Mansi Bakshi

Clarisse Liné

Diana E Bedolla

Ricardo José Stein

Ralf Kaegi

Géraldine Sarret

Ana Pradas

Del Real

Hiram Castillo-Michel

P C Abhilash

Camille Larue
email: camille.larue@ensat.fr

Ana E Pradas Del Real

Assessing the impacts of sewage sludge amendment containing nano-TiO2 on tomato plants: A life cycle study

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Engineered nanoparticles (ENPs, particle having at least one external dimension between 1-100 nm) have many industrial applications ranging from food packaging to remediation of groundwater, wastewater and soil [START_REF] Stark | Industrial Applications of Nanoparticles[END_REF][START_REF] Qu | Applications of Nanotechnology in Water and Wastewater Treatment[END_REF] . Among all ENPs, nano-TiO2 are one of the most produced alongside with Ag and ZnO [START_REF] Sun | Dynamic Probabilistic Modeling of Environmental Emissions of Engineered Nanomaterials[END_REF] . Their photocatalytic activity, high stability and anticorrosion properties make them suitable for a wide range of applications [START_REF] Shi | Titanium Dioxide Nanoparticles : A Review of Current Toxicological Data[END_REF][START_REF] Skocaj | Titanium Dioxide in Our Everyday Life; Is It Safe?[END_REF][START_REF] Riu | Nanosensors in Environmental Analysis[END_REF] . Nano-TiO2 can be used in cosmetics and skin care products like sun blocks, toothpastes, food, pharmaceuticals, paints, water treatment processes and cleaning air products [START_REF] Weir | Titanium Dioxide Nanoparticles in Food and Personal Care Products[END_REF][START_REF] Trouiller | Titanium Dioxide Nanoparticles Induce DNA Damage and Genetic Instability in Vivo in Mice[END_REF][START_REF] Ruffini Castiglione | The Effects of Nano-TiO2 on Seed Germination, Development and Mitosis of Root Tip Cells of Vicia Narbonensis L. and Zea Mays L[END_REF][START_REF] Kim | Characterization and Environmental Implications of Nano-and Larger TiO2 Particles in Sewage Sludge, and Soils Amended with Sewage Sludge[END_REF] . As bulk material (possibly containing a fraction of ENPs), TiO2 is widely used as a pigment [START_REF] Weir | Titanium Dioxide Nanoparticles in Food and Personal Care Products[END_REF] . With a constant rise in the production of ENPs, they will unavoidably make their way to the environment. Agricultural soil, in particular, is a very sensitive compartment and a main sink for ENPs [START_REF] Sun | Dynamic Probabilistic Modeling of Environmental Emissions of Engineered Nanomaterials[END_REF] . Two major pathways are responsible for ENP input into agricultural soil: firstly, ENPs contained in sewage sludge applied as fertilizer in agriculture results [START_REF] Suppan | Nanomaterials In Soil[END_REF][START_REF] Keller | Predicted Releases of Engineered Nanomaterials: From Global to Regional to Local[END_REF] . Indeed, many studies have already evidenced ENP presence in wastewater treatment plants [START_REF] Westerhoff | Occurrence and Removal of Titanium at Full Scale Wastewater Treatment Plants: Implications for TiO2 Nanomaterials[END_REF][START_REF] Kiser | Titanium Nanomaterial Removal and Release from Wastewater Treatment Plants[END_REF] and further in biosolids [START_REF] Kim | Characterization and Environmental Implications of Nano-and Larger TiO2 Particles in Sewage Sludge, and Soils Amended with Sewage Sludge[END_REF] . Secondly, the use of ENPs in plant protection products (nanoencapsulated pesticides, nanoformulations and nano-based carrier systems) leads to their intentional input in soil [START_REF] Ghormade | Perspectives for Nano-Biotechnology Enabled Protection and Nutrition of Plants[END_REF][START_REF] Gogos | Nanomaterials in Plant Protection and Fertilization : Current State , Foreseen Applications , and Research Priorities[END_REF][START_REF] De Oliveira | Application of Nanotechnology for the Encapsulation of Botanical Insecticides for Sustainable Agriculture: Prospects and Promises[END_REF][START_REF] Parisi | Agricultural Nanotechnologies: What Are the Current Possibilities?[END_REF] . A probabilistic material flow analysis predicted nano-TiO2, among other ENPs, to be present in the highest concentrations in the environment [START_REF] Sun | Dynamic Probabilistic Modeling of Environmental Emissions of Engineered Nanomaterials[END_REF] . In particular, the modelled concentration of nano-TiO2 in sewage sludge is predicted to be 1.60 g/kg while for sludge amended soil the concentration is estimated up to 61 mg Ti/kg. This study indicates that the concentrations of nano-TiO2 in biosolids and effluent treated soils have increased 30-40 times between 2005 and 2012 [START_REF] Sun | Dynamic Probabilistic Modeling of Environmental Emissions of Engineered Nanomaterials[END_REF] , highlighting the importance of studying the fate of nano-TiO2 in sewage sludge fertilized soil.

Sewage sludge is widely used in agricultural fields as an alternative to chemical fertilizers due to its high nutrient quality. This use of sewage sludge represents a highly sustainable strategy, since it addresses two important global challenges: efficient agriculture production thanks to inputs in micro and macronutrients and organic matter, as well as disposal of large quantities of waste. However, this entails the risk of toxicity and food chain contamination due to the presence of heavy metals and organic pollutants in the sewage sludge [START_REF] Jośko | The Influence of ZnO and TiO 2 Nanoparticles on the Toxicity of Sewage Sludges[END_REF] . The European Union (Directive 86/278/EEC) and the U.S.A. (Title 40 Code of Federal Regulations, Part 503) have legislations defining permissible limits for heavy metal in sewage sludge used for land application [START_REF] Yuan | Sewage Sludge-Based Functional Nanomaterials: Development and Applications[END_REF][START_REF] Inglezakis | European Union Legislation on Sewage Sludge Management[END_REF] . While ENPs are emerging as possible pollutant with unknown risks, no regulation exists so far for governing their release in the environment 22- 24 .

Despite the risk of food chain transfer and biomagnification through uptake, translocation and accumulation of ENPs in plants and the wide use of sewage sludge as amendments, there is limited research on the impact of ENPs on plants through application of sewage sludge amended soil. Recently, several studies have investigated the phytotoxicity of pristine nano-TiO2 on plants [START_REF] Ruffini Castiglione | The Effects of Nano-TiO2 on Seed Germination, Development and Mitosis of Root Tip Cells of Vicia Narbonensis L. and Zea Mays L[END_REF][START_REF] Clément | Toxicity of TiO2 Nanoparticles to Cladocerans, Algae, Rotifers and Plants -Effects of Size and Crystalline Structure[END_REF][START_REF] Raliya | TiO2 Nanoparticle Biosynthesis and Its Physiological Effect on Mung Bean (Vigna Radiata L.)[END_REF][START_REF] Gao | Effects of Nano-TiO2 on Photosynthetic Characteristics of Ulmus Elongata Seedlings[END_REF][START_REF] Fan | Effects of Nano-TiO2 on the Agronomically-Relevant Rhizobium-Legume Symbiosis[END_REF][START_REF] García-Sánchez | Early Response to Nanoparticles in the Arabidopsis Transcriptome Compromises Plant Defence and Root-Hair Development through Salicylic Acid Signalling[END_REF] . Du et al. (2011) [START_REF] Du | TiO2 and ZnO Nanoparticles Negatively Affect Wheat Growth and Soil Enzyme Activities in Agricultural Soil[END_REF] observed that nano-TiO2 decreased plant biomass in wheat and penetrated cell walls, plant epidermis via apoplastic pathway and inhibited soil enzyme activity. Servin et al. (2013) [START_REF] Servin | Synchrotron Micro-XRF and Micro-XANES Confirmation of the Uptake and Translocation of TiO 2 Nanoparticles in Cucumber ( Cucumis Sativus ) Plants[END_REF] reported root-to-fruit translocation of nano-TiO2 in cucumber grown on a spiked soil. More recently, we observed no acute phytotoxicity in terms of growth, biomass and chlorophyll concentration on wheat plants grown in nano-TiO2 spiked soil, but a significant Ti uptake in sandy soils [START_REF] Larue | Influence of Soil Type on TiO 2 Nanoparticle Fate in an Agro-Ecosystem[END_REF] . While some studies have explored the toxicity of nano-TiO2 on soil related organisms, studies investigating the impacts of nano-TiO2 resulting from sewage sludge applications on sewage sludge microbial communities, soil macro-organisms and plants are very few [START_REF] García | Effect of Cerium Dioxide, Titanium Dioxide, Silver, and Gold Nanoparticles on the Activity of Microbial Communities Intended in Wastewater Treatment[END_REF][START_REF] Chen | Toxicogenomic Responses of the Model Legume Medicago Truncatula to Aged Biosolids Containing a Mixture of Nanomaterials (TiO₂, Ag, and ZnO) from a Pilot Wastewater Treatment Plant[END_REF][START_REF] Chen | Effects of Biosolids from a Wastewater Treatment Plant Receiving Manufactured Nanomaterials on Medicago Truncatula and Associated Soil Microbial Communities at Low Nanomaterial Concentrations[END_REF] . Thus, the predicted increase in nano-TiO2 production volumes [START_REF] Robichaud | Estimates of Upper Bounds and Trends in Nano-TiO 2 Production As a Basis for Exposure Assessment[END_REF] warrants further investigations on fate of nano-TiO2 in sewage sludge and associated implications for food safety.

