support until the formation of a homogenous paste. Finally, this paste was dried overnight at 120°C and crushed afterward. The powder was calcined at 250°C under N2 flow and reduced at 350°C under H2 flow in a tubular oven. The corresponding catalysts were named Ru/TiO2-A and Ru/TiO2-R.

Catalyst characterization

X-ray diffraction (XRD) was performed at Centre de Diffraction Henri Longchambon (CDHL) on a diffractometer Bruker D8 Advance with 2θ until 90°. Infrared (IR) was performed at IRCELYON in absorbance mode using a Vector 22 apparatus on a Nicolet IS5 equipped with an ID7-ATR accessory with diamond crystal from Thermo Scientific with a spectral range of 4000 cm -1 to 525 cm -1 . N2 physisorption was performed on a Micrometrics ASAP 2010 apparatus at -196°C; samples were pre-treated at 350°C under vacuum for 4 hours before physisorption. The acid site concentration in water of each catalyst was measured using a potentiometric titration following the method described by Yu et al. 31 Briefly, 250 mg of catalyst was dried at 120°C and then was stirred at room temperature in 50 mL of a 0.1 M NaCl solution under sparging N2. The suspension was titrated by a NaOH 0.1 M solution using a Mettler Toledo G20S titrator equipped with a DGI 115 SC electrode. Three equivalence points were determined for TiO2-A and only one for TiO2-R. At each equivalence point, a pKa and an acid sites concentration were determined using the Gran plot method. 31- 33 ICP analysis was performed by two external laboratories (IRCELYON and SOCOR) after mineralization of catalysts in a closed vessel. Hydrogen chemisorption studies were carried out in a BELJAPAN BELSORP-max system. In each analysis, ca. 0.4 g of Ru/TiO2 catalyst was used. The sample was first reduced under H2 flow at 350°C for 3 h (ramp of 3°C.min -1 ). After reduction, the sample was evacuated at 350°C for 3 h. The chemisorption measurements were performed at 75°C. The number of available Ru atoms (RuA) was calculated from total adsorption of H2 with a stoichiometry H:Ru equal to 2:1. 34 µ .

-1 = 2 , µ 1 STEM-HAADF (Scanning Transmission Electronic Microscopy -High Angle Annular Dark Field) images were taken by a JEOL 2100F microscope, with a 200 kV acceleration tension. These characterizations were performed at "Centre Technologique des Microstructures" (CTµ, Villeurbanne, France). Samples were dispersed in ethanol using ultrasound prior the deposition of a few drops on holey carbon films on copper grids. Histograms and mean particle diameters were determined with 200 counts with ImageJ software. Temperature programmed reduction (TPR) was performed in a TPD/R/O 1110 Catalytic Surfaces Analyzer apparatus from Thermo Scientific equipped with a TCD detector and a cold trap to remove water before detection. The sample of Ru/TiO2 catalyst was pre-oxidized 30 min at 200°C under 5%O2/He flow, swiped with pure He at room temperature for 30 min and reduced under 5%H2/Ar from room temperature to 800°C.

Catalytic hydrogenation

The catalytic hydrogenation of xylose was performed in a 120 mL Top Industrie autoclave stirred tank reactor heated by an electrical jacket and equipped with a gas-inducing Rushton turbine and four baffles. The reactor is thermo-regulated and when necessary cooling is ensured by a Ranque-Hilsch vortex device. The reactor is operated in batch mode for liquid (and solid) phase and semi-batch mode for the gas phase. In order to work at constant pressure, H2 contained in a pressurised gas tank was continuously added in the reactor via a pressure regulator. 80 mL of 50 g.L -1 xylose solution (0.33 M) and 1.2 g of catalyst (molar ratio Ru/xylose of 0.45 mol.%) were introduced in the reactor, which was closed and purged with N2 before heating to the reaction temperature. Time zero was determined as the moment when the reaction mixture reached the programmed temperature and the reactor was pressurized with 40 bar H2. Samples were taken regularly through a sampling valve and filtered with 0.2 µm syringe filters. The absence of external mass transfer limitations at 1600 rpm stirring rate was verified experimentally by varying the stirring rate (ESI -Fig S1)

and the absence of internal mass transfer limitations was evaluated through estimations of the Weisz-Prater criterion (see ESI for more details).

Analytical methods

HPLC analysis was performed on a Shimadzu apparatus equipped with a RID detector and a Phenomenex Rezex RPM column at 80°C with pure filtered water as a mobile phase, 0.6 mL.min

% = ! " - ! # ! " 2 $ % % = & % ! # ! " 3 ( % = & % ! # ! " - ! # 4 * * % = ∑ , % ! # × . / 0 × 1 / 0 ! # × . / 234567 × 1 / 5
With initial concentration and concentration at time t (mol.L -1 ), . / 0 atoms is the number of carbon atoms, MC is the molar mass of carbon (12 g.mol -1 ). Product can be Xylitol or other byproducts (Lyxose, Xylulose, Ribulose, Ribitol or Arabitol).

Compound can be Xylose, Xylitol or other by-products (Lyxose, Xylulose, Ribulose, Ribitol or Arabitol). Calculations of kinetic constants, energy of activation (Ea) and Turn-Over Frequency (TOF) are detailed in ESI.

