Ali Saad

Khouloud Jlassi

Manef Abderrabba

Mohamed Chehimi

Dimethoxytriazine-Triazole Linked Mesoporous Silica Hybrid Sorbent for Cationic Dyes Adsorption

Keywords: mesoporous SBA-15, triazine/triazole, 1, 3-dipolar cycloaddition, adsorption, dye removal

Organic dye has always been considered one of the most serious water pollutants. Therefore, the great challenge nowadays is to synthesize effective adsorbents for removal of these contaminants. Herein, we prepared successfully a novel sorbent based on dimethoxytriazine/triazole groups functionalized via a 1,3-dipolar cycloaddition click reaction on mesoporous silica SBA-15. The resulting sample denoted DMT-Tr-SBA-15 was characterized by FTIR spectra, X-ray photoelectron spectroscopy (XPS), solid-state 13 C NMR spectroscopy and nitrogen adsorption/desorption isotherm. The resulting hybrid silica material was applied for the recovery of four cationic dyes in aqueous media. It showed high adsorption efficiency and a fast removal rate. Dye adsorption was monitored versus time; using a 40 mg/L of adsorbent in neutral aqueous solution. The adsorption equilibria were reached within 40 min. The methylene blue adsorption capacity of the functionalized mesoporous silica was found to be 1.848 mmol g -1 (591 mg.g -1 ) at room temperature and pH=7. These results indicate the great potential of clickable mesoporous silica to diversify the active sites for adsorption, thus demonstrating a significant interest for environmental applications.

Introduction

Organic dye pollutants have been extensively used for a long time in various industrial fields such as printing, textile, and cosmetics [START_REF] Han | Facile synthesis of mesoporous silica derived from iron ore tailings for efficient adsorption of methylene blue[END_REF]. Some of these dyes can cause various diseases and carcinogenic effects and negative impacts on the environment [START_REF] Boukoussa | Adsorption behavior of cationic dye on mesoporous silica SBA-15 carried by calcium alginate beads: Experimental and molecular dynamics study[END_REF]. To address this concern, many processes have been developed for dyes removal such as; photocatalysis [START_REF] Sunkara | SnO 2 /Fe 2 O 3 /Ag nanocomposite via hydrothermal approach: a novel highly Efficient photodegradation of eosin yellow and brilliant green dyes under Visible light irradiation[END_REF][START_REF] Ouanji | Ca-hydroxyzincate: synthesis and enhanced photocatalytic activity for the degradation of methylene blue under UV-light irradiation[END_REF], membrane filtration [START_REF] Hea | Treatment of methylene blue simulated wastewater by a cost-effective microscale biochar/polysulfone mixed matrix hollow fiber membrane: performance and mechanism studies[END_REF], ultrafiltration [START_REF] Parakala | Effective separation of methylene blue dye from aqueous solutions by integration of micellar enhanced ultrafiltration with vacuum membrane distillation[END_REF] and adsorption [START_REF] Afshari | Synthesis of new imine-linked covalent organic framework as high efficient absorbent and monitoring the removal of direct fast scarlet 4BS textile dye based on mobile phone colorimetric platform[END_REF][START_REF] Baig | DyxMnFe 2x O 4 nanoparticles decorated over mesoporous silica for environmental remediation applications[END_REF][START_REF] Teymoorian | S doped carbon quantum dots inside mesoporous silica for effective adsorption of methylene blue dye[END_REF]. Among them, adsorption has emerged as potential, low cost, simple and efficient alternative [START_REF] Beagan | Investigating Methylene Blue Removal from Aqueous Solution by Cysteine-Functionalized Mesoporous Silica[END_REF]. Therefore, the synthesis of new convenient adsorbent with high adsorption capacity is extremely significant.

Ordered mesoporous silica (OMS) have large surface area and accessible pore volume [START_REF] Saad | Polypyrrole/Ag/mesoporous silica nanocomposite particles: Design by photopolymerization in aqueous medium and antibacterial activity[END_REF][START_REF] Saad | Mesoporous silica/polyacrylamide composite: Preparation by UV-graft photopolymerization, characterization and use as Hg (II) adsorbent[END_REF] which make them suitable materials for the efficient adsorption of a wide range of pollutants [START_REF] Czepa | Graphene oxide-mesoporous SiO 2 hybrid composite for fast and efficient removal of organic cationic contaminants[END_REF]. However, the mesoporous silica with its surface containing inert silanol groups cannot be used directley without modification [START_REF] Ferré | Recyclable organocatalysts based on hybrid silicas[END_REF]. Therefore, organic ligands can be immobilized on mesoporous silica via functionalization of those silanol groups ≡Si-OH [START_REF] El-Nahhal | Silica, Mesoporous Silica and Its Thiol Functionalized Silica Coated MgO and Mg(OH)2 Materials[END_REF][START_REF] Kodeh | Exploring of Potential Antibacterial Activity of Hypochlorite and Chloroamine Adsorbed Ammonium Functionalized Mesoporous SBA-15 Silica[END_REF]. To date, several functional groups containing donor atoms (i.e., O/N/S/P) were successfully attached to OMS and used for organic pollutants adsorption and heavy metal extraction, namely: Bis (2 benzimidazolyl)pyridine (bbp) [START_REF] Badiei | Novel fluorescent chemosensor acssembled with 2,6-Bis(2-Benzimidazolyl)Pyridine functionalized nanoporous silica-type SBA-15 for recognition of Hg 2+ Ion in aqueous media[END_REF], thenoyltrifluoroacetone (TTA) [START_REF] Gök | Efficient adsorption of Th(IV) from aqueous solution by modified SBA-15 mesoporous silica[END_REF], benzene-1,3,5triamido-tetraphosphonic acid [START_REF] Ravi | Benzene triamido-tetraphosphonic acid immobilized on mesoporous silica for adsorption of Nd 3+ ions in aqueous solution[END_REF], carboxylic acid [START_REF] Tsai | Functionalization of cubic mesoporous silica SBA-16 with carboxylic acid via one-pot synthesis route for effective removal of cationic dyes[END_REF] and phosphoryl functionalized OMS [START_REF] Xue | Phosphoryl functionalized mesoporous silica for uranium adsorption[END_REF].

Among the wide variety of possible reactions for functionalization, copper (I) catalyzed azide-alkyne cycloaddition (CuAAC) click reaction is a common approach to introduce electron-rich heterocycles on mesoporous silica [START_REF] Arslan | Click chemistry in macromolecular design: complex architectures from functional polymers[END_REF][START_REF] Ziarani | Advances in click chemistry for silica-based material construction[END_REF][START_REF] Yadav | Click chemistry route to covalently link cellulose and clay[END_REF] which received remarkable interest in various fields such as removal of antibiotics [START_REF] Gao | Azide-functionalized hollow silica nanospheres for removal of antibiotics[END_REF], catalyst supports [START_REF] Nakazawa | Discrete complexes immobilized onto Click-SBA-15 silica: Controllable loadings and the impact of surface coverage on catalysis[END_REF][START_REF] Saad | Triazole/Triazine-functionalized mesoporous silica as a hybrid material support for palladium nanocatalyst[END_REF], immobilization of enzymes [START_REF] Malvia | Encapsulation of enzyme in large mesoporous material with small mesoporous window[END_REF] and biosensing applications [START_REF] Malvi | One pot glucose detection by [FeIII(biuretamide)] immobilized on mesoporous silica nanoparticles: an efficient HRP mimic[END_REF]. Recently, a huge number of significant researchers on triazole-modified mesoporous silica have been reported in literature as adsorbents (Table 1). Electron-rich heterocycles attached to silica surface allow the hybrid material to interact easily with organic dyes by variety of possible adsorption mechanisms such as π-π hydrophobic, electrostatic interaction, van der Waals interactions, hydrogen bonding or coordinative bond that makes them suitable for the adsorption and/or catalytic application [START_REF] Babaei | A sulfonated triazinebased covalent organic polymer supported on a mesoporous material: a new and robust material for the production of 5-hydroxymethylfurfural[END_REF][START_REF] Hosseini | Gold nanoparticles stabilized on SBA-15 functionalized NNN-pincer ligand; highly effective catalyst for reduction of nitroarenes in aqueous medium[END_REF]. For instance, in our previous work we constructed triazole/triazine-functionalized SBA-15 through CuAAC and subsequently we immobilized the palladium nanoparticles used efficiently for the catalytic reduction of 4-nitrophenol [START_REF] Saad | Triazole/Triazine-functionalized mesoporous silica as a hybrid material support for palladium nanocatalyst[END_REF]. In this context, Taheri and coworkers [START_REF] Taheri | Nanospace engineering and functionalization of MCM-48 mesoporous silica with dendrimer amines based on [1, 3, 5]-triazines for selective and pH-independent sorption of silver ions from aqueous solution and electroplating industry wastewater[END_REF] reported dendrimer amines based on 1,3,5-triazines modified MCM-48 as efficient silver ions removal from aqueous solution and electroplating industry wastewater.

Taking advantage of the outstanding properties of OMS and electron-rich heterocycles, herein, we selected N-rich linker dimethoxy 1,3,5-triazine complexes as ligands to synthesize a novel hybrid material denoted DMT-Tr-SBA-15. The schematic process to produce the ligand-modified mesoporous silica is displayed in anhydrous pentane, anhydrous toluene, copper sulfate (CuSO 4 ), sodium ascorbate, dimethylformamide (DMF), HCl, ethanol, and propan-2-ol. Water was deionized using the Milli-Q system of Millipore.

Synthesis of SBA-15 mesoporous silica

SBA-15 mesoporous silica was prepared along the method previously described by Zhao et al. [START_REF] Zhao | Triblock copolymer syntheses of mesoporous silica with periodic50-300 Angstrom Pores[END_REF]. Briefly, Pluronic P123 was mixed with HCl (3g /16.5 ml); the mixture was diluted using 112 ml of deionized water and stirred for three hours at 40 °C, then TEOS (7.427 g) was added to the mixture. After 2h (at 40 °C) a gel was formed, and the mixture was transferred for condensation at 90 °C for 24 h. Following drying and washing, the organic template was removed by calcination at (550 °C with a heating rate set at 2°C min -1 , under air and at set final temperature the solid was left 6h).

Preparation of dimethoxytriazine/triazole linked covalent mesoporous SBA-15

Synthesis of the azidopropyltriethoxysilane-functionalized SBA-15

The used 3-azidopropyltriethoxysilane coupling agent (AZPTES) was fabricated as described in [START_REF] Malvi | Clickable'' SBA-15 mesoporous materials: synthesis, characterization and their reaction with alkynes[END_REF]. The as prepared silane (AZPTES) was colorless liquid. 1 S1). Functionalization was performed by dispersing 1g of SBA-15 in 50 mL of toluene, then 1.96 g of AZPTES were added under nitrogen atmosphere, and subsequently the mixture was stirred for 24 h at 80 °C. After the completion of reaction, the mixture was cooled, filtered and washed with toluene and ethanol.

Synthesis of 2-Propargy-4, 6-dimethoxy-1, 3, 5-triazine (PDMT)

Firstly, (1.2 g, 6.9 mmol) of 2-Chloro-4,6-dimethoxy-1,3,5-triazine was added to a mixture of propargylamine (0.45 g, 8.2 mmol) and triethylamine (0.57 g, 6.9 mmol) in 120 mL acetone.