The objective of the present study was to develop a realistic exposure scenario simulating the growth of edible plants on agricultural soil treated with sewage sludge containing nano-TiO2.

Sewage sludge was produced in a pilot wastewater treatment plant treating municipal wastewater at the Swiss Federal Institute of Aquatic Science and Technology (EAWAG, Dubendorf, Switzerland). Monitoring the entire life cycle of tomato plants grown on this sewage sludge amended soil, we investigated the impact on (i) plant growth and development (ii) biochemical responses (iii) changes in biomacromolecules (lipids, proteins, carbohydrates) using Fourier Transformed Infra-Red spectroscopy (FTIR) (iv) effect on plant elemental concentrations and (v) Ti accumulation by inductively coupled plasma-optical emission spectrometry (ICP-OES).

Materials and Methods

Soil and sewage sludge description

The sewage sludge was produced in a pilot wastewater treatment plant at EAWAG [START_REF] Kaegi | Behavior of Metallic Silver Nanoparticles in a Pilot Wastewater Treatment Plant[END_REF] . This sludge comes from the Dubendorf city, a suburban town of Zurich in Switzerland. During a previous experiment, nano X-ray fluorescence (XRF) performed on ID16 beamline at the European synchrotron radiation facility (ESRF) evidenced the presence of micro-and nano-TiO2 in the sewage sludge (26% ENPs) [START_REF] Elena | Searching for Relevant Criteria to Distinguish Natural vs[END_REF] . X ray absorption spectroscopy (XANES) performed on ID21 (ESRF) showed that Ti was present in the sludge as 55 ± 1% anatase and 45 ± 2% rutile.

The top layer (0-30 cm) of a loamy soil (38% sand, 42% silt and 20 % clay) was collected at La Côte Saint-André (Isère, France) from a field under permanent pasture to serve as "control soil". Control soil contained 37 ± 2% anatase, 49 ± 7% rutile and 14 ± 6% amorphous TiO2 [START_REF] Elena | Searching for Relevant Criteria to Distinguish Natural vs[END_REF] .

Both sludge and soil were previously characterized [START_REF] Del Real | Fate of Ag-NPs in Sewage Sludge after Application on Agricultural Soils[END_REF] and the main physico-chemical properties are given in supplementary information (SI). A mixture of soil and sewage sludge (w/w) was prepared in the ratio of 1:10 (sludge: soil). This was considered as "sludge amended soil". In this condition, 20% of the Ti was in nanoparticulate form (<100 nm) and about 50% in the 100-300 nm range [START_REF] Elena | Searching for Relevant Criteria to Distinguish Natural vs[END_REF] .

Plant exposure

Twelve experimental units were filled with 200 g of sludge amended soil or control soil (six biological replicates per condition). Seeds of tomato plants (Solanum lycopersicum, var. Red Robin, provider: Germinance) were sown and grown until the ripening of tomato fruits with watering ad libitum. This tomato variety was selected because of its small size (dwarf variety) and short life cycle. All experiments were carried out in a growth chamber under controlled conditions: day/night photoperiod (16/8 h), 100 μmol/m²/s, day/night temperature (24/20±1 °C), and day/night relative humidity (70/75%). The development parameters (number of leaves, flowers, fruits, plant height) were monitored throughout the whole plant life cycle.

Chlorophyll level was assessed everyday (SPAD-502 chlorophyll meter, Minolta Camera Co., Japan). After 4 months (120 days), plants were harvested, fresh biomass was recorded and plants were split in three. Fresh leaves for biochemical analyses were weighed, immediately frozen in liquid nitrogen and stored at -80°C until analysis. The remaining leaves along with stems and fruits were dried and ground for determination of elemental concentration by ICP-OES. Finally, for the assessment of biomacromolecular changes by FTIR the dried samples were further grinded using a Fast Prep® grinder.

Analysis of biochemical responses

Tomato leaves were analysed for six different biomarkers. Three biochemical responses were investigated which were oxidative stress (lipid peroxidation), photosynthesis (chlorophyll a, b) and secondary metabolites (phenolic compounds, tannins and flavonoids). Lipid peroxidation was assessed according to Hodges et al. (1999) [START_REF] Hodges | Improving the Thiobarbituric Acid-Reactive-Substances Assay for Estimating Lipid Peroxidation in Plant Tissues Containing Anthocyanin and Other Interfering Compounds[END_REF] . Chlorophyll a and b concentrations were determined by the method given by Lichtenthaler (1987) 41 . Phenolic compounds were estimated following Ainsworth and Gillespie (2007) [START_REF] Ainsworth | Estimation of Total Phenolic Content and Other Oxidation Substrates in Plant Tissues Using Folin-Ciocalteu Reagent[END_REF] . Flavonoids were determined according to Settharaksa et al. ( 2014) [START_REF] Settharaksa | Effect of Solvent Types on Phenolic, Flavonoid Contents and Antioxidant Activities of Syzygium Gratum (Wight) S.N[END_REF] and tannins following El Euch et al.

(2015) [START_REF] Euch | Chemical Composition, Biological and Cytotoxic Activities of Cistus Salviifolius Flower Buds and Leaves Extracts[END_REF] . Detailed methods are described in SI.

FTIR analysis

FTIR was used to probe molecular vibrations in plant samples and thus gain information on the biochemical composition (lipids, carbohydrates, proteins). Dried plant stems, leaves and fruits were analysed on ID21 beamline at ESRF. Each powdered sample was analysed in ATRmode using a diamond crystal (Thermo Nicolet, Nexus, Smart Orbit). Three independent technical replicates from each sample (6 biological replicates per treatment) were acquired.

One spectrum is an average of 64 scans per sample, over a range of 4000 to 400 cm -1 and with a spectral resolution of 4 cm -1 . The full experimental setup for FTIR acquisition is given in SI. OMNIC software was used to export experimental spectra.

FTIR data treatment was performed using Orange software [START_REF] Demšar | Orange: Data Mining Toolbox in Python[END_REF] . Briefly, data were preprocessed which implies selection of the region of interest (here 1755 -455 cm -1 range which contained most of the variability by looking at the average and standard deviation of the absorbance), vector normalization and smoothing by Savitzky-Golay filter. Principal component analyses (PCA) were performed to visualize the data in a new coordinate space optimized for detection of differences between groups. Second derivative was performed to highlight spectral differences detected using PCA.

Elemental concentration in soil, sewage sludge and plants

ICP-OES was used to measure Ti and other micro and macro-elements in plant leaves, stems and fruits as well as in soils according to Larue et al. ( 2018) [START_REF] Larue | Influence of Soil Type on TiO 2 Nanoparticle Fate in an Agro-Ecosystem[END_REF] . Blank (only chemicals) and standard reference material (NIST 1573a, tomato leaves) were used as controls in the digestion process.