Results and discussion

Catalysts preparation and characterization

Ru/TiO2 catalysts were prepared by incipient wetness impregnation followed by calcination under N2 and reduction under H2 and characterised by several techniques (Table 1). Ru loading was verified after preparation by ICP analysis. For Ru/TiO2-A, a value of 0.9 wt.%Ru was found and for Ru/TiO2-R, a value of 0.6 wt.%Ru was found. XRD analysis shows that in Ru/TiO2-A, the support is a pure anatase phase with 11 nm mean crystallite size whereas in Ru/TiO2-R, the support is made of bigger mean crystallite size (228 nm) and contains some anatase impurities (6 %) (ESI -Fig S5). The diffraction peak intensities are much higher for TiO2-R than for TiO2-A, revealing higher degree of crystallinity, in accordance with the crystal sizes measured. Ruthenium is not visible on both diffractograms, probably because of its low loading on each support. corresponds to a type IV isotherm with H3 hysteresis loop typical of mesoporous materials with disordered pore network. 35 For Ru/TiO2-R, the isotherm shape corresponds to minor condensation of N2 in mesopores and major condensation in inter-particles void (mix of Type IV and Type II isotherms), thus indicating a mostly non porous material. The specific BET surface area of TiO2-A is high (more than 100 m².g - 1 ) compared to TiO2-R (less than 2 m².g -1 ). The mean pore diameter of TiO2-A is 11 nm (Table 1), largely superior to the hydrodynamic radius of sugars, estimated to be lower than 4 Å for xylose. 36,37 Hence, both supports exhibit very different bulk properties. FTIR was used to characterize surface groups of both catalysts (Figure 1). Three main bands can be observed on FTIR spectra of catalysts: broad stretching band corresponding to surface O-H bonds around 3300 cm -1 , a small band at 1625 cm -1 corresponding to adsorbed water, and an intense band between 400 and 500 cm -1 corresponding to Ti-O-Ti stretching bond. 38,39 Spectra corresponding to TiO2 and corresponding to Ru/TiO2 are identical. However, the comparison of TiO2-A and TiO2-R spectra shows some differences: the band corresponding to adsorbed water at 1625 cm -1 is only visible on TiO2-A, indicating that this support is more favourable to water adsorption, i.e. is more polar; the band corresponding to O-H group is also more intense on TiO2-A. The band corresponding to Ti-O-Ti is present on both spectra. The presence of -OH groups on anatase and rutile was described in the early 1970's by Primet et al. 40 It was demonstrated that these groups could have a weak basic or a medium acidic behaviour. Moreover, electronic vacancies on the surface of TiO2, and particularly on TiO2 anatase, creates strong Lewis acid sites, which can also form Brønsted acid sites in the presence of adsorbed water. Potentiometric titration of TiO2-A and TiO2-R supports was performed to evaluate their acidity. This method has the advantage to measure acid sites in the presence of liquid water, therefore in environmental conditions close to the reaction. TiO2-A exhibited 3 different types of acid sites with different strengths. The most numerous ones are the weaker ones, with a concentration of 249 µmol.g -1 and pKa 9.3. Stronger acid sites with a concentration of 152 µmol.g -1 and pKa 8.6 and then concentration of 152 µmol.g -1 and pKa 6.2 were also detected.

On the contrary, TiO2-R only bears acid sites with pKa 8.6 in a concentration of 27 µmol.g -1 .

These results are in agreement with the analysis of acid sites in gas phase described in literature: 41 anatase bears medium Brønsted acid sites and strong Lewis acid sites and rutile is much less acidic than anatase. In total, Ru/TiO2-A holds 466 µmol.g -1 and TiO2-R 27 µmol.g -1 acid sites. Both supports have very different specific surface area, as stated before. The density of acid sites on this surface is 3.8 µmol.m -2 for TiO2-A and 13 µmol.m -2 for TiO2-R, evidencing that the acidity of TiO2-A support is mainly a consequence of its large specific surface area. Given these differences in supports properties, one could expect different dispersions of ruthenium and different metalsupport electronic interactions on each TiO2 support. Concerning metal dispersion, STEM analysis revealed major differences between both catalysts (Figure 2, Table 1). Ru/TiO2-A exhibits small nanoparticles, visible as white nanospheres on the gray support, with a homogeneous dispersion in size and on the support. On the contrary, Ru/TiO2-R exhibits a larger range of particle sizes, from small nanoparticles to large aggregates. The dispersion on TiO2-R support is very heterogeneous with large zones of support where ruthenium is absent and small zones with a high density of ruthenium, as shown by EDX mapping on Fig S7 (ESI). Histogram of nanoparticles is centred on 2-2.5 nm for Ru/TiO2-A, with 2.4 nm mean diameter, and on 7-9 nm for Ru/TiO2-R, with 12.5 nm mean diameter. TPR analysis was performed on pre-oxidised samples of both Ru/TiO2 catalysts (Figure 3). Different reduction profiles were observed. For Ru/TiO2-R, a first peak was observed at 89°C, corresponding to RuO2 that have no interaction with the support 30 and a second smaller and broader peak around 150°C corresponds to RuOx in strong interaction with TiO2. 30 Therefore, for Ru/TiO2-R catalyst, RuO2 would correspond to large Ru particles and represent 68% of reductible species and RuOx corresponding to small particles in strong interaction with support corresponds to 32% of reductible species. For Ru/TiO2-A, two reduction peaks are visible at low temperature (57°C and 88°C) and a large, broader peak centred around 320°C. The latter represents 56% of reducible species on the sample. Peaks at low temperature should be similar to RuO2 species observed on Ru/TiO2-R whereas the peak at high temperature is an indication of RuOx nanoparticles as the dominant Ru species. The difference in temperature reduction for RuOx species between both catalysts (+170°C for Ru/TiO2-A) shows that metal-support interactions are much stronger on Ru/TiO2-A than on Ru/TiO2-R. In summary, TiO2-A and TiO2-R supports differs by their physical-chemical properties. TiO2-A is a mesoporous material with relatively high specific surface area, the main crystalline phase is anatase, with a low crystallinity. This support contains acidic sites, which could correspond to Lewis acid sites Ti 4+ on TiO2 surface. On the other hand, TiO2-R is a poorly porous material with low specific surface area; the main crystalline phase is rutile, with a high crystallinity and quite neutral surface properties. Ruthenium impregnation on both supports gave different catalysts. Ru/TiO2-A holds small, homogeneously dispersed Ru nanoparticles, most of them in strong interaction with TiO2 support, but the amount of surface ruthenium atoms able to activate H2 is low. Ru/TiO2-R bears also small nanoparticles in strong interaction with TiO2 support (although this interaction is weaker than for Ru/TiO2-A) and larger aggregates with weak metal-support interaction and with a heterogeneous dispersion; the amount of surface ruthenium atoms able to activate H2 corresponds to the dispersion of ruthenium observed by microscopy. Finally, the amount of available ruthenium atoms (RuA) is similar for both catalysts: 8.99 µmol.g -1 and 8.61 µmol.g -1 for Ru/TiO2-R and Ru/TiO2-A, respectively. Normalized with the specific surface area of each support, the surface density of ruthenium atoms is 4.2x10 17 Ru.m -² for Ru/TiO2-A and 18x10 19 Ru.m -² for Ru/TiO2-R, based on ruthenium content, which explains that ruthenium was much more easily dispersed in small nanoparticles on TiO2-A than on TiO2-R, resulting in smaller nanoparticles in the former case. On the contrary, the density of active ruthenium atoms (RuA, as measured by chemisorption) is 7.3 x10 8 Ru.m -² for RuA/TiO2-A and 4.3 x10 6 Ru.m -² for RuA/TiO2-R. It can be concluded that on the large surface area of TiO2-A, the availability of ruthenium is limited, because of strong metalsupport interactions.