The solution was then heated, filtered, then dried under vacuum. The final isolated product is a white powder. (0.1M), and 0.77 g of sodium ascorbate solution (0.1M) was added to a solution of 2propargy-4,6-dimethoxy-1,3,5-triazine (0.47g, 1M) in 50% aqueous propan-2-ol (20 ml). The obtained mixture was maintained under stirring for 24 h at room temperature. After filtration, washing and vacuum drying, the final product was isolated and abbreviated DMT-Tr-SBA-15.

Adsorption experiments

30 mg of the adsorbent DMT-Tr-SBA-15 were dispersed in 500 mL of aqueous organic dye solution and the mixture was stirred at 300 rpm, at 25°C for a predefined period of time. After reaching equilibrium time, the mixture filtered using membrane filter to separate the solid and liquid phases. The dye concentration in the filtrate was determined by UV-Vis spectrophotometry at the maximum absorbance of each dye. The removal percentage of each dye was calculated as follows:

(

where C 0 and C eq are the initial and equilibrium concentration of organic dye (mg.L -1 ), respectively. The amount of each dye adsorbed onto DMT-Tr-SBA-15 (q t ) was calculated using:

(

) 2
where q t is the amount of dye adsorbed and C t is the dye concentration in the solution at time t and m amount of sorbent (g).

The adsorption capacity expressed in mmol/g was calculated as follows:

(3)

Characterization

The 1 H, and 13 C MAS NMR spectra were acquired, using Bruker AVANCE 300 wide bore spectrometer using deuterated chloroform as the solvent and internal standard. Fourier transform infrared (FTIR) spectra were recorded using an EQUINOX 55 (Bruker) spectrometer ranging from 4000 to 400 cm -1 . X-ray photoelectron spectroscopy (XPS) measurements were made using a K Alpha (Thermo) fitted with a monochromatic Al K X-ray source (spot size: 400 µm). The spectra were calibrated against the C1s component set at 284.9 eV. Thermogravimetric analyses were performed using a SETSYS evolution 16 apparatus from SETARAM by heating up the sample from room temperature up to 800°C under air flow (55 cm 3 (STP)/min) with a heating rate of 10 °C min -1 . Nitrogen adsorption and desorption studies were carried using an autosorb IQ Quantachrome. The specific surface (S BET ) values were obtained using the Brunauer-Emmett-Teller (BET) method, [START_REF] Brunauer | Surface area measurements of activated carbons, silica gel and other adsorbents[END_REF] while the mesopore size study of the materials was carried out using the Barrett-Joyner-Halenda (BJH) method [START_REF] Barrett | The determination of pore volume and area distributions in porous substances. I. computations from nitrogen isotherms[END_REF].

Results and discussion

3.1. Physicochemical properties of materials. Figure 2b shows the synthesis of (3-azidopropyl) triethoxysilane from 3chloropropyltriethoxysilane. One can clearly observe that after modification, a new peak appears at 2095 cm -1 ; it can be ascribed to the azido groups [START_REF] Gao | Azide-functionalized hollow silica nanospheres for removal of antibiotics[END_REF][START_REF] Sun | Triazole-forming waterborne polyurethane composites fabricated with silane coupling agent functionalized nano-silica[END_REF].

Figure 2c shows the spectra of SBA-15-based materials. All samples exhibited a band at 960 cm -1 is a characteristic peak of asymmetric stretching vibrations Si-O-Si [START_REF] Raji | Study of Hg (II) species removal from aqueous solution using hybrid ZnCl 2 -MCM-41 adsorbent[END_REF]. The associated Si-O-Si bands are observed at 1274, 1050, 910, 797, and 448 cm -1 [START_REF] Malvi | Clickable'' SBA-15 mesoporous materials: synthesis, characterization and their reaction with alkynes[END_REF]. After azidosilanization, an obvious band at 2110 cm -1 is observed and assigned to the characteristic stretching vibration of organic azides [START_REF] Gao | Azide-functionalized hollow silica nanospheres for removal of antibiotics[END_REF]. After the click reaction, the intensity of the adsorption peak corresponding to the azido group decreases significantly and disappears. New

13 C solid-state NMR analysis

To confirm FTIR studies above, we have employed the Solid-state 13

X-ray Photoelectron Spectroscopy Surface Analysis.

XPS was used to monitor the stepwise modification of surface chemical composition of the mesoporous silica adsorbent. Survey spectra of pristine and modified mesoporous silica are shown in Figure . 4. In addition to Si2p (∼105 eV), Si2s (∼155 eV) and O1s (∼532 eV), SBA-15 exhibits a very low intensity C1s peak due to adventitious hydrocarbon contamination.

After chemical reaction with AzPTES the survey spectrum shows the appearance of the N1s feature centered at ∼399 eV it is strong supporting evidence of the attachment of azidopropyl groups onto surface. This is accompanied by an attenuation of Si2p and Si2s peaks. After CuAAC reaction, C and N contents increase drastically for DMT-TrSBA-15 adsorbent, which accounts for the attachment of the triazine ring to the azido-functionalized silica via 1,3-Huisgen cycloaddition (Table S1, Supporting Information). This modification is noted for the surface, which is important for the interaction with dyes. However, it would also be interesting to determine the total nitrogen loading to silica by elemental analysis [START_REF] Khalifa | Application of mesoporous silica nanoparticles modified with dibenzoylmethane as a novel composite for efficient removal of Cd (II), Hg (II), and Cu (II) ions from aqueous media[END_REF] and to compare surface and bulk compositions of the DMT-TrSBA-15 adsorbent. The N1s of N 3 -SBA-15 (Figure 5b) was fitted in three components; assigned to quaternized -C-N=N + =N -(∼404.5 eV), -N=N + =N -/N-C (∼401 eV) and N-H/N-C (399-400 eV) [START_REF] Saad | Triazole/Triazine-functionalized mesoporous silica as a hybrid material support for palladium nanocatalyst[END_REF]. The positively charged nitrogen from the azido group coexists in -N=N + =N -↔ -N --N + ≡N mesomeric structures [START_REF] Saad | X-ray induced degradation of surface bound azido groups during XPS analysis[END_REF]. The main peak centered at ~401 is due to the two other nitrogen atoms which can be negatively charged (see mesomeric structures). After the click reaction, the azide is transformed into triazole and the N + peak component, disappeared indicating the successful reaction. The surface analysis results are in line with the FTIR findings which showed the disappearance of the peak related to the organic azides attached to the surface (Figure 2). The sp 3 nitrogen atom has two peaks located at 401.1 eV ascribed to NH + groups and/or triazole N + atoms respectively. The N=N atoms have a single component centered at 399.8 eV. The first peak at 398.9 eV is assigned to N=C bonds in triazole and triazine rings.

Thermal Analysis.

Further to confirm the successful modification, of OMS with the functional groups, thermal stability of all the synthesized materials was studied by thermogravimetric analysis (TGA) under N 2 atmosphere from room temperature to 800 °C under a rate of 10 °C min -1 . The plots of pristine are also reported which does not show any significant weight loss due to its higher thermal stability imparted by the strong Si-O-Si bonds. As shown in Figure 6 the first weight loss stage below 190°C correlated to adsorbed physical water, with a total weight loss of 2.06% and 6.95 % for N 3 -SBA-15 and DMT-Tr-SBA-15, respectively. Above 250 • C a massive thermal decomposition is observed for N 3 -SBA-15 and DMT-Tr-SBA-15 with a total weight loss of 13.5% and 24.6%, respectively. The second weight loss is due to decomposition of the residual surfactant for the pristine and decomposition of the functional groups decorated the OMS network. It is important to note that the weight loss difference between N 3 -SBA-15 and DMT-Tr-SBA-15 is about 11.1% which is an indication of substantial anchoring of triazole/triazine rings to the mesoporous silica surface. To further investigate the textural characteristics of the as-prepared samples N 2 adsorptiondesorption isotherms were measured and displayed Figure . 7. The isotherms indicate that our samples display a type-IV sorption isotherm with a sharp inflection step at P/P 0 ranging from 0.55 to 0.75 typical for OMS [START_REF] Tenorio | Deposition of Pd into mesoporous silica SBA-15 using supercritical carbon dioxide[END_REF]. The pore-size distribution, is shown as inset the Figure7.

The specific surface area, total pore volume, and pore size summarized in Table 2. The pristine OMS display a specific surface area (S BET ) around 733.8 m 2 g -1 , an average pore size (D p BJH ) about 6.81 nm and a pore volume (V p ) about1.15 cm 3 g -1 . After azido-silanization, S BET , the V p and D p BJH decreased to 375.1g -1 , 0.529 cm 3 g -1 and 5.83 nm, respectively. The decrease in S BET is exacerbated after cycloaddition reaction to 276.9 m 2 g -1 , the D p BJH was decreased to 5.06 nm, and the V p was decreased to 0.367 cm 3 g -1 . This noteworthy decrease in porosity, S BET , and V p is, may be caused by the partial blockage of the pores, after the grafting of organic groups on SBA-15 inner walls [START_REF] Sharma | Synthesis of a recyclable and efficient Pd (II) 4-(2-pyridyl)-1,2,3-triazole complex over a solid periodic mesoporous organosilica support by "click reactions" for the Stille coupling reaction[END_REF]. This in line with previous publications, for instance Badiei et al. [START_REF] Badiei | Novel fluorescent chemosensor acssembled with 2,6-Bis(2-Benzimidazolyl)Pyridine functionalized nanoporous silica-type SBA-15 for recognition of Hg 2+ Ion in aqueous media[END_REF] reported the decrease of an initial porosity of 7.1 to 5.7 nm after modification with chloropropyltriethoxysilane then to 4.4 nm after modification with 2,6bis(2-benzimidazolyl) pyridine in second step. a Mean pore diameter determined using the BJH method b Total pore volume determined at P/P 0 = 0.97