Soil exchangeable and extractable Ti concentrations were determined through BaCl2 and HCl extractions, respectively. Briefly, for soil exchangeable elements, 3 g of soil were mixed with 10 mL of unbuffered 0.01 M BaCl2, shaken horizontally at 150 rpm overnight at room temperature. For extractable elements, 1 g of dry soil was extracted with 10 ml of 0.1M HCl, shaken horizontally at 150 rpm for 30 min at room temperature. Soil mixtures were then filtered through Whatman filter paper, dried and digested before analysis. Analyses were performed on an ICP-OES Spectro Arcos (Ametek).

Statistical analysis

Data reported were averages of six biological replicates ± standard deviations (SD). Data were checked for normality (Shapiro's test) and homoscedacity (Bartlett's test). When assumptions were met for parametric analyses, a Student t-test was used. Otherwise, a Wilcoxon test was applied. In all cases the statistical significance was based on a p value < 0.05. Additionally, ICP-OES results were analysed using PCA to identify different profiles. All statistical analyses were performed using the R statistical software (version 3.1.3) [START_REF] Team | A Language and Environment for Statistical Computing[END_REF] and vegan package [START_REF] Oksanen | Community Ecology Package. R package version[END_REF] for PCA.

Results and Discussion

Titanium concentration in soil

Titanium concentration for the sludge amended soil was 819 ± 62 mg/kg and for control soil 801 ± 102 mg/kg (Figure S1 for other element concentrations). The exchangeable Ti concentration for control soil and sludge amended soil was 0.004% and 0.006%, respectively.

The extractable soil fraction for both soils was 0.08%. No significant difference was found between the control and sludge amended soil for the total, extractable and exchangeable Ti (p>0.05).

Despite the fact that the control soil and sludge amended soil had similar Ti concentrations, Ti origin was different for both. While the control soil had geogenic Ti concentration from natural soil, the sludge amended soil had 9/10 natural Ti and 1/10 coming from the sludge which was most probably engineered TiO2. Those two Ti forms might have different behaviour in the environment and different toxicity potentials. Indeed, natural TiO2 has a rough surface and is included in organo-mineral soil aggregates (with Si, Al, Fe), whereas TiO2 observed in the sludge is composed of homo and heteroaggregates dominated by organic matter [START_REF] Elena | Searching for Relevant Criteria to Distinguish Natural vs[END_REF] .

Properties such as organic matter content and soil texture (clay content) have been cited as important parameters controlling ENP behaviour in soil, influencing their mobility [START_REF] Fang | Stability of Titania Nanoparticles in Soil Suspensions and Transport in Saturated Homogeneous Soil Columns[END_REF] . The presence of high organic matter content in the soil (8.8%) and in the sludge amended soil (10.9%) thus is a probable explanation for low fractions of both exchangeable and extractable Ti in the two media. Also, the agricultural soil was rich in clay fraction (20%) which further elucidates our results. Indeed, in our recent work investigating TiO2 ENP fate in 4 different types of soil (from sandy to clayey); clay content and organic carbon were found to influence the transfer of Ti, with the soil with highest clay content (42%) displaying no ENP mobility compared to sand and silty sand soils [START_REF] Larue | Influence of Soil Type on TiO 2 Nanoparticle Fate in an Agro-Ecosystem[END_REF] . Likewise, Gogos et al. (2016) [START_REF] Gogos | Vertical Transport and Plant Uptake of Nanoparticles in a Soil Mesocosm Experiment[END_REF] reported that leached Ti amount in a brown earth soil (sandy loamy to loamy fine fraction) was found to be 10 -4 % of the initial spiked Ti amount even at the highest concentration of 1000 mg/kg.

Plant growth and developmental parameters:

Plants grown in sewage sludge amended soil significantly increased biomass production for all organs (stem: +92 %, leaves: + 142% and fruits: + 102 %, p<0.050) as well as the number of leaves (20 per plant for sludge amended soil vs. 15 for control soil: +33%, p=0.047) and flowers (23 per plant in amended soil vs. 10 for control soil: +130%, p=0.012) compared to plants grown in the control soil (Figure 1A,B). Fruit yield was also significantly increased with an average of 4.4 fruits per plant amounting for 21.50 g in the sludge amended soil and 2.2 fruits per plant for a total weight of 10.63 g in the control soil (p=0.010 for fruit number and p=0.005 for fruit biomass). The average plant height for sludge amended soil was 20.68 ± 1.42 cm which was 43% higher than plants grown in the control soil (14.46 ± 0.45 cm, p=0.012, data not shown). The monitoring of developmental parameters through the entire plant life cycle showed that after an average of 50 days of experiment, plants in the amended soil started exhibiting greater development compared to the control plants (Figure S2A,B,C).

In this study, we found that sewage sludge amendment containing nano-TiO2 stimulated plant growth and tomato yield. Sewage sludge was rich in organic matter and contained substantial amount of nutrients. Total carbon, nitrogen, phosphorus and sulphur concentrations in sludge amended soil were multiplied by 3.4, 6.8, 1.8 and 23.6, respectively in comparison with control soil, thereby contributing to the fertilizer effect. The fact that sewage sludge application is an effective mean for improvement of plant vegetative growth and soil properties has been widely reported [START_REF] Singh | Potential Benefits and Risks of Land Application of Sewage Sludge[END_REF][START_REF] Koutroubas | Growth, Grain Yield and Nitrogen Use Efficiency of Mediterranean Wheat in Soils Amended with Municipal Sewage Sludge[END_REF][START_REF] Kołodziej | Use of Sewage Sludge in Bioenergy Production-A Case Study on the Effects on Sorghum Biomass Production[END_REF] . Some studies in the literature have explored the phytotoxicity of nano-TiO2 using developmental parameters with contrasting results. nano-TiO2 were found to induce longer roots in cucumber plants at a concentration of 750 mg/L 31 as well as increased germination rates in Arabidopsis thaliana seedlings (500 mg/L) [START_REF] Tumburu | Phenotypic and Genomic Responses to Titanium Dioxide and Cerium Oxide Nanoparticles in Arabidopsis Germinants[END_REF] . However, Song et al. (2013a) [START_REF] Song | Functional Analysis of TiO2 Nanoparticle Toxicity in Three Plant Species[END_REF] found no effects of nano-TiO2 treatment on oilseed rape, lettuce and kidney bean at concentration range of 100 to 5000 mg/L. More recently, we evidenced no impact on wheat seedlings grown on different types of soils after exposure to 500 mg/kg [START_REF] Larue | Influence of Soil Type on TiO 2 Nanoparticle Fate in an Agro-Ecosystem[END_REF] . Finally, in a sewage sludge study, Josko and Oleszczuk (2013) [START_REF] Jośko | The Influence of ZnO and TiO 2 Nanoparticles on the Toxicity of Sewage Sludges[END_REF] reported that inclusion of 10 mg/kg TiO2 ENPs in sewage sludge amended sandy soil inhibited root growth in plants Lepidium sativum and Sinapis alba.

Figure 1: (A) Plant fresh biomass for stem, leaves and fruits and (B) number of leaves, flowers and fruits for tomato plants grown for 120 days on control soil and sludge amended soil.

Asterisks represent significant difference (p<0.05) as indicated by Student T test (n=6).

Still it is unclear how TiO2 ENPs affect plants; it seems that in most studies there was no clear dose-response effects and that those effects are modulated by the soil properties. However, bulk Ti has been considered as a biostimulant for a long time and has been used in commercial foliar fertilizers for improving plant growth and development [START_REF] Lyu | Titanium as a Beneficial Element for Crop Production[END_REF] . For instance, use g Ti /ha [START_REF] Kleiber | TYTANIT[END_REF] . In our study we concluded that the overall improved yield of tomato plants after sewage sludge amendment was attributed to the nutritive value of sludge, while we cannot be certain on the positive or negative effects of nano-TiO2. .