Xylose hydrogenation over Ru/TiO2 catalysts

Xylose hydrogenation over Ru/TiO2-R at 120°C produced xylitol with 100 % selectivity (Figure 4a). The conversion was complete after 2 h. No xylitol degradation was observed after the end of the reaction. On the contrary, xylose hydrogenation over Ru/TiO2-A led to 61 % conversion after 4 h, indicating a reaction rate much lower than for Ru/TiO2-R (Figure 4b). Xylitol was produced with low selectivity around 52 % at 10 % conversion and decreasing to 17 % at 60 % of conversion. This continuous decay in selectivity with increasing conversion seems to indicate that the catalyst favours side-reactions producing unexpected by-products. Moreover, in a comparable blank experiment, the supports were tested without ruthenium. TiO2-R exhibited a negligible activity in xylose conversion whereas TiO2-A led to an important conversion of xylose (Figure 5) and to the formation of various by-products, some of them corresponding to the products obtained with Ru/TiO2-A. This point will be discussed in detail below. By comparison, blank experiments without any solid catalyst nor support gave only a negligible level of conversion at 120°C after 4 h (less than 10 %).

Effect of temperature on xylose hydrogenation

The effect of temperature on catalytic performances was investigated for both catalysts between 100°C and 140°C (Figure 6). For Ru/TiO2-R, an increase in temperature led to an increase in xylose conversion rate and xylitol selectivity remained unchanged and superior to 90 mol%, whatever the temperature. For Ru/TiO2-A, an increase in temperature led to an increase in xylose conversion but also to a sharp decrease in xylitol selectivity. This indicates the predominance of sidereactions at high temperatures with Ru/TiO2-A catalyst.

Formation of by-products during xylose hydrogenation over Ru/TiO2-A

Numerous by-products were observed on HPLC chromatograms when Ru/TiO2-A was used as a catalyst (see ESI, Figure S2). Xylulose, ribulose, arabitol and ribitol were identified xylose hydrogenation on Ru/TiO2-A and TiO2-A alone. Ribose and arabinose were not detected. The evolution of xylose conversion products with Ru/TiO2-A catalyst is represented on Figure 7a. Xylulose and lyxose are isomer and epimer of xylose, respectively. Ribulose can be formed by epimerisation of the C3 carbon on xylulose. When TiO2-A alone was used as a catalyst, xylulose was formed selectively at the very beginning of the reaction, and then converted into other products (Figure 7b). Lyxose was formed as a secondary product, most probably by isomerisation from xylulose, even if a small contribution of direct epimerisation from xylose cannot be discarded. Ribulose was observed in minor amount after 1 h of reaction, most probably from xylulose. All pentoses also seem to degrade into other products over the course of reaction. When Ru/TiO2-A was used as catalyst, xylulose is also present with a high selectivity at the beginning of the reaction, and lyxose as a secondary product when conversion increases. However, the presence of ruthenium leads to the hydrogenation of pentoses to form arabitol, from lyxose and xylulose, and ribitol, most probably from xylulose via a ribulose intermediate (Figure 8). Ribulose itself was not observed in this case. At high conversion, all selectivities decrease, indicating the formation of degradation products. The interconversion of aldoses can occur through different mechanisms. In the presence of a base or a Lewis acid catalyst, isomerization of aldoses into ketoses and reversely can occur through a Lobry de Bruyn-Alberda van Ekenstein mechanism (LdB-AvE) through an enediol intermediate and an intramolecular hydrogen shift. [45][46][47][48] This reaction is well known for producing fructose from glucose over Sn-BEA zeolite catalysts. 49 The isomerisation of xylose into xylulose and to a lesser extent, lyxose (formed by the reverse isomerization of xylulose) as well of arabinose into ribose was also reported on Sn-BEA zeolites or Nb2O5 catalysts. [49][50][51] The direct epimerisation of xylose into lyxose (or arabinose into ribose) can also occur through a Bilik reaction with a carbon shift and a rotation of C2-C3 bond. 47 It was described in the presence of a Lewis acid catalyst and a complexation agent (e.g. borate, calcium), [52][53][54] or using molybdenum-based catalysts. 55 Ti-doped zeolite was also active for isomerisation and epimerisation of glucose, the activity was attributed to Ti 4+ Lewis acid sites. 52,[START_REF] Moliner | Proc. Natl. Acad. Sci[END_REF] The temperature used for isomerisation of sugars ranges between 60 to 120°C, which is compatible with the experimental conditions applied in our catalytic experiments. The acidity of TiO2-A and TiO2-R was investigated above. The presence of acid sites on TiO2-A was observed and attributed to -OH groups and Ti 4+ species on TiO2 surface. These sites are assumed responsible for isomerisation and/or epimerisation activities. As these reactions are catalysed only by Lewis acid sites, it is assumed that a large part of the acid sites detected on TiO2-A are Lewis acid sites. On the contrary, TiO2-R does not bear enough acid sites and to be active for xylose conversion into other pentoses. Moreover, all pentoses can undergo hydrogenation to three corresponding polyols: arabitol, ribitol, xylitol, as described on Figure 8. Therefore, apparent Ru activity is not limited to xylitol production but also include ribitol and arabitol production; indeed, the overall reaction system is a complex network of parallel and consecutive reactions. Although this was not the point of this research, this result is also, to the best of our knowledge, the first report of ribitol production from ribulose over a Ru/TiO2 catalyst. Finally, it is worth noting that the carbon balance is decreasing during xylose reaction over Ru/TiO2-A (reaching 68 % after 240 min at 120°C) or TiO2-A (reaching 57 % after 240 min at 120°C), indicating the formation of unidentified products through successive reactions (see ESI, Figure S4). This phenomenon was already described in literature (cf. 47,50 ) and the degradation products were identified as glycoaldehyde, glyceraldehyde, dihydroxyacetone, and at higher temperatures lactic acid, HMF, furfural. Traces of lactic acid, acetic acid and furfural were detected in the products of xylose hydrogenation over Ru/TiO2-A in our study but in amounts too low to be quantified. The formation of humins, i.e. unidentified carbonaceous compounds, is assumed and corroborated by the change in colour of the solution from colourless to light-brown.