Adsorption of selected cationic dyes

Four cationic dyes; namely rhodamine B (Rhd B), rhodamine 6G (Rhd 6G), methylene blue (MB), and methyl orange (MO); were employed as adsorbates in order to investigate the adsorption capacity of the DMT-Tr-SBA-15 hybrid material and its potential selectivity towards one of these dyes. Their molecular structures and chemical properties are reported in Table 2. A series of batch adsorption experiment were carried out at room temperature, using the same amount of the adsorbent (30 mg) and in a neutral aqueous solution. For instance, the normalized concentration curve of methylene blue in the liquid phase and solid phase versus contact time with an initial concentration of 40 mg/L and in neutral solution are plotted in Figure .8a. It can be noticing that at the initial adsorption stage, the adsorbent surface contains a lot of available active sites to adsorbate concentration in the liquid phase which led to a significantly increase of the capacity of adsorption q eq . This result is accompanied by a considerable decrease of the intensity of the maximum UV absorption wavelength as displayed Figure 8b. Consequently, a significant evolution of the color of the aqueous solution for both of MB and Rhd B dyes (inset Figure 8a-and Figure S3 supporting information). In the second adsorption stage, it can be seen after 12 min that the evolution of the capacity of adsorption became slowly till reaching the equilibrium time. This could be due to: (i) a decrease of the dye concentration in liquid phase, (ii) a beginning of the saturation of the available adsorption sites in solid phase, (iii) a combination of these factors [START_REF] Rabti | Ferrocenefunctionalized carbon nanotubes: an adsorbent for Rhodamine B[END_REF]. On the other hand, the adsorption rate decreased gradually with increasing contact time and a maximum adsorption of MB was achieved 1.848 mmol g -1 at 24 h. Therefore, the minimum contact time of 40 min could be considered for an optimum adsorption of methylene blue. In order to study the selectivity of the DMT-Tr-SBA-15 toward the adsorption of these cationic dyes, Figure 9 display the removal efficiency histograms and adsorption capacity of all of selected dyes in the same conditions (30 mg mass of the adsorbent, 40 min as equilibrium time and initial dye concentration: 40 mg/L). The capacity of adsorption q eq of for MB, Rdh B, Rdh 6G and MO at the equilibrium time is 1.848, 1.036, 0.952, and 1.606 mmol.g -1 , respectively. Based on the q eq results, it is clear that the novel adsorbent presents a good removal efficient (74 % for Rhd B, 68% for Rhd 6G, 73% for MO, and 88%

for MB) and excellent adsorption capacity for all of selected dyes can be attributed to the surface-active sites for adsorption. The adsorption capacity of DMT-Tr-SBA-follows the sequence MB > MO> Rhd B> Rhd 6G. The higher removal efficient for MB adsorbates could be explained by the favored/strong interactions of its positive functional groups and the high density of negative active sites onto adsorbent surface. In the light of the achieved results, it is clear that the DMT-Tr-SBA-15 has excellent capacity and efficiency for cationic dyes adsorption compared to other mesoporous silica-based adsorbents reported in the literature (see Table 3), with a maximum adsorption capacity of 1.848 mmol.g -1 , corresponding to 591 mg.g -1 , at neutral pH and reached within 40 min. It would be interesting to conduct additional studies in order to determine adsorption isotherm model and apparent adsorption kinetic constant [START_REF] Beagan | Amine and Sulfonic Acid Functionalized Mesoporous Silica as an Effective Adsorbent for Removal of Methylene Blue from Contaminated Water[END_REF].

Conclusion

To sum up, a novel dimethoxytriazine/triazole linked SBA-15 has been synthetized by CuAAC click reaction. Azido-silanization was conducted in order to achieve silicaheterocycles interface with excellent adhesion. An arsenal of techniques (FTIR spectroscopy, XPS, TGA, 13 C solid-state NMR N 2 and sorption measurement isotherms) was employed for structural characterization of the hybrid material. Given the strong electrostatic attraction between its negative surface and cationic charges of adsorbates, this novel hybrid was evaluated to remove four selected cationic dyes from aqueous media. The new hybrid material showed high adsorption efficiency, over 88% for MB and a fast removal rate. The adsorption was monitored versus time; the equilibrium was reached within 40 min for a concentration of 40 mg/L. The maximum adsorption capacity for methylene blue at room temperature and pH 7 was 1.848 mmol.g -1 (591 mg.g -1 ) So far, adsorption of four cationic dyes, with distinct chemical structures, was investigated, one could thus anticipate the DMT-Tr-SBA-15 adsorbent to be efficient for numerous organic dyes.

Brὃnsted basic functions Electronic properites

Lewis basic functions H-bond acceptors

Figure 1 .

 1 The first step consists in the modification of the SBA-15 by an azido-propylsilane. Then in second step the propargyl-4,6dimethoxy-1,3,5-triazine N-rich ligand was synthesized from 2-Chloro-4,6-dimethoxy-1,3,5triazine by reaction witch propargylamine. As a third step, we used the conventional click coupling reaction between azido groups attached to SBA-15 (N 3 -SBA-15) and the alkyne group from modified triazine ligand in the presence of copper species as catalyst. Finally, the hybrid material DMT-Tr-SBA-15 was evaluated as adsorbent of four cationic dyes from an aqueous media. The hybrid material showed high adsorption efficiency and rate.The novelty of our approach lies in the unique use of click chemistry reaction to modify a high surface area pristine material (SBA-15) by different active sites (triazine and triazole moieties) for high adsorption capacity of cationic dyes adsorbates.

Figure . 1 .

 1 Figure .1. Molecular view of dimethoxytriazine/triazole linked covalent mesoporous silica preparation.

  1 H NMR (CDCl 3 , 400 MHz): δ (ppm) = 5.7 (H, N-H); δ (ppm) = 4.15 (dd, 2H, N-CH 2 -C); δ (ppm) = 3.9 (d, 6H, O-CH 3 ); 2.2(t, H, alkyne C-H) (see FigureS2).

2. 3 . 3

 33 Preparation of DMT-Tr-SBA-15 4, 6-dimethoxy 1,2,3-Triazole -1,3,5-Triazine (DMT-Tr) ligand decorated SBA-15 was obtained by 1,3-dipolar cycloaddition reactions. First, 1.22 g of N 3 -SBA-15, 0.43g of CuSO4

3. 1 . 1 .

 11 Fourier transform infrared spectroscopic analysis.The dimethoxy-triazine ligand FT-IR changes upon modification are presented in Figure2a.The spectrum of pure 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) exhibits the characteristic absorption bands in the 478-1662 cm -1 range i.e. 1562, 1460, 1300, 818 and 558 cm -1 that can be assigned to aromatic stretching modes in triazine ring, C=N, C-O stretching, C-N stretching, and C-H outer-bending vibrations, respectively. After reaction with propargylamine, the characteristic bands of the triazine ring appear. The new peaks centred at at 2109, 2923, 2961, 3128 and 3244 cm -1 , can be assigned to the triple bond CC,  sy CH in (-CH 2group),  as CH (in -CH 2group), N-H secondary amine and the C-H stretch on the terminal alkyne, respectively.

  bands are observed in the spectrum of DMT-Tr-SBA-15 between 1589 and 1376 cm -1 which can be assigned to ring stretching vibration of conjugated C=C, C=N and C-N in triazine and triazole rings, giving evidence of the successful functionalization of the OMS pristine.

Figure. 2 .

 2 Figure.2. FT-IR spectra of: (a) dimethoxy-triazine ligand before and after modification with propargylamine, (b) (3-azidopropyl)triethoxysilane (AZPTES) and 3chloropropyltriethoxysilane (CPTES), and (c) SBA-15-based materials.

  C CP/MAS NMR. Experimental conditions were as follow: contact time of 2 ms and a spinning rate of 10 kHz. NMR spectra of N 3 -SBA-15 and DMT-Tr-SBA-15 are displayed in Figure.3. N 3 -SBA-15 spectra show three peaks at δ= 9.4, 22.5 and 54 ppm can be attributed to the first carbon (C1) attached to Si, the middle carbon (C2) and the third carbon (C3) attached to the azido groups, respectively [44]. One can note the presence of two additional peaks signals can be associated to residual P123 template and EtO-groups (59 and 17 ppm) [45]. However, after azide-alkyne modification, extra newly peaks are clearly observed at δ= 120, 145, 165 and 170 ppm, which corresponds to the carbons present in the rings of triazole/triazine attached to the surface. The appearance of smaller peak at 37 ppm in the 13 C NMR spectrum of DMT-Tr-SBA-15 corresponds to carbon attached to the oxygen atom in the methoxy group of triazine heterocycles.

Figure. 3 .

 3 Figure.3. Solid-state 13 C CP/MAS NMR spectra of N 3 -SBA-15 and DMT-Tr-SBA-15.*Surfactant signals (P123).

Figure. 4 .

 4 Figure. 4. XPS survey spectra for different SBA-15 materials.

Figure 5 .

 5 Figure 5. Peak-fitted C1s and N1s regions from N 3 -SBA-15 (a, b), and DMT-Tr-SBA-15 (c,

Figure. 6 . 15 3. 1 . 5 .

 61515 Figure. 6. Thermogravimetric analysis of (a) pristine mesoporous silica SBA-15, (b) N 3 -SBA-15, and (c) DMT-Tr-SBA-15

Figure. 7 .

 7 Figure.7. Nitrogen sorption isotherms at 77 K for the N 3 -SBA-15, and DMT-Tr-SBA-15 samples.

Figure 8 .

 8 Figure 8. (a) Equilibrium adsorption isotherm of MB onto the DMT-Tr-SBA-15 at 25°C; inset shows optical images of dyes solutions MB with the DMT-Tr-SBA-15 adsorbent. (b) Temporal evolution of UV-Vis absorption spectra of MB.

Figure. 9 .

 9 Figure. 9. Removal efficiency of selected dyes by the DMT-Tr-SBA-15 adsorbent (30 mg) at room temperature, after optimum contact time and initial dye concentration of 40 mg/L.

Figure 10 displays

 10 Figure 10 displays the chemical structure of the adsorbent. It accounts for the possible molecular interactions with the cationic dyes. Triazine could be protonated and interact with -COO -group from Rhd B or -SO 3 -from MO. In addition, the cationic sites S + in MB and N + in Rhd 6G and Rhd B [57] favor adsorption on the surface which has silanol groups that are known to deprotonate at pH ~2 to yield SiO -. Indeed, these dye molecules are completely ionized and become positively charged in the solution. Moreover, DMT-Tr-SBA-15 adsorbent favorably captures aromatic organic molecules via π-π interactions on top of electrostatic, anion-cation interactions [58]. MB is smaller and flat dye;it could interact favorably with the triazine and triazole groups via π stacking in addition to H-bonding. Rhd 6G and Rhd B bear ethyl groups (MB has methyl groups) which limit specific interactions and thus account for weak London interactions, hence the lower adsorption capacity for these Rhodamine type dyes compared to MO and particularly MB.

Figure. 10 .

 10 Figure. 10. Schematic illustration of the available adsorption sites onto the DMT-Tr-SBA-15 adsorbent.

Table 1 :

 1 Recent triazol linked mesoporous silica via azide-alkyne cycloaddition to generate hybrid adsorbents.

	N 3 -Mesoporous silica	Grafted molecules	Sorbates	ref
	N 3 -SBA-15	phenylacetylene	Phenols	[30]
	N 3 -SBA-15	propargylamine	Niobium and copper species [31]
		propargylsulfone
	N 3 -HNS (Hollow nanostructured silica)	1-heptyne	Antibiotics	[25]
		2-butyne-1,4-diol
		propargyl alcohol
		phenylacetylene
	N 3 -Silica	10-undecynoic acid	Neutral and acidic drugs	[32]
	N 3 -SBA-15	3-ethynyl-1-methylimidazole	5-amino-4-hydroxy-7-	[33]
			sulfonaphthalene-2-
			sulfonate anion (H-acid)
	N 3 -SBA-15	propargyl alcohol	Copper	[34]

Table 2

 2 Summary of Nitrogen Sorption Results.

	Material	SBET (m 2 g -1 ) Dp BJH (nm) a	Vp (cm 3 g -1 ) b
	SBA-15	733.8	6.81	1.15
	N3-SBA-15	375.1	5.83	0.529
	DMT-Tr-SBA-15	276.9	5.06	0.367

Table 2 .

 2 Chemical properties of selected dyes.

	Dye	M.W.	UV	Type	Molecular structure
			absorption
			wavelength
			(nm)
	MB	319.85	664	Cationic dye
	Rhd B	479.01	551	Cationic dye
	Rhd 6G	479.01	496	Cationic dye
	MO	305.35	466	Cationic dye

Table 3 .

 3 Comparison of the maximum uptake capacity (q max ) values of MB in the present study and of other adsorbents reported in literature.