Biochemical responses

No significant difference was observed in leaves for all the biomarkers related with secondary metabolites except tannins (Table 1). Leaf tannins significantly decreased from 3.09 ± 0.95 in control to 1.77 ± 0.13 mg CE/g f.wt in plants exposed to sewage sludge (p=0.022). Flavonoid concentration in control soil was 13.24 ± 5.04 and in sludge amended soil 12.02 ± 0.23 mg CE/g f.wt. The concentration for phenolic compounds for control soil and sludge amended soil was 10.54 ±4.48 and 10.79 ± 3.56 µg GAE/g f.wt. respectively.

Among secondary metabolites, tannins, flavonoids and phenolic compounds are widely distributed in plants, and play a prominent role in general defence strategies (against herbivores, microbes, viruses, or competing plants), as well as contributing to food quality [START_REF] Swanson | Encyclopedia of Food Sciences and Nutrition[END_REF] .

Usually, a high tannin concentration favours plant growth in poor soil, in which the same plant species with a low tannin concentration could not grow [START_REF] Hässig | Flavonoids and Tannins: Plant-Based Antioxidants with Vitamin Character[END_REF][START_REF] Salunkhe | Dietary Tannins: Consequences and Remedies[END_REF] . A decrease in secondary metabolites might weaken the defence system of the plants and be a threat to plant survival in a more competitive environment (vs. in growth chamber). So far a very limited number of studies have investigated the impact of ENPs in general, and nano-TiO2 in particular, on secondary metabolites. For photosynthesis assessment, SPAD chlorophyll meter measurements recorded along plant growth revealed no major difference in chlorophyll content (data not shown). This result was further confirmed by assessing photosynthetic pigments in plant leaves at the end of plant exposure: the average concentration of chlorophyll a and b was 1.53 ± 0.52 (p=0.520) and 0.97 ± 0.34 mg/g f.wt. (p=0.623) respectively. Previous studies have also reported similar results where chlorophyll content in wheat [START_REF] Larue | Accumulation, Translocation and Impact of TiO2 Nanoparticles in Wheat (Triticum Aestivum Spp.): Influence of Diameter and Crystal Phase[END_REF] [START_REF] Koce | Oxidative Potential of Ultraviolet-A Irradiated or Nonirradiated Suspensions of Titanium Dioxide or Silicon Dioxide Nanoparticles on Allium Cepa Roots[END_REF] found no significant effect on anti-oxidative enzymes (glutathione reductase, ascorbate peroxidase, guaiacol peroxidase, catalase) and lipid peroxidation levels in Allium cepa in a 24 hr study using a wide range of nano-TiO2 concentration (0.1-1000 mg/L).

MDA is the ultimate product of lipid peroxidation damage caused by generation of free radicals. Flavonoids and tannins are reported to exert inhibition of lipid peroxidation and scavenging of oxygen radicals [START_REF] Hässig | Flavonoids and Tannins: Plant-Based Antioxidants with Vitamin Character[END_REF] . Therefore, a decrease in tannin and flavonoid concentrations and a subsequent increase in MDA content might be indicative of a correlation between decreasing scavenging activity and increasing reactive oxygen species (ROS) concentration.

Tomato plants might thus have gone through a moderate oxidative stress during plant growth as suggested by the significant decreased in tannin concentration and trend in increased MDA content (however, not significant upon harvest). The lack of biochemical response might also be associated with the duration of exposure. Indeed, a longer contact duration has been found to favour interactions between ENPs and sewage sludge components (e.g. organic matter) thereby reducing their bioavailability (and toxicity) through adsorption or aggregation [START_REF] Jośko | The Influence of ZnO and TiO 2 Nanoparticles on the Toxicity of Sewage Sludges[END_REF][START_REF] Chen | Effect of Natural Organic Matter on Toxicity and Reactivity of Nano-Scale Zero-Valent Iron[END_REF] . Also it is possible that initially plants might have experienced stress but later adapted to it such that no significant difference was observed later. Dosing of ROS and antioxidative enzymes during exposure would be interesting to further understand implied processes.

Impact on plant biomacromolecules: FTIR analysis

In this study, FTIR was used to identify changes in specific functional groups in tomato plants grown in control soil and sludge amended soil (Figure 2A, B, C and Figure S3). During data preprocessing, on the absorbance spectra, it was noted that most of the variability observed in the average spectra of each group arose from the carbohydrate/protein/aromatic/saccharide region (1755-455 cm -1 ), while the PCA on the lipid region (between 3000 and 2800 cm -1 ) failed to separate the groups in this region and the region in-between (2800-1755 cm -1 ) was mainly background noise.

We thus selected the range from 1755 to 455 cm -1 , also known as fingerprint region, for further processing. After PCA treatment, PC1 (62%) separated clusters corresponding to the stems, leaves and fruits, while on PC4 (5%) data segregated clearly in two groups (control vs.

sludge amended soil) for leaf samples, to a lesser extent for stem samples but not for fruits (Figure 2A). These results highlight thus no impact of the sewage sludge treatment on the biomacromolecules of the edible part of tomato. Also, when comparing the average absorbance of control and sludge amended soil for the fruits (Figure S3), there is no clear difference between the spectra, as reflected in the PCA analysis. Excluding the fruits from the analysis, leaves and stems were further investigated to identify the main spectral differences.

As previously mentioned, the major difference between control and sludge was found in the leaves. These differences were highlighted with PC1 explaining 79% of the variance between leaf samples, and changes were evidenced in the second derivative (Figure 2B). The majority of the differences were associated with spectral bands of carbohydrate (900-1200 cm -1 ) attributed to C-O stretching vibrations and OH deformation vibrations. Differences were also detected in the region 1480-1180 cm -1 where CH2 wagging vibrations occur that may correspond to aromatic and ring vibrations, followed by changes in the amide, phenolic and aromatic band contributions (1700-1500 cm -1 ) [START_REF]Infrared and Raman Characteristic Group Frequencies: Tables and Charts, 3rd Edition[END_REF] where compounds such as lignin or tannin (that are mainly aromatic) could be highly contributing. Indeed, the band at 1515 cm -1 is quite characteristic of lignin [START_REF] Servin | Synchrotron Verification of TiO 2 Accumulation in Cucumber Fruit: A Possible Pathway of TiO 2 Nanoparticle Transfer from Soil into the Food Chain[END_REF] . In particular in leaves, the relative intensities of bands 1029, 1050, 1075 and 1144 cm -1 mainly characteristic of carbohydrates were increased in sludge plants while bands for lignin (666, 760, 780, 1260, 1315, 1370, 1416, 1515, 1540, 1606 cm -1 ) were stronger in control plants.

The spectral region 1200-1610 cm -1 contains many contributions that are characteristic of tannins 66 and lignin [START_REF] Liu | Study on Biodegradation Process of Lignin by FTIR and DSC[END_REF] . This result is in agreement with the decrease of tannins observed when measuring biochemical biomarkers.

In stems, the differences were significantly smaller (Figure 2C), to separate the control from the sludge condition, it was necessary to look into the loadings of the PC4 explaining 10.6%

of the variance of the samples. The major differences occured in the amide, aromatic regions (1680-1500 cm -1 ) and in the 1500-1300 cm -1 region, typical of polysaccharides and cellulose.

A decrease at 1417 and 1372 cm -1 and a slight shift from 1614 to 1620 cm -1 was observed in the sludge condition. This decrease may also be reflecting the changes in concentration of tannin and lignin but in a less evident manner. Indeed, when looking at the band at 1515 cm - 1 , no apparent change is observed.

With all these observations in mind, we can state that fruits were not affected by the exposure to the sewage sludge treatment, while some changes were observable in the stems and leaves. Changes are in the same spectral regions, but the differences are greater in the leaves.

Those results imply that impact of TiO2 ENP contamination on food nutritional value of leafy crop should be investigated more closely. Additionally, impact on fruits cannot be excluded since a study from Servin et al. ( 2013) [START_REF] Servin | Synchrotron Verification of TiO 2 Accumulation in Cucumber Fruit: A Possible Pathway of TiO 2 Nanoparticle Transfer from Soil into the Food Chain[END_REF] evidenced changes in cucumber fruits upon TiO2 ENP exposure.