First approach of kinetics of sugars hydrogenation

The detailed analysis of reaction products led to a detailed reaction mechanism (Figure 8). In this mechanism, several sugars are hydrogenated in several polyols. A basic kinetic model was built to determine simply but quantitatively the hydrogenation activity of both ruthenium catalysts (Figure 9). The model is based on a simplified mechanism were the four identified pentoses are reasonably lumped together ("sugars") as well as the three polyols products ("polyols"). Therefore, two reactions were included in the model: the hydrogenation of sugars into polyols, with a kinetic constant kH, and the degradation of sugars into unknown products, kD. The following hypotheses were made: i) reaction orders were assumed to be 1 for sugars and 0 for H2 (i.e. H2 concentration was assumed constant because the system is constantly fed with gaseous H2 to maintain constant H2 pressure), ii) volumes of liquid and solid were assumed constant, iii) kinetic constants are apparent pseudo-first order rate constants including a factor corresponding to the catalyst concentration. Details on kinetic modelling are given in ESI. Figure 10 depicts the experimental results and the modelling results obtained at 120°C for both catalysts. A good agreement between experimental and modelling results was observed at all studied temperatures, as shown on parity plot (Figure S3 in ESI). At 120°C, kinetic constant for hydrogenation rate kH was 20 times higher for Ru/TiO2-R than for Ru/TiO2-A, resulting in a 20fold increase in initial reaction rate. This illustrates the superior catalytic activity of Ru/TiO2-R. Kinetic constant for degradation rate kD was negligible for Ru/TiO2-R, which corresponds to the high selectivity for xylitol observed earlier. On the other hand, degradation rate for Ru/TiO2-A was almost twice higher than hydrogenation rate, which explains the low selectivity for xylitol. As Ru/TiO2-R exhibited a higher accessibility of Ru atoms, its superior activity in xylose hydrogenation was expected. Turnover Frequency (TOF) values were calculated to normalize ruthenium catalytic activity by the number of surface ruthenium atoms able to activate H2 (cf ESI). TOF reaches 0.990 s -1 for Ru/TiO2-R and only 0.052 s -1 for Ru/TiO2-A at 120°C, which indicates a lower activity of ruthenium catalytic sites on Ru/TiO2-A (Table 3). Therefore, Ru/TiO2-A's low activity cannot be completely explained by the low amount of accessible ruthenium atoms but also by a lower intrinsic activity of catalytic sites for hydrogenation. As a comparison, Lee et al. measured a initial TOF of 0.688 s -1 at 100°C for xylose hydrogenation over 3%Ru/Al2O3 21 whereas our Ru/TiO2-R catalyst reached 0.469 s -1 at the same temperature and similar operating conditions.

With the same methodology, TOFdeg for degradation reaction were calculated from acid sites concentration (see Table 3) and the initial rate of degradation reaction at 120°C. The values obtained were 0.003 s -1 for Ru/TiO2-R and 0.002 s -1 for Ru/TiO2-A. Therefore, the degradation activity per acid sites is similar for both catalysts. The high selectivity for degradation products in the presence of TiO2-A is thus a consequence of its large specific surface area. Activation energies were calculated following the Arrhenius law (see ESI). For Ru/TiO2-R, activation energy determined from hydrogenation rate was 83.7 kJ.mol -1 . In literature, activation energies for xylose hydrogenation vary from 32 kJ.mol -1 (over Raney nickel) 11 to 53 kJ.mol -1 57 or 82 kJ.mol -1 58 (over ruthenium). Our results are in accordance with the values reported. For Ru/TiO2-A, activation energy determined from hydrogenation rate was 18.5 kJ.mol -1 , indicating a mechanism different for polyols production, in accordance with the reaction network shown on Figure 8, and activation energy determined from degradation rate was 106.2 kJ.mol -1 . These values of Ea are consistent with the low xylitol selectivity at 140°C observed for Ru/TiO2-A (Figure 6): at high temperature, degradation is favoured over hydrogenation, whereas at low temperature, xylitol production is favoured over sugars degradation. In summary, the support TiO2-A is responsible for two phenomena in xylose reactivity: i) A decrease in xylose conversion, i.e. a decrease in catalytic activity, which is linked with a decrease in ruthenium sites active for hydrogenation; ii)