	Adsorbent	Maximum adsorption	pH	Equilibrium	Reference
		capacity q max (mmol.g -1 )		time (min)
	β-Cyclodextrin -SBA-15	5.61	-	25	[53]
	SBA-15	1.19	8.0	-	[54]
	Poly(GDMA)/ MCM-41	0.35	7.0	20 min	[55]
	MCM-41	0.206	7.0	60 min	[56]
	CuO/MCM-41	0.27	7.0	60 min	[56]
	COOH-SBA-16	1.85	9.0	200 min	20
	DMT-Tr-SBA-15	1.848	7.0	40 min	This work

Acknowledgements

AS wishes to thank the Tunisian Ministry of Higher Education and Scientific Research for the provision of travel grants to conduct research at ITODYS (Université de Paris) and ICMPE (CNRS) research labs.

Declaration of interest: the authors declare no conflict of interest.


Julie Bourilhon

Claire Olivier

Hana You

Antoine Collomb-Clerc

David Grabli

Hayat Belaid

Yannick Mullie

Chantal François

Virginie Czernecki

Brian Lau

Chantal Franc

Fernando Pérez-García

Eric Bardinet

Sara Fernandez-Vidal

Carine Karachi

MD Marie-Laure Welter
email: marielaure.welter@icm-institute.org.

Results from a Randomized Double-Blind Clinical Trial

Keywords: Mesencephalic locomotor region, Parkinson's disease patients, freezing of gait, postural instability, deep brain stimulation 131

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INTRODUCTION

Freezing of gait (FOG) and falls represent the dominant motor disabilities in advanced Parkinson's disease (PD). These signs worsen over time, are associated with increased morbidity, mortality, poor quality of life and high public health cost [START_REF] Kempster | Relationships between age and late progression 593 of Parkinson's disease: a clinico-pathological study[END_REF][START_REF] Nutt | Freezing of gait: moving forward 597 on a mysterious clinical phenomenon[END_REF][START_REF] Bloem | Prospective assessment of falls in 601 Parkinson's disease[END_REF][START_REF] Nonnekes | Freezing of gait: a practical approach to 604 management[END_REF],

and become unresponsive to dopaminergic treatment or deep brain stimulation (DBS) of the subthalamic nucleus [START_REF] Nonnekes | Freezing of gait: a practical approach to 604 management[END_REF][START_REF] Fasano | Axial disability and deep brain stimulation in patients 607 with Parkinson disease[END_REF][START_REF] Bloem | Influence of dopaminergic medication on automatic postural responses and balance impairment in Parkinson's disease[END_REF] or internal part of the globus pallidus [START_REF] Rodriguez-Oroz | Bilateral deep brain stimulation in Parkinson's disease: a multicentre study with 4 years follow-up[END_REF][START_REF] Tsuboi | Long-term clinical outcomes of bilateral GPi deep brain stimulation in advanced Parkinson's disease: 5 years and beyond[END_REF]. Their pathophysiology is poorly understood, but imaging and post-mortem studies suggest a causal role of cholinergic dysfunction within the pedunculopontine nucleus (PPN) [START_REF] Snijders | Gait-related cerebral alterations in patients with Parkinson's disease with freezing of gait[END_REF][START_REF] Sebille | Normal and pathological neuronal distribution of the human mesencephalic locomotor region[END_REF][START_REF] Karachi | Cholinergic mesencephalic neurons are involved in gait and postural disorders in Parkinson disease[END_REF][START_REF] Fasano | Neuroimaging of Freezing of Gait[END_REF], located in the mesencephalic locomotor region (MLR), also including the cuneiform nucleus (CuN) dorsally [START_REF] Grillner | On the descending control of the lumbosacral spinal cord from the "mesencephalic locomotor region[END_REF][START_REF] Garcia-Rill | Locomotion-inducing sites in the vicinity of the pedunculopontine nucleus[END_REF][START_REF] Takakusaki | Role of basal ganglia-brainstem systems in the control of postural muscle tone and locomotion[END_REF].

In mice, optogenetic stimulation of cholinergic and glutamatergic PPN neurons modulates and produces slow exploratory locomotion, respectively, whereas stimulation of glutamatergic CuN neurons produces high-speed running [START_REF] Xiao | Cholinergic mesopontine signals govern locomotion and reward through dissociable midbrain pathways[END_REF][START_REF] Caggiano | Midbrain circuits that set locomotor speed and gait selection[END_REF]. In cats, PPN or CuN stimulation modifies axial tone or elicits locomotion, respectively [START_REF] Takakusaki | Role of basal ganglia-brainstem systems in the control of postural muscle tone and locomotion[END_REF]; and in monkeys, cholinergic PPN lesions provoke increased hindlimb and tail tone with impaired locomotion, whereas CuN lesions increased forelimb and neck tone and gait speed [START_REF] Takakusaki | Role of basal ganglia-brainstem systems in the control of postural muscle tone and locomotion[END_REF].

In humans, neuronal recordings and imaging studies suggest that imagined and simulated gait evoke MLR activation [START_REF] Karachi | Cholinergic mesencephalic neurons are involved in gait and postural disorders in Parkinson disease[END_REF][START_REF] Marchal | Deep brain activation patterns involved in virtual gait without and with a doorway: An fMRI study[END_REF][START_REF] Jahn | Supraspinal locomotor control in quadrupeds and humans[END_REF][20][21][22][23][24], with the CuN more involved in initiated high speed gait and the PPN in the automaticity for paced walking [25].

Based on this and on the correlation between PPN cholinergic cell death and falls in PD [START_REF] Sebille | Normal and pathological neuronal distribution of the human mesencephalic locomotor region[END_REF] neurons and GABAergic neurons of the PPN and CuN [START_REF] Sébille | Post mortem high resolution diffusion MRI for large specimen imaging at 11.7 T with 3D segmented echoplanar imaging[END_REF], adjusted to fit the specific geometry of each patient's brain MRI (Fig. 2). The trajectory was chosen in order to have an electrode which passes through We performed an assessment 1 month after surgery (month 2, Fig. 1). The stimulators were activated, and parameter settings determined for the double-blind period. We randomly assigned patients 1 month later (month 3) to the 3 DBS conditions. At the end of the 6 month double-blind period, as previous studies have demonstrated a link between cholinergic cell death and gait and balance disorders of PD patients [START_REF] Snijders | Gait-related cerebral alterations in patients with Parkinson's disease with freezing of gait[END_REF][START_REF] Sebille | Normal and pathological neuronal distribution of the human mesencephalic locomotor region[END_REF][START_REF] Karachi | Cholinergic mesencephalic neurons are involved in gait and postural disorders in Parkinson disease[END_REF][START_REF] Fasano | Neuroimaging of Freezing of Gait[END_REF], all patients received PPN-DBS for the subsequent 3

213

LG6-4-1) and walked for 5-m at a self-paced speed, 214 making a half-turn and returning to the initial position

215

(n = 15-20 trials, Fig. 3). The gait initiation, straight 216 forward walking and turn events were manually 217 identified and the following parameters calculated: On-dopa, comprising the "axial" subscore) [34]; the gait and balance scale (GABS) [START_REF] Thomas | Clinical gait and balance scale (GABS): validation and utilization[END_REF], also assessed both Off and On-dopa; the Rating Scale for Gait Evaluation (RGSE) [START_REF] Martinez-Martin | A new clinical tool for gait evaluation in Parkinson's disease[END_REF]; the Tinetti scale [START_REF] Esculier | Home-based balance training programme using Wii Fit with balance board for Parkinson's disease: a pilot study[END_REF]; the freezing of gait questionnaire (FOG-Q) [START_REF] Giladi | Construction of freezing of gait questionnaire for patients with Parkinsonism[END_REF]; the Activities-Balance Confidence (ABC) scale [START_REF] Powell | The Activities-specific Balance Confidence (ABC) Scale[END_REF]; the Parkinson's Disease Questionnaire (PDQ-39); the Mattis Dementia Rating Scale (MDRS) [START_REF] Mattis | Dementia Rating Scale[END_REF]; the Comprehensive Psychopathological Rating Scale [START_REF] Asberg | Construction of a new psychiatric rating instrument, the Comprehensive Psychopathological Rating Scale (CPRS)[END_REF], which includes the Montgomery and Asberg Depression rating Scale (MADRS) [START_REF] Montgomery | A new depression scale designed to be sensitive to change[END_REF] and Brief Anxiety Scale (BAS) [START_REF] Tyrer | The brief scale for anxiety: a subdivision of the comprehensive psychopathological rating scale[END_REF].

We also assessed the differences in gait initiation and clinical parameters between inclusion and the end of each 2-month double-blind period (inclusion vs. month 5-7 or 9); between inclusion and the end of the open-label period (inclusion vs. month 12); and between inclusion and post-surgery (inclusion vs. month 3) to assess the effects of the surgery itself.

We assessed safety in all patients who were 263 enrolled and received MLR-DBS surgery. We clas-264 sified any new symptoms as adverse events. We

Electrode locations and stimulation parameters

Electrodes were accurately implanted in the PPN and the CuN on both sides except for one side in P04 (Fig. 2, Table 1). We used bipolar DBS using adjacent contacts as anode and cathode, for both CuN and PPN targets with 30 Hz stimulation frequency, 60 s pulse width and 1.7 to 4.2mA amplitude. During the randomized period, Off-dopa, we found that the first step length was higher with PPN-DBS relative to Sham-DBS (Fig. 6); and that the number of FOG episodes was lower with CuN-relative to PPN-DBS (Supplementary Figure 2). In the On-dopa condition, we found that the APAs, double-stance and turn durations were lower with Sham-relative to both PPN-and CuN-DBS (Fig. 6, Supplementary Table 1, Supplementary Figure 2), and the mediolateral CoP displacement during the APAs, step width, cadence, and turn amplitude were higher (Fig. 6, Supplementary Table 1, Supplementary Figure 2).

Changes in gait parameters

On-dopa, the first step length, velocity, and mediolateral CoP displacements were also significantly higher (Fig. 6, Supplementary Table 1) and the gait cycle duration lower (Supplementary Figure 2) with CuNrelative to PPN-DBS. The first step length (On-dopa) was also significantly higher with CuN-relative to Sham-DBS (Fig. 6) and the number of FOG lower with Sham-relative to PPN-DBS (Supplementary Figure 2). Lastly, we also found a significant interaction between DBS conditions and the effects of the dopaminergic treatment on gait parameters during the randomized period (p < 0.001, Supplementary Material, Results). Comparing DBS conditions to before surgery, Off-dopa, we found that the APAs mediolateral CoP displacement, first step length and width were decreased with both Sham and PPN-DBS, and the velocity also decreased with PPN-DBS relative to before surgery (Fig. 6). On-dopa, we found that the APAs and double-stance durations were significantly higher and the APAs mediolateral CoP displacement and first step width lower with both CuN-and PPN-DBS relative to before surgery (Fig. 6). On-dopa, the APAs mediolateral CoP displacement, first step length and velocity were also significantly lower with PPN-DBS relative to before surgery; and the first step length significantly lower with Sham-DBS relative to before surgery (Fig. 6). Lastly, the first step length and velocity (On-dopa) were also significantly decreased one year after surgery with open-label PPN-DBS rel-375 ative to before (Fig. 6).

376

Lastly, looking for differences in the clinical scores with no significant change On vs. Off-dopa for the "axial", GABS and Tinetti scales (Table 2).