Effect on plant elemental concentrations

Plant macro (S, Ca, Mg, P, K) and microelements (Al, B, Cu, Fe, Mn, Na, Si, Zn) were determined in the different organs, to assess the impact of sewage sludge amendment on plant ionome (Figure S4A,B,C). Tomato leaves were again found to be the most impacted plant organ which is in line with the above discussed FTIR results.

Sewage sludge amendment significantly increased the translocation (concentration in the leaves/concentration in the soil) of Fe, B, and Na in all plant organs (stem, leaves, fruits) while S, P increased only in leaves and stem (Figure 3A, B, C). Mn and Si translocation was also consistently lower in sewage sludge exposed plants. Those elements are of primary importance for plant development. For instance, B is an essential micronutrient influencing good seed set and fruit development as it plays a vital role in growth of the pollen tube during flower pollination [START_REF] Jehangir | Micronutrients for Crop Production: Role of Boron[END_REF] . Na is a functional nutrient regulating stomatal opening and closing [START_REF] Pilon-Smits | Physiological Functions of Beneficial Elements[END_REF] .

Sulfur is also a key component of defense compounds cysteine, glutathione (GSH) and phytochelatins (PCs) where it appears in the sulfhydryl (-SH or thiol) group. These thiol compounds synthesised in plants under metal stress represent a very important detoxification pathways for plant tolerance and survival [START_REF] Rausch | Sulfur Metabolism: A Versatile Platform for Launching Defence Operations[END_REF] . Also, according to Amtmann and Armengaud (2009) [START_REF] Amtmann | Effects of N , P , K and S on Metabolism : New Knowledge Gained from Multi-Level Analysis[END_REF] changes in nutrient content can impact the overall plant nutritional quality and taste of crops.

Studies in the literature report both sewage sludge 72 and Ti as stimulants of increased nutrient absorptions in plants [START_REF] Kleiber | TYTANIT[END_REF] . While it is likely that the increase in many elements in plants is due to the effect of the sludge, it can partly also be associated with the presence of TiO2 functioning as promoter of plant nutrition. In particular, TiO2 ENPs have been reported to increase P uptake in several studies on different plant species (lettuce [START_REF] Zahra | Metallic Nanoparticle (TiO2 and Fe3O4) Application Modifies Rhizosphere Phosphorus Availability and Uptake by Lactuca Sativa[END_REF] , wheat [START_REF] Rafique | Dose-Dependent Physiological Responses of Triticum Aestivum L. to Soil Applied TiO 2 Nanoparticles: Alterations in Chlorophyll Content, H 2 O 2 Production, and Genotoxicity[END_REF] , tomato [START_REF] Tiwari | Nanotitania Exposure Causes Alterations in Physiological, Nutritional and Stress Responses in Tomato (Solanum Lycopersicum)[END_REF] ). Likewise, Ti has also been associated with enhancement of Fe uptake [START_REF] Lyu | Titanium as a Beneficial Element for Crop Production[END_REF] . In a hydroponic study of two weeks, nano-TiO2

(concentration of 0.5 to 2 g/L) also significantly increased S, Mg and Fe contents in tomato leaves and roots [START_REF] Tiwari | Nanotitania Exposure Causes Alterations in Physiological, Nutritional and Stress Responses in Tomato (Solanum Lycopersicum)[END_REF] . Similarly, application of Ti in the form of Tytanit® foliar fertilizer increased the N, Ca, and Mg content of tomato plants [START_REF] Kleiber | TYTANIT[END_REF] . The decrease in Mn concentration after TiO2 ENP application has also been evidenced before [START_REF] Jacob | Uptake and Translocation of Ti from Nanoparticles in Crops and Wetland Plants[END_REF] .

Therefore, we assume the elemental impacts to be a cumulative effect of both sludge and nano-TiO2. More studies are needed to distinguish the sludge from the ENP effect, for example by spiking different concentrations of TiO2 ENPs in the sewage water before treatment.

Presence of Ti in plant parts

Ti concentrations were 1.19 ± 0.51 and 1.03 ± 0.35 mg/kg in stems, 2.59 ± 1.62 and 1.14 ± 0.31 mg/kg in leaves, and 2.31 ± 1.67 and 0.88 ± 0.30 mg/kg in fruits for plants grown on control soil and sludge amended soil, respectively (Figure 4). While Ti concentration was similar for both soils Ti uptake was reduced in plant organs in sludge amended soil with significant reduction in leaves (p=0.032). Further µXRF analysis was performed to investigate Ti distribution in leaf cross sections (Figure S5). However, Ti was below the detection limit in both samples (description of the experimental set-up and results in SI).

Our results are in agreement with the literature that has reported low uptake of Ti based ENPs The lower translocation of Ti in the leaves of plants grown on sewage sludge amended soil might be due to the presence of a significantly higher amount of organic matter (+24%) consisting of functional groups that readily form complexes with ENPs, compared to the control soil, thereby possibly, reducing the bioavailability of Ti. Indeed, studies have reported that pH and organic matter directly influence the bioavailability of ENPs in soil [START_REF] Pachapur | Behavior and Characterization of Titanium Dioxide and Silver Nanoparticles in Soils[END_REF] . Similarly, we evidenced that Ti exhibited no significant root internalization and no upward translocation to leaves in wheat plants grown in soils with organic matter content above 1.5%.

So far there is no defined range for deficiency, sufficiency or toxicity for Ti in plants [START_REF] Kužel | The Effect of Simultaneous Magnesium Application on the Biological Effects of Titanium[END_REF] .

However, Kabata-Pendias and Pendias (2001) [START_REF] Kabata-Pendias | Trace Elements in Soils and Plants[END_REF] suggested that Ti content ranging from 50 to 200 mg kg -1 in mature leaves of plants could be excessive or toxic while Lyu et al. (2017) [START_REF] Lyu | Titanium as a Beneficial Element for Crop Production[END_REF] proposed a Ti threshold of 50 mg kg -1 in leaf tissues. So, based on our results, Ti concentrations in plant parts were far lower than these proposed concentrations, thus suggesting no risk of toxicity.

Another issue to consider is that nano-TiO2 exhibit a high potential to adsorb toxic metals on their surfaces as well as nutrients [START_REF] El-Deen | Immobilisation of TiO 2 -Nanoparticles on Sewage Sludge and Their Adsorption for Cadmium Removal from Aqueous Solutions[END_REF][START_REF] Kim | Characterization and Environmental Implications of Nano-and Larger TiO2 Particles in Sewage Sludge, and Soils Amended with Sewage Sludge[END_REF][START_REF] Thomas | Synthesis of Ag Doped Nano TiO2 as Efficient Solar Photocatalyst for the Degradation of Endosulfan[END_REF] . Over the years, repeated applications of nano-TiO2 through sewage sludge might lead to the release of metals sorbed on ENP surface into the environment becoming readily available for crop plants [START_REF] Kim | Characterization and Environmental Implications of Nano-and Larger TiO2 Particles in Sewage Sludge, and Soils Amended with Sewage Sludge[END_REF] and the decreased bioavailability of nutrients such as Ca and Fe [START_REF] Larue | Innovative Combination of Spectroscopic Techniques to Reveal Nanoparticle Fate in a Crop Plant[END_REF] . According to Hartmann and Baun (2010) [START_REF] Hartmann | The Nano Cocktail: Ecotoxicological Effects of Engineered Nanoparticles in Chemical Mixtures[END_REF] the interaction between ENPs and toxic compounds in environmental mixtures can either amplify or alleviate toxicity. But it is still unclear whether the co-contamination of ENPs and metals would lead to an enhance toxicity of the mixture (Trojan horse effect) or rather a decreased one.