A decrease in xylitol selectivity, which corresponds to the formation of by-products through undesired reactions and therefore to the presence of different catalytic sites on the support. Therefore, contrary to the study of Hernandez-Mejia et al. on Ru/TiO2 support effects during xylose hydrogenation, 27 we attribute the higher activity and selectivity of Ru/TiO2-R not to the presence of smaller Ru nanoparticles but rather to the higher ability of ruthenium active sites to activate H2 on Ru/TiO2-R.

Conclusions

The role of TiO2 support in the hydrogenation of xylose over Ru/TiO2 catalysts was investigated by preparing Ru catalysts with two different TiO2 materials: an anatase TiO2 support with a large specific surface area, large pore volume and medium surface acidity, and a rutile TiO2 support without porosity and negligible surface acidity. The synthesis of Ru/TiO2 catalysts from these two supports gave two different catalytic materials: Ru/TiO2-R bears large ruthenium nanoparticles in weak interaction with TiO2 support, whereas Ru/TiO2-A bears small ruthenium particles in strong interaction with TiO2 support. Whereas one would expect that small, well-dispersed nanoparticles would result in higher catalytic activity for hydrogenation reaction, the opposite happened. Ru/TiO2-A was less efficient to activate H2, as evidenced by chemisorption measurements. It was also less active for xylose hydrogenation, with initial hydrogenation TOF 20 times slower than for Ru/TiO2-R. This lack of activity was attributed to strong metal support interactions between small ruthenium nanoparticles and TiO2 anatase support. Moreover, the selectivity of the reaction is also impacted by the type of support. Ru/TiO2-R led to a xylitol selectivity close to 100 % at all studied temperatures. In presence of Ru/TiO2-A xylitol selectivity decreased with conversion. The low selectivity in presence of TiO2 anatase was explained by the formation of multiple by-products on TiO2 catalytic sites, including several pentoses isomers of xylose: xylulose, lyxose, ribulose, and the corresponding polyols: arabitol, ribitol. The presence of Lewis acid sites on TiO2 surface is assumed responsible for this particular reactivity. In conclusion, TiO2 support plays an important role in the reactivity of Ru/TiO2 catalysts. It has been demonstrated that TiO2 rutile, even with a small specific surface area, is the best option for xylitol production. Alternatively, Even if TiO2 anatase present textural properties can compatible with better active phase dispersion, it also produces various pentoses and polyols from a single sugar, xylose due to its acidic character. This work underlines the importance of choice of support in metalsupported catalysis and for its appropriate and fine characterization. Although the formation of xylose isomers and polyols is due to serendipity, it can represent a new approach in the synthesis of rare sugars and rare polyols from available resources. We present here a new reactivity of TiO2, which is a simple, robust and commercial material, as an alternative for the production of xylulose and lyxose from xylose. Moreover, the production of ribitol and arabitol from these pentoses was also evidenced in the presence of ruthenium.

- 1 .

 1 Additionally, a Phenomenex Rezex ROA column and a Phenomenex Rezex RCM column at 50°C with acidified water (0.005 N H2SO4) were used to confirm the identification of sugars and polyols by comparison with retention times of commercial standards. External calibration with four levels was used for quantification of sugars and polyols. Typical chromatograms are presented in ESI -Fig S2. The catalytic tests were duplicated and relative errors values were calculated from the HPLC analysis results and represented below as error bars. The following calculations were used in this work, based on concentrations in mol.L -1 :

Figure 1 .

 1 Figure 1. FTIR spectra of Ru/TiO2-A (a) and Ru/TiO2-R (b).

Figure 2 .

 2 Figure 2. TEM pictures of Ru/TiO2-R (up) and Ru/TiO2-A (down), with the corresponding histograms of Ru nanoparticles size.

Figure 3 .

 3 Figure 3. Reduction profiles of Ru/TiO2 catalysts.

Figure 4 .

 4 Figure 4. Xylose hydrogenation over Ru/TiO2-R (a) and Ru/TiO2-A (b). Reaction conditions: 120°C, 40 bar H2, 0.33 M xylose, molar ratio Ru/xylose 0.45%.

Figure 5 .

 5 Figure 5. Xylose hydrogenation over TiO2-R and TiO2-A. Reaction conditions: 120°C, 40 bar H2, 0.33 M xylose.

Figure 6 .

 6 Figure 6. Effect of temperature during xylose hydrogenation over Ru/TiO2-R on xylose conversion (a) and on xylitol selectivity (b) and over Ru/TiO2-A on xylose conversion (c) and on xylitol selectivity (d). Reaction conditions: 100-140°C, 40 bar H2, 0.33 M xylose, molar ratio Ru/xylose 0.45%.

Figure 7 .

 7 Figure 7. By-products molar selectivities during xylose hydrogenation over Ru/TiO2-A (a) and TiO2-A alone (b). Reaction conditions: 120°C, 40 bar H2, 0.33 M xylose, molar ratio Ru/xylose 0.45%.

Figure 8 .

 8 Figure 8. Reaction network during xylose hydrogenation.

Figure 9 .

 9 Figure 9. Simplified reaction network of xylose hydrogenation.

Figure 9 .