Changes in gait parameters and clinical scales with PPN-DBS during the open-label period

At the end of the open-label follow up period (month 12) with PPN-DBS vs. before surgery, On-dopa, the first step length and velocity were sig-405 nificantly lower (Fig. 5), with no other significant 406 change in gait parameters or clinical scales (Table 2).

407

At the individual level, 3 patients (P01, P03, and P06) 408 showed higher FOG-Q and lower ABC scale scores 409 at month 12, relative to inclusion; with small or no 410 change in the three remaining patients (Fig. 7).

411

Adverse events 412

All patients were ambulatory the day after surgery.

413

Three serious adverse events were reported in 3 414 patients (Table 3). One was related to surgery with one 415 electrode displacement of one hemisphere (patient 416 P04). One patient had recurrent falls two months after 417 surgery with the occurrence of a subdural hematoma revealed by a mental confusion that necessitated hospitalization and reduction of the apormorphine daily dosage and resolved spontaneously (P03). One patient had an abdominal wall abscess necessitating surgery 7 months after DBS surgery (P01). Twentyseven non-serious adverse events were reported (Table 3). Falling was the most common adverse event (24/27) and reported in all patients, leading to shoulder injury in one patient (P02), and scalp or face wound in 3 patients (P02, P05, and P07).

Lastly, comparing gait parameters before (inclusion) vs. post-surgery (month 3), we observed significant 430 decreases in the step length (On-dopa Fig. 4), width 431 and velocity (On-dopa, not shown). Data are the number of adverse events, with the patients who had the adverse events in parentheses. * 3 serious adverse events occurred in these 6 patients. ‡ The safety committee advocated no reimplantation. $ 29 non-serious adverse events occurred in these 6 patients.

digital histological atlas [START_REF] Sébille | Post mortem high resolution diffusion MRI for large specimen imaging at 11.7 T with 3D segmented echoplanar imaging[END_REF]. We found better antero- [52] Fins JJ, Kubu CS, Mayberg HS, Merkel R, Nuttin B,

  , MLR-DBS has been attempted to alleviate FOG and falls for PD. About half of patients reported a subjective reduction of FOG and falls after MLR-DBS [26-29], but randomized controlled studies including a small number of PD patients generally failed to demonstrate 71 significant objective clinical effects [28-30]. Using 72 gait recordings in a controlled study, we previously 73 observed a small improvement of objective gait initi-74 ation parameters in 4 patients with DBS applied in the 75 dorsal MLR, i.e., just above the pontomesencephalic 76 junction (PMJ) [29], and in a postmortem series of 2 77 patients with progressive supranuclear palsy, a possi-78 ble better result was also obtained with DBS applied 79 in the dorsal MLR [31]. However, a reduction of FOG 80 during turning was reported in 7 patients with DBS 81 applied in the more ventral MLR, below the PMJ 82 [32]. Finally, the heterogeneous clinical outcomes 83 obtained in previous studies could be due to 1) the 84 unknown optimal target within the MLR, namely the 85 CuN or the PPN that may control different walk-86 ing parameters; 2) the difficulty in assessing episodic 87 phenomenon such as FOG and falls under experimen-88 tal conditions, and 3) the unknown PD population that 89 would be the best candidates [33]. 90 Here, we aimed to evaluate the effects of DBS of 91 both PPN and CuN in PD patients with dopa-resistant 92 FOG and/or falls, in a crossover, double-blind ranrandomized, double-blind, 97 crossover trial with a 6-month blinded phase, we 98 recruited patients from the Pitié-Salpêtrière Hospital. 99 All patients were assessed at the Clinical Investiga-100 tion Center at the Paris Brain Institute and operated at 101 the Neurosurgery department of the Pitié-Salpêtrière 102 Hospital. Patients were eligible for inclusion if they 103 fulfilled the criteria for idiopathic PD diagnosis based

Fig. 1 .

 1 Fig. 1. Study design. The study included three 2-month treatment phases (month 3 to month 9). Patients were evaluated at inclusion, less that 2 months before surgery; 2 months after surgery, before sham or active stimulation; 4, 6 and 8 months after surgery, after 2 months with active PPN, active CuN or sham stimulation; and 11 months after surgery after 3 months with open-label active PPN stimulation. The optimal stimulation settings for the randomization period were determined 1 month after surgery.

  Randomization and blinding old and spent the same time adjusting each patient's 143 stimulator at the start of both active and sham stimula-144 tion periods. The DBS parameters were not modified 145 during each double-blind period. The primary and 146 secondary outcomes were assessed by the same 147 blinded investigators throughout the study. 148 Procedures 149 Patients were assessed at inclusion (baseline), fol-150 lowed 1 month later by bilateral implantation of DBS 151 electrodes into the MLR (Fig. 2). For each patient, we 152 determined the targets after superimposing our 3D 153 histological atlas providing a map of PPN cholinergic

Fig. 2 .

 2 Fig. 2. Reconstruction of the PPN and CuN nuclei DBS electrodes location. Left panel: Location of the highest density of cholinergic neurons within the mesencephalic locomotor region corresponding to the pedunculopontine nucleus (PPN, red area). Middle and right panels: Location of contacts used for DBS and reported on 3-D views after fusion with the three-dimensional MRI image (middle panel: posterior view; right panel: right and left sagittal views). The pink and light-blue outline the cholinergic PPN and GABAergic CuN neurons, respectively, with the dashed line delimiting the dorsal part of the CuN. The active contacts used for PPN-DBS during the double-blind period are represented in green and those for CuN DBS in yellow, showing that the electrodes all penetrate the PPN and CuN areas.

these 2

 2 MLR areas, with at least one contact located within the PPN and another more dorsally within the CuN. Implantation of the two electrodes with 8 in-line contacts (model DB-2201, contact spacing: 2 mm, diameter: 1.3 mm, Boston Scientific). Surgery was performed under general anesthesia in 3 patients and under sedation in 3 patients, with two microelectrodes recordings on each side, with one along the central trajectory and one along the anterior, posterior, or medial trajectories depending on the individual patient's anatomy. According to per-operative physiological and anatomical checks using X-ray, the central trajectory was chosen for implanting the definitive DBS electrode in 9 cases, the anterior trajectory in 2 cases and the posterior trajectory in one case. A post-operative CT-scan was performed to check the absence of complications and determine electrode locations (Fig. 2A). The pulse generator was implanted 1-4 days after (Vercise; Boston Scientific).

  months in an open-label fashion in order to assess 188 more specifically the effects of PPN-DBS over a 189 longer period and with the possibility to adapt param-190 eters settings (month 9 to month 12). 191 We recommended that patients' medical treat-192 ments for PD remain stable over the study period. 193 We carefully recorded treatment adjustments neces-194 sitated by patients' neurological conditions. 195 Outcomes 196 The primary outcome was the difference in the 197 posterior center of foot pressure (CoP) displacement 198 during the anticipatory postural adjustments (APAs) 199 phase Off-dopa between the end of each 2-month 200 period of the randomized double-blind period, i.e., 201 with Sham-vs. PPN-vs. CuN-DBS. We chose this 202 parameter as 1) it has been widely reported to be 203 decreased in PD patients, and more specifically in 204 PD patients with FOG [36], 2) was recorded using 205 validated procedures and calculated independently, 206 and 3) we have already shown it to be modified with 207 MLR-DBS [29]. 208 For this, gait parameters were recorded at each visit 209 using the VICON system with Plug-In-Gait model 210 with markers positioned on the arm and leg joints. The 211 patients, barefoot, initiated gait on a force platform 212 (0.9 × 1.8 m, Advanced Mechanical Technology Inc.

Fig. 3 . 1 )

 31 Fig. 3. Recordings of gait initiation, straight-line walking and turn using the force platform and VICON system. Top row: The subject stands motionless on the force plate (Grey square) and initiates gait with his right leg (Gait initiation), and then walks straight forward self-paced, makes a half-turn to a landmark taped to the ground and comes back to the starting position. The standardized gait alternation is represented with green and purple filled ellipses when the foot is in floor contact and a dotted outline ellipse during the swing phase of the foot. A freezing of gait episode is represented by the two feet on the ground. Bottom row: Illustration of the AP and ML CoP displacements during the gait initiation phase (left). Events of the gait initiation task are represented with black arrows and grey and white columns. At bottom right, illustration of both pelvis, knee, and ankle kinematics (upper rows) and soleus EMG signal (lower rows) for right and left legs, during gait initiation, straight-line forward walking, and U-turn. A FOG episode is characterized by an absence of forward movement, with attempts to lift the left foot from the ground, and bilateral simultaneous soleus muscle activity. AP, Anteroposterior; APAs, Anticipatory Postural Adjustments; CoP, Centre of foot Pressure; DS, double-stance phase; FC1, Foot Contact of the starting leg; FO1, Foot Off of the starting leg; FOG, Freezing of Gait; ML, Mediolateral; Sol, Soleus; t0, time of the first biomechanical event corresponding to the beginning of the APAs.

  265classified an adverse event as serious if the patient 266 required hospital admission, if sequelae were present, 267 or if the clinician considered the event to be serious. 268 Statistical analysis 269 This is a phase II study, and all outcomes are 270 exploratory. We performed statistical analyses inclu-271 ding all randomly allocated patients who received 272 sham or active DBS during the double-blind period. 273 All quantitative data are described with mean and 274 SDs. 275 To assess a difference in the primary outcome, we 276 used the non-parametric Friedman rank sum test with 277 stimulation target (Condition, i.e., sham-, PPN-or 278 CuN-DBS) as the treatment or grouping variable and 279 patient as the blocking factor. We examined the differ-280 ences in other gait parameters and clinical scores by 281 modelling each outcome using a linear mixed-model. 282 We included treatment condition (baseline, month 3, 283 Sham-, CuN-, or PPN-DBS, and month 12) and a 284 period effect as fixed effects, and patient intercepts 285 as random effects. We used the R package (version 286 3.3.1, R Core Development Team) for the statistical 287 analysis, with the LmerTest package for the linear 288 mixed-effect model testing. We used a significance 289 threshold of 0.05. Post-hoc comparisons were FDRand June 2018, we assessed 294 9 patients for eligibility, enrolling 8 in the study 295 (Fig. 4). Two patients were withdrawn before surgery 296 because of non-inclusion criteria: one with dementia 297 (MMSE = 18) and one showed dopaminergic respon-298 sive axial motor signs (improvement of 72%). Thus, 299 6 patients were operated, randomly allocated and 300 completed the study (Table 1). Medication was held 301 constant during the double-blind period except in one 302 patient (P03). Patients inclusion was stopped after 303 completion of the randomized double-blind period by 304 the sixth patient following a decision by the indepen-305 dent supervisory committee. This decision was based 306 on the absence of an objective or subjective individ-307 ual improvement in clinical scores in the 6 operated 308 patients.

  Fig.5).

Fig. 4 .

 4 Fig. 4. Trial profile. DBS, deep brain stimulation; CuN, cuneiform nucleus; MLR DBS, mesencephalic locomotor region deep brain stimulation; PPN, pedunculopontine nucleus.