Our results indicate that sewage sludge amendment containing nano-TiO2 improved the plant yield due to its high organic matter and nutrient content and did not lead to significant changes in the edible part of tomato. However, over time nano-TiO2 in sewage sludge is expected to increase while organic matter content may decrease if the sludge application stops. This may affect Ti availability in the long run. Another aspect to take into consideration is the impact of such amendment on soil bacterial communities along time.

  a Ti based foliar fertilizer, significantly improved the fruit yield of tomato at 960

Figure 2 :

 2 Figure 2: (A) PCA including FTIR spectra from all samples (leaves, stems, fruits) of tomato

Figure 3 :

 3 Figure 3: Principal component analysis (PCA) based on elemental translocation factors

  from soil.Du et al. (2011) [START_REF] Du | TiO2 and ZnO Nanoparticles Negatively Affect Wheat Growth and Soil Enzyme Activities in Agricultural Soil[END_REF] in their study of 7 months on wheat found no significant difference in the Ti content of wheat grain between nano-TiO2 treated soil (10 g/kg TiO2) and control soil classified as loamy clay. Burke et al. (2014)[START_REF] Burke | Titanium Oxide Nanoparticle Effects on Composition of Soil Microbial Communities and Plant Performance[END_REF] also found very low levels of Ti (<2.5 mg/kg) in the above ground tissue of soybean and maize plants after six week exposure to 200 mg/kg TiO2 ENPs.

Figure 4 :

 4 Figure 4: Ti concentrations in in the stems, leaves and fruits for tomato plants grown for 120

Table 1 : Values for flavonoids, phenolic compounds, tannins, chlorophylls a, b and malondialdehyde (MDA) in leaves of tomato plants grown for 120 days on control and sludge amended soils. Asterisks represent significant difference (p<0.05) as indicated by Student T test

 1

	Biomarker	Control soil	Sludge amended soil
	Flavonoids (mg CE/g f.wt.)	13.24 ± 5.04	12.02 ± 0.23
	Phenolic compounds (ug GAE/g f.wt.)	10.54 ±4.48	10.79 ± 3.56
	Tannins (mg CE/g f.wt.)	3.09 ± 0.95	1.77 ± 0.13*
	Chlorophyll a (mg/g f.wt.)	1.37 ± 0.31	1.62 ± 0.62
	Chlorophyll b (mg/g f.wt.)	0. 89 ± 0.20	1.20 ± 0.40
	MDA (nmol/g f.wt.)	3.84 ± 2.43	4.86 ± 1.64

(n=6). CE: catechin equivalent, GAE: gallic acid equivalent

Acknowledgements

We acknowledge ESRF for provision of synchrotron radiation facilities and we would like to thank the staff of ID21 for assistance in using beamline. Mansi Bakshi is thankful to University Grant Commission, New Delhi for awarding UGC-NET-SRF. Authors would also like to thank David Baqué, R&D Engineer on the ICP platform of EcoLab, for operating the ICP-OES instrument. This project received funding from the Toulouse Tech InterLab programme under project name SPECPLANP.


Lalli

Alphonsine Ngo Ndimba

T Roisnel

G Argouarch

C Lalli

Harvesting New Chiral Phosphotriesters by Phosphorylation of BINOL and Parent bis-Phenols

Keywords: chiral phosphotriesters, chiral phosphates, BINOL, bis-phenols, phosphorylation

Asymmetric catalysis represents one of the most efficient way to prepare enantiopure building blocks to be exploited in modern industry for the synthesis of pharmaceuticals, agrochemicals, polymers, and even new materials with different properties. [START_REF] Jacobsen | Comprehensive Asymmetric catalysis[END_REF] To achieve this goal big efforts have been done in the synthesis of libraries of chiral catalysts and ligands for metal coordination. Among the plethora of chiral backbones 1,1'-bi-2-naphthol (BINOL) and the partially hydrogenated H8-BINOL are widely used in the development of chiral catalysts and ligands for a huge number of catalytic asymmetric transformations. 2 In this field, chiral phosphorus compounds have proved to be an ubiquitous class of catalysts, as evidenced by many reviews, [START_REF]Phosphorus(III) Ligands in Homogeneous Catalysis. Design and Synthesis[END_REF] and phosphorus-based BINOL architectures such as phosphanes, 4 phosphites, 4b phosphoric acids or phosphate metal salts, 5 and phosphoramidites 6 have undoubtedly a place of honor in asymmetric catalysis.

On the other hand, pure phosphotriesters (phosphates) are surprisingly much less used in transition metal catalysis, as only three examples were reported to date. 7 Miura's group investigated the Pd/phosphate-catalyzed oxidative coupling between arylboronic acids and alkynes, 7a while the Zn/triphenylphosphate (TPP)-catalyzed hydrosilylation of ketones together with the Ti/bisphosphate-promoted diethylzinc addition to aldehydes were reported by us. 7b Moreover only two examples of chiral phosphates appeared as organocatalysts. 8 Ishihara's group exploited chiral BINOLderived triaryl phosphates as nucleophilic catalysts for the Niodolactonisation of 4-arylmethyl-4-pentenoic acids. 8a Luo and co-workers reported a latent concept for asymmetric carbocation catalysis with chiral BINOL-derived trityl phosphate, and its application to Friedel-Crafts, inverse electron-demanding hetero-Diels-Alder, and carbonyl-ene reactions. 8b In continuation with our interest in organocatalytic enantioselective transformations 9 and in transition metal catalysis, 10 we took in consideration the possibility to further exploit the Lewis base P=O coordination site of P(V) compounds by designing novel chiral (S)-BINOL-derived mono-and bisphosphotriester architectures such as A and B, respectively (Figure 1). In the literature, beyond the phosphorylation of biological molecules, 11 there are several available methods to synthesize phosphate esters starting from phosphoric acids derivatives, 12 which are summarized in Figure 2. The access to the target compounds can be direct through either esterification in the presence of different additives (eq. 1, Figure 2), 12a-d or alkylation with alkyl halides (eq. 2, Figure 2). 12e Alternatively the phosphotriesters can be synthesized via a stepwise procedure involving the formation of the corresponding P(O)Cl followed by Template for SYNTHESIS © Thieme Stuttgart • New York 2020-11-25 page its nucleophilic substitution in the presence of alcohols (eq. 3, Figure 2). 12f-h

Figure 2 Typical procedures for the synthesis of phosphates.

In line with our preliminary report, 7b we started to investigate the reactivity of (S)-BINOL ( 1) and (S)-H8-BINOL (2) with different chlorophosphates in the presence of a base and without additives. It has to be pointed out that, compared to what has been previously described in the literature, this represents the first study dedicated to the systematic study on the phosphorylation of BINOL and analogous bis-phenol-based structures that showed an unexpected reactivity ascribable to the combination of two different parameters: the hydroxyl group acidity on one hand, and the leaving group nature within the phosphorylating agent of general formula R 1 R 2 P(O)Cl on the other hand.

First of all we wondered how to selectively obtain monophosphates of general formula A (Figure 1). Without any surprise they were readily obtained by reaction between precursors 1-4 and dichlorophosphates. Thus phosphotriesters 6, 13 7, 8 [START_REF] Panmand | Compound 8 has already been described[END_REF] and 9 [START_REF] Voropai | Compound 9 has already been described[END_REF] were isolated in very good yields, from 96% to > 99%, by reacting (S)-BINOL ( 1), (S)-H8-BINOL (2), [START_REF] Pousse | For the synthesis of[END_REF] 2,2'biphenol (3) and 1,8-dihydroxynaphthalene (4) respectively with the commercially available phenyl dichlorophosphate (5) in basic conditions (Scheme 1). The structures of compounds 8 and 9 have been unambiguously confirmed by single crystal Xray analysis (Figure 3). 17 We next attempted the esterification of the commercially available diphenyl phosphoryl chloride (10) with 1-4 in order to synthesize bisphosphates of general formula B (Figure 1). To this end we let (S)-BINOL (1) react with an excess of 10 in the presence of Et3N as proton scavenger (Scheme 2). Surprisingly the desired compound was not recovered from the reaction mixture but monophosphate 6 was again isolated in 90% yield, together with a reasonable amount of triphenyl phosphate (TPP), arising from the trapping of the excess of 10 by the phenol released in the course of the reaction. From 3 and 4 we similarly obtained the corresponding monophosphates 8 and 9, in 95% and 89% yields respectively, along with TPP. In parallel we investigated the reaction between (S)-H8-BINOL (2) and 10 under the same reaction conditions and we were surprised to find out that this time the expected bisphosphate 11 could be isolated in 84% yield.