 9 Figure 9. Concentration profiles for xylose hydrogenation over Ru/TiO2 catalysts: Ru/TiO2-R (a) and Ru/TiO2-A (b). Dots correspond to experimental data and lines correspond to kinetic modelling data. Reaction conditions: 120°C, 40 bar H2, 0.33 M xylose, ratio Ru/xylose 0.45%.

  k, TOF and Ea are presented in ESI.

Table 1 .

 1 Characterization of Ru/TiO2 catalysts.

		Catalyst	Ru/TiO2-A	Ru/TiO2-R
	XRD	Crystalline phase	100% anatase	94% rutile -6% anatase
	ICP-OES	Ru loading (wt.%)	0.86 ± 0.03	0.61 ± 0.07
	Physisorption	BET specific surface area (m².g -1 )	122 ± 12	2 ± 0.2
		Pore volume (mL.g -1 )	0.45 ± 0.05	0.01 ± 0.001
		Mean pore size* (nm)	11 ± 1	n.d.
	FTIR	IR bands	1625 cm -1 (H2O-ads)	Weak 3300 cm -1 (ν(O-H))
			3300 cm -1 (ν(O-H))	400-500 cm -1 (ν(Ti-O-Ti))
			400-500 cm -1 (ν(Ti-O-Ti))
	TEM	Ru nanoparticles	Small nanoparticles, homogeneously	Heterogeneous dispersion, from 1 nm to
			dispersed.	100 nm, presence of aggregates.
			2.4 nm mean diameter	12.5 mean diameter
	H2 Chemisorption	Ru accessibility (number of available	8.61 µmol.g -1	8.99 µmol.g -1
		Ru atoms, RuA)

*Calculated using the BJH method N2 physisorption isotherm of Ru/TiO2-A (ESI -Fig S6)

  38,4 1

Table 2 .

 2 Acidity of TiO2-A and TiO2-R supports.

	Support	TiO2-A	TiO2-R
	Weak acid sites
	Concentration (µmoleqH+.g -1 )	249.67	-
	pKa	9.3	-
	Medium acid sites
	Concentration (µmoleqH+.g -1 )	152.59	27.18
	pKa	8.6	8.6
	Strong acid sites
	Concentration (µmoleqH+.g -1 )	63.35	-
	pKa	6.2	-
	Total acid sites
	Concentration (µmoleqH+.g -1 )	465.61	27.18
	(Fig 2)

Table 3 .

 3 Kinetic parameters -sugars hydrogenation.

Acknowledgements

Ruben Vera (CDHL) is acknowledged for his assistance on XRD analysis. Antoinette Boréave (IRCELYON) is thanked for her help on TPR analysis. Stéphanie Pallier (LGPC/CP2M) is acknowledged for her assistance on solid characterizations. Laurent Djakovitch, Noémie Perret and Antonio Hernandez Mañas (IRCELYON) are thanked for their help in FTIR analysis and HPLC analysis. Alain Favre-Réguillon is acknowledged for scientific discussion. This work was supported by National Research Agency (ANR) with the funding of project CHICHE (ANR-18-CE43-0004). The authors thank Axelera cluster for the chemical and environmental sectors.

Notes and references

Author Contributions

Conflicts of interest

There are no conflicts to declare.


Sylvestre

Maurice Mallet

Blaise

Fluorogenic and genetic targeting of a red-emitting molecular calcium indicator

Sylvestre P.J.T. Bachollet, a Nicolas Pietrancosta, a,b Jean-Maurice Mallet a and Blaise Dumat a *

We introduce a strategy for the fluorogenic and genetic targeting of a calcium indicator by combining a protein fluorogen with the BAPTA sensing group. The resulting dual-input probe acts like a fluorescent AND logic gate with a Ca 2+ -sensitive red emission that is activated only upon reaction with HaloTag with a 25-fold intensity enhancement and can be used for wash-free calcium imaging in Hela cells. The modular all-molecular design relying on a well-estalished protein self-labeling tag opens future possibilitie for tuning the photophysical properties or target different analytes.

To decipher biological processes of increasing complexity using fluorescence imaging, the development of "smart" probes able to report on biological analytes or processes with good selectivity and appropriate spatial and temporal resolutions is crucial. Ca 2+ imaging is used extensively by biologists as an indirect measure of the electrical activity of cells to follow neuronal communication. It is a complex endeavor since it aims at recording localized and transient concentration spikes and is a typical example where sophisticated probes are required. 1 Calcium probes essentially fall into two categories:

(i) Genetically-encoded calcium indicators (GECIs) based on fluorescent proteins 2 and (ii) molecular fluorescent calcium probes. [3][4][5] Currently, the field is dominated by GECIs, owing to the ability to selectively express them in a chosen cell type or sub-cellular compartment.

2

The genetic targeting of small chemical indicators using selflabeling protein tags (SLP tags) has emerged as a promising strategy to combine the selectivity of GECIs with the diversity of molecular probes. [6][7][8] The most commonly used SLP tags, SNAP-tag and HaloTag, have for instance been used to control the subcellular localization of Ca 2+ or Zn 2+ probes. 9-12 However, even with an efficient targeting reaction, one has to get rid of the unbound probes that may yield an unspecific signal. Metal cations sensors often involve cell-impermeant carboxylate chelating groups masked as ester functions and the excess of dye cannot always be easily washed away after esterase hydrolysis in cells. One way to circumvent this issue is to use fluorogenic targeting where the fluorescence is only activated upon binding to a target protein. 13,14 Although there is large body of work on the development of single-input fluorogenic reporters for high-contrast protein imaging (Figure 1A), 15-17 there are much fewer instances of fluorogenic activation of chemical sensors via protein binding. The fluorogenic protein targeting of a functional chemical probe indeed makes for a difficult molecular design, since it requires the combination of two fluorescence activating mechanisms (fluorogenic protein reaction AND analyte sensing ability, Figure 1B) that may work well independently but lose efficiency when nested within the same molecule. Lavis and coworkers coupled a HaloTagtargeted fluorogenic Si-rhodamine probe with the calcium chelator BAPTA. 18 Once coupled to BAPTA, it afforded a far-red targetable calcium indicator, but the probe lost most of its fluorogenic character with only a 2.4-fold increase in intensity upon reaction with HaloTag. To build-up hybrid fluorogenic and chemogenetic indicators, researchers have thus resorted to alternative strategies where the analyte detection is achieved via the protein moiety 19,20 or using a two-step labeling reaction involving a fluorogenic tetrazine ligation 21 but the combination of fluorogenic protein targeting and analyte sensing within the same molecular probe has only rarely been achieved with satisfying results. In this work, we have set out to combine a fluorogenic molecular rotor scaffold with the calcium sensing group BAPTA to build the hybrid chemogenetic and fluorogenic probe Ca-DIP (Calcium Dual-Input Probe) with a dual-input emission in the red range activated by reaction with the protein selflabeling tag HaloTag and by binding to Ca 2+ cations (Figure 1B).