377

  and gait parameters with exclusion of the patient with 378 unilateral MLR-DBS (P05), we found no differences 379 in the results obtained, except a lower step length with 380 Sham-relative to CuN-DBS (On dopa, not shown). 381 Clinical scales 382 We found no significant differences in the MDS-383 UPDRS, "axial" subscore, GABS, RSGE, ABC, 384 FOG-Q, PDQ-39, MDRS, MADRS, and BAS scores 385 at the end of each DBS condition (Off and On-386 dopa conditions,

*

  The axial score is the sum of the following items: "arising from chair", "gait", "freezing of gait", "postural stability", and "posture" DBS, deep brain stimulation; FOG-Q, freezing of gait questionnaire; GABS, gait and balance scale; LED, levodopa-equivalent daily dosage; MDRS, Mattis Dementia Rating Scale; MDS-UPDRS, Movement Disorders Society-Unified Parkinson's Disease Rating. Electrodes coordinates are expressed in the lateral (X, relative to the midline, absolute values), anteroposterior (Y, relative to the posterior commissure) and depth (Z, relative to the ponto-mesencephalic junction with negative values indicating electrodes placed below the PMJ) axes, in mm, relative to the fourth ventricle floor and the PMJ line.

Fig. 5 . 4 Fig. 6 .

 546 Fig. 5. Effects of MLR DBS on posterior APAs displacement of the center of foot pressure. Posterior displacement of the CoP during the APAs of the gait initiation in the 6 PD patients before surgery (Preop), 3 months after surgery without active DBS (M3); and during the randomized double-blind period with Sham, CuN-DBS, or PPN-DBS, and at the end of the open label period with PPN-DBS (M12); OFF (left graph) and ON-dopa (right graph). Each color point represents an individual patient. One patient (P05, blue circle) could not perform gait initiation Off-dopa during the randomized and open-label periods. Horizontal black lines represent the estimated marginal mean for each condition, and the upper and lower bounds of the rectangles represent the corresponding 95% confidence intervals. APAs, anticipatory postural adjustments; CoP, centre of foot pressure; M, month; PPN, pedunculopontine nucleus; CuN, cuneiform nucleus. * p < 0.05; * * p < 0.01; * * * p < 10 -4

432 DISCUSSION 433 Fig. 7 .

 4337 Fig. 7. Effects of CuN, PPN and sham DBS on freezing of gait and feelings of imbalance scores. Effects of surgery (M3), Sham, CuN and PPN-DBS during the randomized controlled period and after 3 months with PPN-DBS during the open-label period (M12) on the FOG severity and imbalance feelings. FOG-Q, freezing of gait questionnaire; ABC, Activities Balance Confidence. Table 3 Adverse events Before Active PPN Active CuN Sham Open-label PPN randomization stimulation stimulation stimulation stimulation Serious adverse events (n = 3) * Surgery-related Electrode displacement 1 (P05) ‡ 0 0 0 0 Disease-related Falls with subdural hematoma 1 (P03) 0 0 0 0 Other Abdominal wall abscess 0 1 (P02) 0 0 0 Non-serious adverse events (n = 29) $ Surgery-related 0 0 0 0 0 Stimulation-related 0 0 0 0 0 Disease-related Falls 3 (P02, P03, P08) 2 (P03 and P05) 4 (P02, P03, P07) 1 (P01) 2 (P02 and P05) Falls with injury 1 (P05) 2 (P02 and P07) 0 0 0 Urinary retention 0 0 0 0 1 (P07) Orthostatic hypotension 0 1 (P03) 1 (P03) 1 (P07) 0 Visual hallucinations 0 1 (P05) 0 0 0 Other Ear pain 0 0 0 1 (P03) 0 Leg oedema 0 0 0 0 1 (P02) Urinary infection 1 (P05) 0 0 0 0 Prostatitis 0 1 (P05) 0 0 0 Car accident without injury 0 0 0 1 (P05) 0 Anxiety 0 0 0 1 (P08) 0 Muscular pain 0 1 (P08) 0 0 0

  and significant lower step length after open label PPN-DBS. This result is not in accordance with decreased time and increased cadence during the turn task found in 7 PD patients with MLR-DBS applied below the PMJ, with however, no significant change in spatiotemporal parameters of straight-line gait[32]. The DBS electrodes in that study were also located more deeply within the pons, thus possibly modulating the activity of the descending MLRspinal pathways, also involved in locomotion and postural controls in mammals[START_REF] Grillner | On the descending control of the lumbosacral spinal cord from the "mesencephalic locomotor region[END_REF]. The absence of improvement with PPN-DBS found in our patients could also be related to the differences in the patient selection, with On-dopa FOG and falls suggesting a high proportion of cholinergic cell death preventing to obtain benefit using DBS[START_REF] Sebille | Normal and pathological neuronal distribution of the human mesencephalic locomotor region[END_REF] 33]. Finally, one should also take into consideration that even though the surgery was well tolerated, MLR surgery has potential risks. The fact that these advanced PD patients had recurrent falls represent an additional risk factor with implanted electrodes and one of our patients indeed presented a non-surgical subdural hematoma following falls. Differences in DBS programming may also explain the variability of MLR-DBS clinical effects, and the parameter settings used during the double-blind period could be not completely optimal. The duration of DBS with a 2-month period may not be long enough, a sustained improvement or no aggravation of FOG/falls being reported two years after surgery suggesting a possible gradual alleviation [26, 46, 49]. In our study, 4/6 patients reported a subjective improvement in the hours following DBS parameters changes, with two patients after PPN-DBS and two after CuN-DBS (not shown). A short, 30-minute duration of PPN-DBS has been reported to induce positive effects on gait [32] and such subjective short 515 duration effects were also previously reported [28], 516 thus leading the proposal of intermittent or diurnal 517 cycling DBS, [50] but without significant objective 518 improvement. Finally, no evidence of the "good" 519 parameters have been determined up to now [33], and 520 we used low frequency stimulation as also proposed 521 by others teams, with similar amplitude [51]. 522 Limitations 523 Several limitations of this study need to be pointed 524 out. Firstly, these results were obtained in a small 525 group of patients, thus limiting the generalizability 526 of our data. Indeed, at the request of the safety board 527 the study was prematurely halted and only half of the 528 patients previously plan to be included in this study 529 were finally included, randomized, and assessed. This 530 under-powered the study and prevented us to fully 531 test the efficacy of MLR-DBS for these severe PD 532 patients. However, the fact that all the patients pre-533 sented a similar form of the disease, were precisely 534 assessed using specific tools to measure gait and 535 balance controls, and that all the electrodes were pre-536 cisely positioned within both the CuN and PPN using 537 a histological individualized atlas of the MLR, with 538 no dramatic clinical change in gait and balance dis-539 orders at an individual level favored the robustness of 540 these results. Secondly, the occurrence of side effects 541 also rendered the stimulation parameter selection dif-542 ficult, and the double-blind design of our study may 543 also have prevented us from optimally setting the 544 DBS parameters, with stimulation amplitude being 545 potentially too low to produce significant clinical that both PPN-and CuN-DBS 549 are not clinically efficient to improve severe and dopa-550 resistant gait and balance disorders in PD patients. 551 Further research is needed to test other parameter set-552 tings to modulate this particular anatomical area, such 553 as intermittent stimulation and longer double-blind 554 period of stimulation, and using different protocol 555 designs allowing setting optimal parameters before 556 blinded assessments or parallel group study to com-557 pare the changes in gait and balance disorders over 558 time [52]. Finally, we need to further understand 559 the role of the MLR in gait and balance control in 560 humans using new imaging or neurophysiological 561 approaches, and also its dysfunction in FOG or falls 562 using animal models research.deep brain stimulation for parkinsonian disorders: a case series. Stereotact Funct Neurosurg 99, 287-294. [51] Nosko D, Ferraye MU, Fraix V, Goetz L, Chabardes S, Pollak P, Debû B (2015) Low-frequency versus highfrequency stimulation of the pedunculopontine nucleus area in Parkinson's disease: a randomised controlled trial. J Neurol Neurosurg Psychiatry 86, 674-679.

Table 2 )

 2 , and relative to pre-surgery

387

scores (Table

2

). During the randomized period, 3/6 388 patients had lower scores for FOG-Q with CuN-DBS

Table 1

 1 Baseline demographic and clinical characteristics of 6 PD patients and stimulation parameters setting used during the blinded period

	Patient Age, y/	Disease	MDS-UPDRS Axial score *	GABS	FOG-Q MDRS ABC-	LED	DBS coordinates:
		Sex	duration, y	3 Off/On	Off/On	Off/On			Scale	(mg/d)	X/Y/Z (mm)
	01	65/M	6	29/17	7/6	10/12	29	144	78.1	650	CuN-R: 10/8/3
											CuN-L: 7/8/3
											PPN-R: 7/5/-2
											PPN-L: 6/4/-2
	02	68/M	12	37/19	9/7	29/24	36	142	52.5	2,200	CuN-R: 11/12/3
											CuN-L: 10/10/3
											PPN-R: 8/8/-1
											PPN-L: 7/7/-1
	03	67/F	10	51/28	16/11	40/25	36	141	46.3	860	CuN-R: 8/8/5
											CuN-L: 8/9/4
											PPN-R: 6/8/-2
											PPN-L: 6/8/-1
	04	68/M	17	43/28	15/12	39/30	40	139	42.5	1,650	CuN-R: NA
											CuN-L: 8/10/2
											PPN-R: NA
											PPN-L: 6/7/-2
	05	67/M	12	75/40	19/12	51/19	44	139	43.1	2,175	CuN-R: 9/8/5
											CuN-L: 8/9/5
											PPN-R: 6/5/-1
											PPN-L:5/6/-1
	06	54/M	12	27/12	6/3	11/4	33	139	79.4	1,200

CuN-R: 7/10/4 CuN-L: 7/9/4 PPN-R: 6/8/0 PPN-L: 6/7/0 Mean 64.8 11.5 43.7 (17.7)/ 12.0 (5.3)/ 30.0 (16.6)/ 36.3 140.7 57.0 1,456 (660) (SD) (5.

Table 2

 2 Changes in gait and balance disorders, quality of life, cognition and psychiatric signs in 6 PD patients with PPN, CuN, or sham deep brain stimulation Values are mean ± standard deviation. ABC, Activities Balance Confidence; BAS, Brief Anxiety Scale; FOG-Q, Freezing of Gait Questionnaire; GABS, Gait And Balance Scale; MADRS, Montgomery and Asberg Depression rating Scale; MDRS, Mattis Dementia rating; MDS-UPDRS, Movement Disorders Society-Unified Parkinson's Disease Rating Scale; PDQ-39 SI, Parkinson's Disease Questionnaire Summary Index; RSGE, Rating Scale for Gait and Equilibrium. * p values for the comparison between values with Sham vs. CuN-DBS vs. PPN-DBS, $ p values for the comparison between values at Month 12 with PPN-DBS relative to baseline. £ p < 0.05 On relative to Off-dopa condition.