Template for SYNTHESIS © Thieme Stuttgart • New York 2020-11-25 page

Scheme 2 Synthesis of phosphates 6, 8, 9 and 11 in the presence of diphenyl phosphoryl chloride (10).

Which reaction pathways leading to cyclic monophosphates 6, 8 or 9 from 1, 3 or 4 and to the bisphosphate 11 from (S)-H8-BINOL are To answer this in a more way and to gain insights into the different behaviors of the bisphenols, we decided bring light on their phosphorylation reactions operated by chlorophosphates of general formula R 1 R 2 P(O)Cl.

To this point we went back to the literature and we found out that it was possible to obtain the bisphosphate 13 in 88% yield by phosphorylation of 1 with the commercially available diethyl chlorophosphate (12) in the presence of NaH (Scheme 3). 18 When we tried to change NaH for Et3N a complex mixture was obtained, from which the starting materials, the desired bisphosphate 13 and compound 14 as the major product were isolated. The structure of 14 has been confirmed by single crystal X-ray analysis (Figure 4). 17 Additionally 3 was phosphorylated with 12 to give 52% of bisphosphate 15 19 as the major product, together with the opened monophosphate form 16, following again a trend close to that of BINOL. The analysis of these sets of experiments indicated that the acidity of the protons on the phenolic counter-parts, together with the nucleofuge character of the OR groups installed on the chlorophosphates, have an influence on the reaction outcome leading to a competitive mono-vs bisphosphate formation.

If we try to compare the estimated pKa values of the four compounds we used, we can deduce a scale of decreasing acidity going from 2,2'-biphenol 20 and 1,8-dihydroxynaphthalene, through BINOL, 21 to H8-BINOL, 3≈4<1<2, as depicted in Figure 5. By virtue of this, we suppose that the reaction pathway goes through intermediate C and that in the case of 1, 3 and 4, even with an excess of diphenyl phosphoryl chloride (10), the intramolecular cyclisation to deliver the monophosphate of type A, with the release of a molecule of PhOH, is faster than the coupling with a second molecule of 10, i.e. the displacement of the chloride on a second chlorophosphate (Scheme 4). This principle is not valid for (S)-H8-BINOL (2) which possesses much less acidic phenolic protons, therefore the limit in the acidity scale is reached and in this case only the substitution of chlorides onto the P(V) can take place leading to a bisphosphate of type B (compound 11 on Scheme 2). The formation of the bisphosphates of type B (together with the opened monophosphates 14 and 16) is favored in the case of diethyl chlorophosphate (12) because the phosphorous atom in C' is less electrophilic with respect to 12 and also the nucleofuge character is lowered for the ethoxy group. In other words, by establishing the acidity threshold of the substrates and the nucleofugality scale 22 Cl ->PhO ->EtO -, the reactivity of different diols with chlorophosphates can be modulated in order to guide the reaction towards the preferential formation of mono-or bis phosphotriesters.

Template for SYNTHESIS © Thieme Stuttgart • New York 2020-11-25 page

Scheme 4 Reaction pathway for the strongest acidic substrates in the presence of diphenyl chlorophosphate (10).

With these results in hand we decided to further explore the scope and limitations of such esterification reactions and in particular the access to new chiral bisphosphates. In the phosphorylation reactions below, the idea was to use phosphoryl chlorides whose structures couldn't preclude the reaction to evolve toward the formation of bisphosphates, contrary to diphenyl phosphoryl chloride (10). We started with the synthesis of symmetric architectures; the reaction of (S)-BINOL ( 1) with an excess of its phosphorochloridate 17 23 in the presence of Et3N smoothly led to the formation of the tris-BINOL bisphosphate 18 (BINOPHAT) in 83% yield (Scheme 5). The structure of the new BINOPHAT 18 has been confirmed by single crystal X-ray analysis (Figure 6). 17 Similarly when (S)-H8-BINOL (2) was reacted with the corresponding phosphorochloridate 19 [START_REF] Furuno | For the synthesis of H8-BINOL phosphorochloridate 19[END_REF] in the presence a stronger base such as NaH to offset the lower acidity of 2, the tris-H8-BINOL bisphosphate 20 (H8-BINOPHAT) was obtained in 88% yield. Finally the same strategy was successfully applied to the synthesis of the previously reported bisphosphate 22, 7b which was obtained in 72% yield starting from 3 and its corresponding chlorophosphate 21.

Scheme 5 Improved phosphorylations leading to single motif bisphosphates. We wondered next if it was possible to obtain symmetrical nonhomo-BINOL bisphosphates, incorporating a non-chiral central unit such as 2,2'-biphenol (3) or 1,8-dihydroxynaphthalene (4). Thus we carried out the reaction between 3 or 4 and an excess of 17 and we were surprised to find out that these combinations readily led to the formation of the unsymmetrical bisphosphates 23 (73%) and 24 (56%) respectively, bearing one BINOL motif in the side position and the other as the central core of the molecules (Scheme 6), as confirmed by single crystal X-ray analysis of 24 (Figure 7). 17 As seen above, replacement of 10 by phosphoryl chlorides 17 and 19 in their reactions with BINOL and other closely related structures allowed us to prepare original bisphosphate architectures. Nonetheless, the question remains of whether the bisphosphate that was expected in place of 6 by the reaction between (S)-BINOL ( 1) and diphenyl phosphoryl chloride ( 10) is accessible. To answer this, we turned our attention towards the synthesis and oxidation of the corresponding phosphite (Scheme 7). According to this method, pre-mixing in basic conditions PCl3 with PhOH delivers the corresponding diphenyl phosphorochloridite (PhO)2PCl, that in turn reacts with (S)-BINOL ( 1) to give the desired bisphosphite 25. [START_REF]For the synthesis of the bisphosphite intermediate 25[END_REF] In this case the intramolecular cyclisation can't take place due to the much less electrophilic character of P(III), therefore 25 is formed exclusively and undergoes in situ oxidation, in mild conditions with a 5% NaOCl aqueous solution, to afford the corresponding phosphotriester 26 in 74% yield. The structure of the new bisphosphate 26 has been confirmed by single crystal X-ray analysis (Figure 8). 17 In conclusion we have reported the phosphorylation of BINOL and other bis-phenols with chlorophosphates and observed an unexpected reactivity due to the pKa of hydroxyl group of four different bis-phenols associated with the leaving group nucleofugal behavior of six different chlorophosphates.

Monophosphotriesters can be selectively obtained by reaction of the four bis-phenols 1-4 with phenyl dichlorophosphate (5), and diphenyl phosphoryl chloride (10) with only one exception for (S)-H8-BINOL (2) which delivers a new bisphosphate 11. This represents thus the threshold in the acidity scale to switch from mono-to bis-phosphotriesters. Bisphosphates 13 and 15 were instead selectively obtained by reaction of 1 and 3 respectively with diethyl chlorophosphate (12), this time under control of the EtO -nucleofuge behavior. New symmetrical homo-BINOL bisphosphates have been obtained in very good yields by reaction of (S)-BINOL ( 1) or (S)-H8-BINOL (2) with their chlorophosphates, while unsymmetrical non-homo-BINOL ones, incorporating a non-chiral side unit, were synthesized from 2,2'-biphenol (3) or 1,8-dihydroxynaphthalene (4) in the presence of 17. A plausible reaction pathway has been proposed based on acidity and nucleofugality scales. Finally a different route to bisphosphate 26 through in situ oxidation of its bisphosphite has been evaluated to overcome the previous methodology limits. Further researches on the substrate scope and investigations of those new chiral Lewis bases as ligands in enantioselective metal-based catalytic transformations, in line with our research interests are underway and will be reported in due course.