Such a dually-conditioned probe can be described as a fluorescent AND logic gate where the target analytes are the inputs and the fluorescence signal is the output. 23 The structure of Ca-DIP is built on Red-Halo2, a fluorogenic HaloTag probe that we recently developed based on a dipolar molecular rotor structures with a viscosity-sensitive emission (Figure 1A). 16,24 Upon reaction with HaloTag, its broad orangered emission is enhanced 156-fold. The polarity and viscositysensitive fluorescence emission is an inherent feature of a wide variety of flexible dipolar structures, 25 which made us confident that we could make large structural modifications to transform Red-Halo2 into a chemical sensor without losing the protein fluorogenicity. To obtain a dual-input probe with calcium sensitivity (Ca-DIP), the structure of Red-Halo2 was redesigned to incorporate a BAPTA moiety (Figure 1B). BAPTA is the common calcium chelator developed by Tsien on which the vast majority of molecular calcium probes such as Fura-2 or Oregon green BAPTA is built. [3][4][5]26 When non-electronically coupled to a fluorophore, it quenches the fluorescence emission by photoinduced electron transfer (PeT). Calcium binding inhibits the PeT process and thus restores the fluorescence, creating an on/off switch actuated by Ca 2+ cations. Red-Halo2 was built on a julolidine electron-rich group, since the locked aniline derivative inhibits the formation of a twisted intramolecular charge transfer (TICT) which results in higher brightness. 16 Since 2+ and HaloTag (average of two measurements). Input states 0 and 1 correspond to 0 µM Ca 2+ and 39 µM Ca 2+ or to the free and HaloTag-bound probe respectively. An arbitrary intensity limit (dashed line) can be set to distinguish between 0 (low) and 1 (high) output.

the efficiency of PeT is directly related to the distance between the electron donor and acceptor groups, we seeked to position the BAPTA moiety in close proximity to the Red-Halo2 scaffold. Replacing the julolidine of Red-Halo2 by a tetrahydroquinoline had the benefit to maintain the inhibition of TICT while offering a reactive amine function to incorporate the BAPTA moiety (scheme S1). The optical properties of Ca-DIP were first characterized in vitro to study its fluorescent response to HaloTag and calcium.

Ca-DIP free in Ca

2+ -free buffer displays a broad absorption band centered on 508 nm with an absorptivity of 33 000 M -1

•cm -1 and a very weak fluorescence emission (Figure 2A and Table S1 for detailed photophysical properties). The addition of calcium to the free or HaloTag bound probe induces a small hypsochromic shift of the absorption wavelength confirming that the BAPTA is only weakly conjugated to the fluorophore, which is a condition for an efficient PeT process. The addition of calcium is expected to inhibit the PeT-quenching but, thanks to the dual activation, Ca 2+ alone induces only a minute increase of fluorescence intensity. On the other hand, Ca-DIP becomes fluorescent upon reaction with HaloTag with a 25fold increase in intensity and exhibits a Ca 2+ -sensitive emission (Figure 2). In agreement with the rational design, the fluorescence of Ca-DIP is thus simultaneously controlled by two external inputs and it operates a fluorescent AND logic gate able to sense calcium only when bound to the HaloTag protein (Figure 2C). The calcium sensing ability was further studied by performing a calcium titration of Ca-DIP bound to HaloTag. Ca-DIP displays a 6.4-fold increase of fluorescence intensity from 0 µM to 1 mM

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Ca

2+ and a dissociation constant K d of 113 nM (Figure 2B). This affinity ranks it among high affinity calcium indicators and is due to the substitution of the BAPTA with an electron-rich amine. Ca-DIP thus exhibits very promising Ca 2+ -sensing properties that are comparable to those of alternative fluorogenic and chemogenetic calcium indicators, but with an all-molecular approach (Table S2). However, its fluorescence brightness is markedly lower than that of the parent compound Red-Halo2 (Table S1). To try to understand this drop in intensity, we studied the binding of Ca-DIP to HaloTag by molecular dynamics. Ca-DIP complexed with Ca 2+ was covalently attached to the HaloTag crystal structure and we performed a 2 ns dynamic with a 10 ps step, generating 200 conformations. A cluster analysis underlined the presence of two representative structures accounting for 74 % and 26 % of the conformations (Figure 3). In the minor cluster, Ca-DIP does not interact with the protein and adopts a planar conformation suitable for fluorescence emission. In the major cluster, the methyl group of BAPTA is found to interact with a hydrophobic pocket formed by Proline 259, Threonine 252 and Alanine 260 on the surface of HaloTag. These hydrophobic interactions successfully minimize the potential energy of the complex but they twist the molecule in a non-planar conformation unfavorable for fluorescence that may explain the limited brightness of Ca-DIP compared to the parent compound Red-Halo2.