				Double-blind period			Open-label period
		Baseline	Sham-DBS CuN-DBS	PPN-DBS	p *	PPN-DBS	Change during open-	p $
								label PPN DBS
								(month 12 vs. baseline)
	MDS-UPDRS part 1	10.3 ± 6.6	13.2 ± 5.7	13.0 ± 4.6	13.3 ± 7.6 1.00	12.7 ± 4.9	2.3 ± 5.6	0.34
	MDS-UPDRS part 2
	Off-dopa	31.6 ± 10.1	26.2 ± 9.7	27.7 ± 9.9	25.3 ± 9.1 0.88 28.0 ± 10.8	-1.2 ± 3.1	0.41
	On-dopa	19.0 ± 4.2	18.5 ± 7.0	17.3 ± 6.3	17.2 ± 5.4 0.87	17.0 ± 5.7	-2.0 ± 3.0	0.47
	Falls severity (item 13)
	Off-dopa	2.2 ± 0.4	2.2 ± 0.8	2.3 ± 0.8	2.0 ± 1.1	0.87	1.8 ± 1.2	-0.3 ± 1.0	0.75
	On-dopa	2.0 ± 0.6	2.0 ± 1.1	1.8 ± 0.8	2.5 ± 0.8	0.57	2.0 ± 0.9	0.0 ± 1.1	1.00
	FOG severity (item 14)
	Off-dopa	3.2 ± 0.8	3.5 ± 0.5	3.3 ± 0.8	3.3 ± 0.8	0.96	3.2 ± 1.0	0.2 ± 0.4	1.00
	On-dopa	2.8 ± 1.0	2.0 ± 1.3	2.2 ± 0.4	1.5 ± 0.5	0.30	1.7 ± 0.5	-0.2 ± 0.8	0.52
	MDS-UPDRS part 3
	Off-dopa	43.7 ± 17.7 37.2 ± 10.2 37.0 ± 11.7 34.0 ± 6.9 0.93	36.8 ± 9.4	-0.6 ± 8.4	0.64
	On-dopa	24.0 ± 10.1 £ 29.4 ± 11.2 30.0 ± 11.3 29.0 ± 11.0 0.96 28.2 ± 10.2 £	4.2 ± 8.8	1.00
	Axial subscore
	Off-dopa	12.0 ± 3.4	11.0 ± 5.7	11.0 ± 4.8	9.8 ± 4.3	0.85	9.8 ± 4.0	-0.8 ± 3.3	1.00
	On-dopa	8.3 ± 4.0	8.2 ± 4.8	8.5 ± 3.9	7.7 ± 3.4	0.80	7.8 ± 3.5	-0.5 ± 3.7	0.52
	MDS-UPDRS part 4	8.8 ± 1.6	6.7 ± 3.9	7.7 ± 4.2	6.3 ± 3.4	0.69	7.7 ± 4.2	-1.2 ± 3.9	0.68
	GABS
	Off-dopa	30.0 ± 16.6 23.8 ± 12.4 24.8 ± 8.6 24.8 ± 11.5 0.97	23.8 ± 9.4	-2.0 ± 13.9	0.47
	On-dopa	19.0 ± 9.6	17.2 ± 9.6	18.8 ± 7.1	17.5 ± 9.5 0.86	18.8 ± 9.3	-3.3 ± 12.8	0.78
	Tinetti static
	Off-dopa	10.4 ± 5.3	12.2 ± 3.2	12.4 ± 2.3	11.4 ± 2.3 0.82	11.8 ± 2.8	-0.2 ± 2.6	0.85
	On-dopa	13.3 ± 2.9	11.4 ± 4.2	12.8 ± 1.3	13.2 ± 1.7 0.63	12.8 ± 1.6	-0.5 ± 1.9	0.38
	Tinetti dynamic
	Off-dopa	7.2 ± 4.2	7.4 ± 2.8	8.2 ± 3.2	7.8 ± 2.7	0.88	9.6 ± 2.6	1.6 ± 4.0	0.32
	On-dopa	8.8 ± 2.7	10.8 ± 1.1	8.8 ± 2.4	9.8 ± 2.1	0.23	9.3 ± 2.4	0.5 ± 2.7	0.75
	RSGE
	Off-dopa	43.0 ± 13.7 37.3 ± 12.6 33.5 ± 10.4 33.8 ± 7.2 0.94 35.3 ± 11.5	-5.6 ± 11.4	0.27
	On-dopa	23.2 ± 8.2	25.2 ± 8.2	25.7 ± 6.9	22.2 ± 6.9 0.48	26.3 ± 8.9	3.2 ± 8.4	0.52
	FOG-Q	36.3 ± 5.2	33.8 ± 5.3	40.5 ± 6.1	38.3 ± 3.3 0.43	40.0 ± 4.4	3.7 ± 7.2	0.20
	ABC scale	57.0 ± 17.2 51.9 ± 14.2 57.9 ± 16.6 59.8 ± 20.4 0.72 56.3 ± 17.1	-0.7 ± 19	1.00
	PDQ-39 SI	64.7 ± 22.8 62.0 ± 20.9 68.2 ± 20.4 63.7 ± 20.8 0.75 66.0 ± 20.2	1.7 ± 1.5	0.15
	MDRS	140.7 ± 2.1 142.3 ± 1.9 141.0 ± 2.8 140.3 ± 3.7 0.50 142.0 ± 3.1	1.3 ± 3.8	0.23
	MADRS	6.2 ± 4.2	6.0 ± 5.1	4.7 ± 2.1	5.2 ± 3.7	0.97	5.3 ± 4.6	-0.8 ± 7.0	0.66
	BAS	6.0 ± 5.8	3.7 ± 2.3	3.3 ± 2.1	3.0 ± 2.6	0.87	3.5 ± 3.3	-2.5 ± 7.8	0.40

ACKNOWLEDGMENTS

This study was supported by the Institut Na-572 tional de la Recherche Médicale (INSERM) and 573 the 'Investissements d'avenir' program (ANR-10-574 IAIHU-06 and ANR-11-INBS-0006) and grants from 575 the Michael J Fox Foundation for Parkinson's disease 576 (grant number: 10019). JB was supported by a grant 577 from the Fondation pour la Recherche Médicale.

578 YM was supported by a grant from the European 579 Union's Horizon 2020 research and innovation pro-580 gramme under the Marie Slodowska-Curie-Horizon 581 2020 (H2020-MSCA-IF-2019, grant agreement No 582 898265).

CONFLICT OF INTEREST

584

The authors declare to have no conflict of interest 585 relative to the research.

SUPPLEMENTARY MATERIAL

587

The supplementary material is available in the 588 electronic version of this article: https://dx.doi.org/


Dr Xiaojiao Yuan

Dr Kunran Yang

Chloé Grazon

Dr Cong Wang

Dr Lorenzo Vallan

Jean-David Isasa

Dr Pedro M Resende

Dr Fanxing Li

Dr Cyril Brochon

Dr Hynd Remita

Dr Georges Hadziioannou

Eric Cloutet
email: eric.cloutet@u-bordeaux.fr

Dr Jian Li
email: jian.li@epfl.ch

Dr J.-D Isasa

Tuning the Aggregates of Thiophene-based Trimers by Methyl Side-chain Engineering for Photocatalytic Hydrogen Evolution

Keywords: Aggregation, Intramolecular packing, Thiophene, Photocatalysis, Hydrogen Production

Organic π-conjugated semiconductors (OCSs) have recently emerged as a promising alternative to traditional inorganic materials for photocatalysis. However, the aggregation of OCSs in photocatalytic aqueous solution caused by self-assembly, which closely relates to the photocatalytic activity, has not yet been studied. Here, the relationship between the aggregation of 4,7-Bis(thiophen-2yl) benzothiadiazole (TBT) and the photocatalytic activity was systematically investigated by introducing and varying the position of methyl side chains on the two peripheral thiophene units. Experimental and theoretical results indicated that the introduction of -CH3 group at the 3-position of TBT resulted in the smallest size and best crystallinity of aggregates compared to that of TBT, 4-and 5positions. As a result, TBT-3 exhibited an excellent photocatalytic activity towards H2 evolution, ascribed to the shorten charge carrier transport distance and solid long-range order. These results suggest the important role of aggregation behavior of OCSs for efficient photocatalysis.

Introduction

Organic π-conjugated semiconductors (OCSs) with tunable energy levels, excellent chemical stability and broad absorption, have triggered a huge interest in photocatalysis. [1] Substantial efforts such as molecule design, structure regulation and sidechain engineering have recently been invested to improve the photocatalytic performance of OCSs by fine-tuning optical property, band energy levels and conductivity. [2] Because of the insolubility in aqueous solution and intermolecular non-covalent interactions (such as electrostatic, hydrophobic, and 𝜋𝜋-𝜋𝜋 stacking), OCSs usually exhibit distinct aggregation characteristics in aqueous photocatalytic solution, i.e., self-assemble to form nanoor micro-agglomerates. [3] Therefore, the entire photocatalytic process including generation, separation and migration of photogenerated carriers, takes place in aggregates instead of individual organic molecules. On one hand, the size of aggregates, which determines both the number of surface-active sites in term of specific surface area and the transportation distance of electrons to the surface, has a remarkable influence on the photocatalytic performance. [4] On the other hand, the crystallinity of the aggregates critically affects the intrinsic charge separation and transport. A higher crystallinity with fewer recombination centers facilitates more photogenerated charge carrier to diffuse from the bulk to surface for redox reactions. Therefore, judiciously engineering the intramolecular packing to regulate the size and crystallinity of aggregates is essential to achieve efficient photocatalytic performance for OSCs.

Side-chain engineering is an effective strategy to fine-tune the intramolecular packing and crystallinity by alteration of chain length, branching position and size of the branched alkyl chains. [START_REF] Chai | Energy & Environmental Science[END_REF] [6], [7] In this work, methyl groups (-CH3), as the smallest alkyl substitution unit, with minimal competition for space-filling, were selected as side chain groups, which can effectively avoid side chain entanglement caused by long side chain, thus reducing the interference of other factors. Thiophene-based conjugated trimers with donor-acceptor-donor (D-A-D) molecule structure were prepared as an OCS model because the strong intermolecular interactions generated by the D-A moiety favor the selforganization of trimers into ordered structures and close packing with increasing 𝜋𝜋-𝜋𝜋 orbital overlap. [8] The relationships of aggregation-property-activity was systematically studied by introducing -CH3 side chain on different positions of TBT. 4,7bis(3-methylthiophen-2-yl)benzo[c][1,2,5]thiadiazole (TBT-3) which possesses larger steric hindrance than TBT, 4,7-bis(4methylthiophen-2-yl)benzo[c] [1,2,5]thiadiazole (TBT-4) and 4,7bis(5-methylthiophen-2-yl)benzo[c] [1,2,5]thiadiazole (TBT-5) showed the smallest size and the best crystallinity. The smaller aggregates and favorable crystalline properties contribute to the solid long-range order and charge carrier transport of TBT-3, leading to a superior photocatalytic activity for hydrogen production. DFT calculations demonstrated that TBT-3 maintained weak aggregation ability, which enables the reaction on a larger surface area; TBT-3 also exhibited reactive molecular orbitals, making it favorable for hydrogen evolution. The deep understanding of the relationship between the aggregation and photocatalytic activity can provide important insight in design of OCSs for efficient photocatalytic fuel production.

Results and Discussion

The D-A-D conjugated trimers (TBT) were synthesized by Pd (0)catalyzed Suzuki-Miyaura cross-coupling and Stille crosscoupling reactions (Figure 1a and Schemes S1-S4), in which thiophene (T) and benzothiadiazole (B) serve as electron donor and acceptor, respectively. (Figure S1). The chemical structures of these purified trimers were confirmed by 1 H NMR, 13 C NMR, mass spectrometry, elemental analysis and single crystal X-ray diffraction (Figure S2-S10, Table S1). All the prepared trimers are fully soluble in common organic solvents such as dichloromethane, tetrahydrofuran (THF), acetone, dimethyl sulfoxide (DMSO) and ethanol, but insoluble in water, which thus results in the formation of aggregates in aqueous photocatalytic system (H2O/TEOA = 9: 1, v/v). Obviously, TBT-3 presented the best dispersion compared with TBT-4, TBT-5 and TBT (Figure 1b). The aggregates size of the trimers in H2O/TEOA solution was further measured by dynamic light scattering (DLS). Compared to TBT (~400 nm), TBT-4 (~1.2 µm) and TBT-5 (~1.3 µm), TBT-3 exhibited the smallest aggregates size with the average dimeter of ~50 nm (Figure 1c and Figure S11). The similar surface tension for all the samples suggests that the aggregates size is not dominated by the surface tension (Figure S12). Compared to TBT, a blue shift of absorption and emission wavelengths was observed for TBT-3 while a red shift was obtained for TBT-4 and TBT-5 (Figure 1d and Figure S13), consistent with the simulated results (Figure S14). The different absorption shifts of these trimers indicate that the position of the side chains affect the photophysical properties. [5a, 9] S2). Raman spectra was used to characterize the chemical structure of the prepared trimers. The strong peaks around 1377 cm -1 and 1419 cm -1 correspond to the C-C and C=C stretches, respectively, on the bridging thiophene units. The peaks in the range 1324-1360 cm -1 and the peak at 1538 cm -1 correspond to the C-C bands and ring stretch in B unit, respectively. [10] Furthermore, the increased steric hindrance caused by the side chain led to some peaks splitting into two components. Similar phenomenon also occurs in previous research in regio-random P3HT. [10a] Differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), and thermogravimetric analysis (TGA) revealed that all the trimers possess good crystallinity and thermal stability (Figure 2n, Figure S18-19). The diffuse rings in two-dimensional (2D) scattering pattern maps indicated that the crystals were randomly oriented (Figure 2o) and all the trimers were 2D hexagonal structures based on the peak position of (100) Bragg reflection (Figure S20). Unlike TBT-3, a wide peak appeared in the pattern of TBT, TBT-4 and TBT-5, which suggested the presence of an amorphous phase in TBT, TBT-4 and TBT-5 (Figure 2p). The crystalline fraction for TBT-3 was further determined to be 88.1%, which was much higher than that of TBT (65.1%), TBT-4 (74.6%) and TBT-5 (70.0%) (Figure S21). Evidently, TBT-3 displayed the best crystallinity compared with TBT-4, TBT-5 and TBT, possibly due to the enhanced steric hinderance which prevent the tight packing of molecules and increase the intermolecular spacing, leading to an improved crystallinity. [11] Therefore, the introduction of methyl side-chains at the 3-position of TBT improves the crystalline properties, which contribute to solid long-range order and charge carriers' transport in photocatalysis. [8a, 12] The optical absorption properties of the solid trimers were investigated by UV-Vis diffuse reflectance spectroscopy (DRS) and simulated DFT calculations. The results showed that all the trimers exhibit broad light absorption (Figure 3a and Figure S22). Notably, different stacking modes in the solid state contribute differently to the absorption properties. [3,13] TBT-5 and TBT show red-shifted absorption compared to that of TBT-3 and TBT-4, which may be ascribed to more planar backbone of TBT and TBT-5. [14] The optical band gaps (Eg) of the trimers were calculated and found to be 2.32 eV (TBT-3), 2.17 eV (TBT-4), 2.04 eV (TBT) and 1.99 eV (TBT-5) from the Tauc plots (Figure 3b). The HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) energy levels of trimers were determined by electrochemical cyclic voltammetry (Figure 3c-d and Figure S23). The HOMO/LUMO energy levels of TBT, TBT-3, TBT-4 and TBT-5 were calculated to be 0. As discussed above, photocatalytic process takes place in aggregates instead of individual organic molecules, (Figure 4a). The photocatalytic hydrogen production of the prepared trimers was investigated in TEOA/H2O (VTEOA/VH2O = 1: 9) solution without any cocatalysts modification under both UV-Vis light (λ ≥ 360 nm) and visible light (λ ≥ 420 nm) irradiation. No hydrogen was measured without trimers in the solution or under dark conditions. Large amounts of H2 were produced for all the trimers under UV and visible light illumination (Figure 4b-c, Table S3). Due to the electron donating effect of the alkyl group, TBT-3, TBT-4 and TBT-5 presented higher H2 production than TBT. TBT-3 displayed the best activity although it has larger band gap and blue-shifted absorption compared to TBT-4 and TBT-5, which was attributed to its better crystalline properties and smaller aggregation size. It should be noted that the activity of TBT-3 was comparable to other OCSs even without cocatalyst and residual Pd (Table S4). The photocatalytic stability of the TBT-3 was also tested, and no obvious change was observed during 25 h test (Figure 4d). FTIR analysis of the recycled TBT-3 revealed the high stability of its chemical structure and composition after photocatalytic tests (Figure S24). To further study the dynamics of photo-excited carriers of the aggregates, transient absorption spectroscopy (TAS) was carried out (Figure S25). Notably, the charge-transfer signal in TBT-3 was much more pronounced and persisted for a longer timescale than that in TBT, TBT-4 and TBT-5. The lifetime was estimated to be ∼1.2 ns, which possibly reflects the fewer structural defects in TBT-3 which effectively decelarate the charge trapping and recombination. These results demonstrated that the smaller size and higher crystallinity of TBT-3 enabled it with much enhanced charge carrier dynamics, which possibly promotes the light-driven electron transfer from aggregates to bulk solvent for H2 production. [15] Density functional theory (DFT) was further employed to provide molecular-level understanding on these trimers. Figure 5a showed the energy change (ΔE) of the aggregation process from separated molecules. Compared with TBT, TBT-4 and TBT-5, TBT-3 released the smallest amount of energy, suggesting that the TBT-3 had the lowest tendency to aggregate. In addition to the aggregates, the energy change for the formation of crystal structures from separated molecules was also determined (Figure S26). The ΔE of forming the crystal structures of TBT-3, TBT-4 and TBT-5 were -1.04 eV, -1.20 eV and -1.14 eV per molecule, respectively, consistent with the trend in Figure 5a. The smaller aggregate size of TBT-3 created a smaller reaction platform for HER, which facilitates the reaction on a larger specific surface area and shortens the transportation distance of photogenerated carriers to the surface of the aggregates. The decreased thermodynamic driving force for aggregation of TBT-3 was further confirmed by the lower densities of TBT-3 crystals (Figure 5d). In addition, the calculated HOMO-LUMO gaps are generally consistent with the optical gaps determined experimentally (Figure 5b). Despite the large HOMO-LUMO gap of TBT-3, TBT-3 exhibited a superior activity as indicated from its large density of electrons at the HOMO state (Figure S27). Electrons are mainly distributed around the S atoms of the thiophen-group and the N atoms of the thiadiazole-group at both ground and excited states, indicating the existence of intramolecular charge transfer (Figure 5c). In addition, there is an obvious structural deformation at the thiophen side groups after excitation due to the largest steric hindrance effect of TBT-3, which will also contribute to the charge transfer by forming a larger π-conjugated region. In summary, the decreased aggregation tendency of TBT-3 leads to a smaller aggregate size, and its reactive molecular orbitals strongly enhance the photocatalytic HER, which is responsible for the higher H2 production of TBT-3.

Conclusion

In summary, the aggregation size and crystallization properties of TBT were successfully regulated by incorporating and alternating the position of methyl side chains on thiophene units. The introduction of -CH3 at the 3-position of TBT not only increased the crystallinity, but also reduces the degree of agglomeration, which in turn reduced the distance for electrons to migrate to the surface and improved the charge transportation performance, resulting in an enhanced photocatalytic activity. These results demonstrated that the aggregation behavior and crystalline state of OSCs can be effectively tuned by the subtle manipulation of side chain, which opens a new avenue for the design of highperformance OCSs photocatalysts for solar fuels production.

Figure 1 .

 1 Figure 1. (a) Chemical structures of conjugated trimers and (b) Photographs of the trimer samples in THF/H2O/TEOA solution; (c) DLS measurement of trimer aggregates; (d) Normalized UV-Vis absorption spectra of catalysts dissolved in THF and photograph of catalysts in THF solution under ambient light (insert images);

Figure 2 .

 2 Figure 2. TEM images and element mappings of TBT (a-c), TBT-3 (d-f), TBT-4 (g-i) and TBT-5 (j-l); (m) Raman spectra for the trimers; (n) DSC measurement of the trimers; (o) the WAXS 2D pattern and (p) WAXS spectra for the asprepared trimers.

  77/-1.27 eV, 0.98/-1.34 eV, 0.93/-1.24 eV and 0.73/-1.26 eV, respectively, according to the equations: ELUMO = -e [Ered -E(Fc/Fc+) + 4.8] eV and EHOMO = Eg -ELUMO. All the trimers are thermodynamically suitable for driving the reduction of protons to H2 due to their more negative reduction potential than the reduction of proton. The smallest semicircle in a Nyquist plot for TBT-3 demonstrated a better charge transfer ability than TBT, TBT-4 and TBT-5 (Figure3e).

Figure 3 .

 3 Figure 3. (a) DRS absorption spectra of the samples measured in the solid state (intensities normalized); (b) Calculated band gaps from the Tauc plots; (c) Cyclic voltammetry plots (reduction scans) of trimers in acetonitrile solution and 0.1 M tetrabutylammonium perchlorate; (d) Experimentally determined HOMO and LUMO energy levels of samples; (e) Nyquist plot of TBT, TBT-3, TBT-4, and TBT-5.

Figure 4 .

 4 Figure 4. (a) Probable mechanism for the formation of microaggregates and the photocatalytic mechanism. (b) Time course of photocatalytic H2 generation for aggregates under UV-Vis and visible light (λ ≥ 420 nm) irradiation; (c) Normalized H2 production under UV-Vis light. Time-course of photocatalytic H2 generation for trimers under visible light and visible light; (d) H2 generation with cycling.

Figure 5 .

 5 Figure 5. (a) Energy change of aggregating different number of molecules. (b) Calculated HOMO-LUMO gap and the experimental determined gaps for the considered molecules. (c) Electron density distribution of TBT-3, TBT-4, TBT-5 and TBT at ground state and excited state (d) Optimized structures and corresponding densities of TBT-3, TBT-4, TBT-5 and TBT.

Acknowledgements

X. Yuan gratefully acknowledges the financial support for postdoctoral research by European Union's Horizon 2020 research and innovation program under the grant agreement No.800926 (HyPhOE). Catherine Denage (ICMCB-UMR5026) is acknowledged for the ICP-OES measurement. We acknowledge Sabrina Lacomme for the TEM measurements (Bordeaux Imaging Center-Imagerie Electronique, Université de Bordeaux, 33076 Bordeaux Cedex). We thank C. Labrugere (PLACAMAT) for the XPS measurement.

Entry for the Table of Contents

The relationship between the aggregation of TBT-based conjugated trimers and the photocatalytic activity was systematically investigated by methyl side-chain engineering. TBT-3 with the smallest size of aggregates and best crystalline, exhibit the best photocatalytic H2 evolution activity, which is due to the shorten charge carrier transport distance and solid long-range order.