To test the probe in cellular imaging, we synthesized an acetoxymethyl (AM) ester analogue Ca-DIP-AM, following the classical strategy used for making calcium probes cell permeant (Scheme S1). We assessed the ability of the probes to record localized calcium concentration variations following Histamine stimulation in Hela cells transiently expressing HaloTag fusion proteins. In non-excitable cells, Histamine is known to induce an increase in cytoplasmic calcium concentration with periodic oscillations. 27 We first imaged cells expressing a soluble HaloTag protein. The dye was incubated for 60 min leaving the time for cell permeation and AM ester hydrolysis and timecourse imaging over 5 minutes was performed (Figure S1 and SI movie 1). The addition of Histamine induced an enhancement of the fluorescence intensity consistent with an increase in cytosolic calcium (Figure S1C). The monitoring of non-transfected cells (cell #4 and #5 on Figure S1B) showed no significant fluorescence signal and no variation over the course of the experiment (Figure S1D). To further evidence the ability of our probes to selectively image calcium in genetically-defined organelles, we performed a similar experiment in cells expressing a nuclear HaloTag-NLS protein (Figure 4 and SI movie 2). The average intensity projection image shows a clear selective staining of the nucleus even without a washing protocol (Figure 4A). The average intensity measured in the nucleus of 3 different cells shows a clear increase of the fluorescence signal (from 140 to 250 %) following histamine stimulation at 50 s with periodic oscillation visible for one cell with good temporal resolution (Figure 4B, C&E). To determine the maximum fluorescence enhancement in the cellular environment, ionomycin and calcium were then added at 290 s (Figure 4D&E). Ionomycin is a calcium transporter that will saturate the cell in calcium ions and allow Ca-DIP to reach its maximum fluorescence This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins enhancement values that vary between 210 and 400 %. Although we observe a clear selective staining of the nucleus (Figure 4A), it is inevitable to observe some nonspecific signal due to the off-target activation of the probe in hydrophobic regions of the cells. We have thus also monitored the signal intensity in cytoplasmic regions of cells #1 to #3 (Figure 4 and Figure S2). The low nonspecific signal does not show any sensitivity to calcium concentration changes showing that the binding to the target HaloTag protein is necessary to activate the calcium-sensitive emission of the probe. Despite having a moderate sensitivity to calcium in vitro (F/F 0 = 6.4), the cellular performance of the probe in cells is quite satisfactory, allowing targeted calcium imaging with a maximum signal increase up to 400 % that is comparable or superior to the performance of recently reported BAPTA-based or chemogenetic calcium indicators in similar experiments (Figure 4C, Table S2). 19,28,29 In conclusion, we have successfully used a fluorogenic molecular scaffold targeting HaloTag to develop one of the first fluorescent molecular logic gate for genetically targeted calcium imaging. With the help of molecular dynamics calculations, combined molecular and protein engineering can be envisioned to optimize the properties of the sensor/protein complex and in particular its brightness. The versatile design strategy described herein allows future tuning of the fluorescence properties 24 and application to other analytes and/or to different proteins by selecting the proper sensing moieties and ligands. It may thus open many new possibilities for localized functional imaging in genetically-defined cell types or subcellular organelles.

There are no conflicts to declare This work was supported by the Agence Nationale de la Recherche (ANR-18-CE44-0006).

Notes and references

Figure 1 .

 1 Figure 1. Design of the dual-input calcium probe Ca-DIP. (A) previously reported fluorogenic HaloTag probe Red-Halo2 and general principle of fluorogenic protein targeting. (B) Structure of Ca-DIP and general design principle of the fluorescent AND logic gate dually activated by HaloTag and Ca 2+ .

Figure 2 .

 2 Figure 2. in vitro characterization of Ca-DIP. (A) Absorption (dashed lines) and emission (full lines) spectra of Ca-DIP in the presence (39 µM) or absence (0 µM) of Ca 2+ in the free and HaloTag bound form ([Ca-DIP] = 1 µM, λexc = 500 nm). (B) Calcium titration of HaloTag bound Ca-DIP from 0 to 1000 µM Ca 2+ . Inset graph shows the signal expressed as ΔF/F0 where F0 is the initial fluorescence at 0 µM Ca 2+ (average of two measurements). To calculate the dissociation constant, the titration was fitted to a hill function (n = 1) shown in red. (C) Logic gate operation and truth table of Ca-DIP as a function of Ca 2+ and HaloTag. Integrated intensities F normalized to the intensity F0 of the free probe in the absence of Ca2+ and HaloTag (average of two measurements). Input states 0 and 1 correspond to 0 µM Ca 2+ and 39 µM Ca 2+ or to the free and HaloTag-bound probe respectively. An arbitrary intensity limit (dashed line) can be set to distinguish between 0 (low) and 1 (high) output.

Figure 3 .

 3 Figure 3. Molecular dynamics of Halo-Tag-bound Ca-DIP. Representative structures of the two clusters of conformations of Ca-DIP bound to Ca 2+ and HaloTag obtained by molecular dynamics.

Figure 4 .

 4 Figure 4. Calcium imaging of Hela cells transfected with HaloTag-NLS and incubated with 1 µM of Ca-DIP-AM. Time-lapse imaging at 1 frame/s. Histamine (50 µM) was added at 50 s and ionomycin/Ca 2+ (5 µM/10 mM) at 290 s. (A) Average intensity projection fluorescence image merged with the transmission image showing cell numbering. (B,C,D) Intensiometric images obtained by averaging 30 frames before addition of histamine (B), after addition of histamine (C) and after addition of ionomycin (D). (E) Evolution of the average intensity in the nucleus of the cells numbered on panel (A) over the course of the experiment, plotted as a percentage of variation relative to the initial intensity ΔF/F0.