All the reactions were performed in dried glassware, under argon atmosphere, and sealed with a rubber septum. Reagents were obtained from commercial suppliers and used without further purification unless otherwise noted. TLC analyses were performed using precoated Merck TLC Silica Gel 60 F254 plates. Purifications by column chromatography on silica gel were performed using Merck Silica Gel 60 (0,040-0,063 nm). Petroleum ether (PE) used for purifications was the low boiling point fraction (40-60 °C). 1 H and [START_REF] Legrand | Compound 6 has already been synthesized[END_REF] C spectra were recorded on a Bruker Avance 300 instruments using TMS and CDCl3 respectively as internal standard. 31 P NMR spectra were recorded on a Bruker DMX 500. Chemical shifts (δ) are reported in parts per million (ppm) relatively to TMS and residual solvent as internal standards. The following abbreviations are used for multiplicities: s, singlet; d, doublet; t, triplet; dd, doublet of doublets; td, triplet of doublets; m, multiplet. Coupling constants (J) are reported in Hertz (Hz). HRMS analyses were obtained using a Waters Q-TOF 2 or a Micromass ZABSpec TOF or a Bruker Micro-TOF Q II or a LTQ Orbitrap XL instrument for ESI. X-ray crystallographic data were collected on a D8 Venture or a APEXII Bruker AXS diffractometers at 150 K. Optical rotations were recorded on a Perkin Elmer Model 341 polarimeter. Melting points were obtained on a hot bench. IR spectra were recorded on a Perkin Elmer FT-IR Spectrometer UATR Spectrum Two.

General procedure for the synthesis of monophosphates 6, 8, and 9 from phenyl dichlorophosphate (5): Under an argon atmosphere, to a solution of 1, 3, or 4 (1 eq) and Et3N (3 eq) in dry CH2Cl2 (0.15 M), 5 (1.2 eq) was added drop by drop at 0 °C. The mixture was stirred at room temperature overnight, then hydrolyzed with H2O and extracted with CH2Cl2. The organic phase was washed with an aqueous 1N HCl solution and with H2O, then dried over anhydrous MgSO4. The solvent was removed under vacuum and the residue was purified by column chromatography on silica gel using a mixture of petroleum ether/ethyl acetate as eluent. X-ray single quality crystals of compounds 8 and 9 were grown by slow diffusion of pentane in CH2Cl2 solution.

Synthesis of monophosphate 7 with phenyl dichlorophosphate (5):

Under an argon atmosphere, to a solution of 2 (250 mg, 1 eq) in dry THF (0.3 M), NaH (2.5 eq) was added at 0 °C and the mixture was stirred at

Template for SYNTHESIS © Thieme Stuttgart • New York 2020-11-25 page this temperature for 30 min before the addition of 5 (1.2 eq) drop by drop at 0 °C. The mixture was stirred at room temperature for an additional 30 min, then hydrolyzed with H2O and extracted with CH2Cl2.

The organic phase was washed with an aqueous 1N HCl solution and with H2O, then dried over anhydrous MgSO4. The solvent was removed under vacuum and the residue was purified by column chromatography on silica gel using a mixture of petroleum ether/ethyl acetate 7/3 as eluent, to give a white solid, 369 mg, 96% yield. M.p. 89-91 °C; [α] 20 D +75 (c = 1.0, CHCl3); IR (neat, cm -1 ) 2923, 2854, 1589, 1488, 1300, 1190, 1159, 1008, 947, 812, 751 General procedure for the synthesis of monophosphates 6, 8, 9, and 11 with diphenyl phosphoryl chloride (10): Under an argon atmosphere, to a solution of 1-4 (1 eq) and Et3N (3 eq) in dry CH2Cl2 (0.15 M), 10 (3 eq) was added drop by drop at 0 °C. The mixture was stirred at room temperature overnight, then hydrolyzed with H2O and extracted with CH2Cl2. The organic phase was washed with an aqueous 1N HCl solution and with H2O, then dried over anhydrous MgSO4. The solvent was removed under vacuum and the residue was purified by column chromatography on silica gel using a mixture of petroleum ether/ethyl acetate as eluent.

Compound 11: from 2 (590 mg, 1 eq), purified by column chromatography on silica gel using a mixture of petroleum ether/ethyl acetate 7/3 as eluent, to give a colorless oil, 1.28 g, 84% yield. 134.6, 129.9 (d, JCP = 0.6 Hz), 129.8, 129.7, 126.8 (d, JCP = 8.6 Hz), 125.4 (d, JCP = 0.9 Hz), 125.2 (d, JCP = 0.9 Hz), 120.1 (d, JCP = 4.9 Hz), 120.0 (d,JCP = 4.9 Hz),116.4 (d,JCP = 1.9 Hz),29.5,27.3,22.8,22.6 ppm;31 P NMR (202 MHz, CDCl3) δ = -18.40 ppm.

General procedure for the synthesis of bisphosphates 13 and 15 with diethyl chlorophosphate (12): Under an argon atmosphere, to a solution of 1 or 3 (1 eq) in dry THF (0.3 M), NaH (2.5 eq) was added at 0 °C and the mixture was stirred at this temperature for 30 min before the addition of 12 (2.1 eq). The mixture was stirred at room temperature for an additional 30 min, then hydrolyzed with H2O and extracted with CH2Cl2. The organic phase was washed with an aqueous 1N HCl solution and with H2O, then dried over anhydrous MgSO4. The solvent was removed under vacuum and the residue was purified by column chromatography on silica gel using a mixture of petroleum ether/ethyl acetate as eluent.

Compound 13:

[α] 20 D -2.2 (c = 1.2, CHCl3) vs -5.1 (c = 0.85, CHCl3) described.

Compound 15: from 3 (500 mg, 1 eq), purified by column chromatography on silica gel using a mixture of petroleum ether/ethyl acetate 2/3 as eluent, to give a colorless oil, 640 mg, 52% yield. IR (neat, cm -1 ) 3495, 2984, 1477, 1440, 1270, 1208, 1021, 926, 761; ESI-HRMS calculated for C20H28O8NaP2 [M+Na] + 481.1151, found 481.1154; 1 H NMR (300 MHz, CDCl3) δ = 7. 46-7.43 (m, 2H), 7.37-7.33 (m, 4H), 7.24-7.19 (m, 2H), 3.95-3.84 (m, 8H), 1.17 (t, J = 7.2 Hz, 12H) ppm; [START_REF] Legrand | Compound 6 has already been synthesized[END_REF] C NMR (75 MHz, CDCl3) δ = 148.3 (d, JCP = 6.7 Hz), 131.8, 129.3, 129.2, 124.6, 119.6 (d, JCP = 1.5 Hz), 64.4 (d, JCP = 6.7 Hz), 16.0 (d, JCP = 6.7 Hz) ppm; 31 P NMR (202 MHz, CDCl3) δ = -7.16 ppm.

General procedure for the synthesis of bisphosphates 18, 23, and 24: Under an argon atmosphere, to a solution of 1, 3, or 4 (1 eq), and (S)-1,1'-Binaphthyl-2,2'-diyl phosphorochloridate 17 (3 eq) in dry CH2Cl2 (0.15 M), Et3N (3 eq) was added drop by drop. The mixture was stirred at room temperature overnight, then hydrolyzed with H2O and extracted with CH2Cl2. The organic phase was washed with an aqueous 1N HCl solution and with H2O, then dried over anhydrous MgSO4. The solvent was removed under vacuum and the residue was purified by column chromatography on silica gel using a mixture of petroleum ether/ethyl acetate as eluent.

Compound 18: from 1 (200 mg, 1 eq), purified by column chromatography on silica gel using a mixture of petroleum ether/ethyl acetate 7/ 1, 130.9, 130.8, 130.0, 128.8, 128.3, 128.2, 127.4, 127.2, 126.9, 126.6, 126.5, 126.0, 125.9, 125.8, 125.7, 121.6 (d 1, 130.9, 130.8, 130.4, 130.0, 129.9 (d, JCP = 5.8 Hz), 129.4 (d, JCP = 2.5 Hz), 128.7, 128.5, 128.2, 128.0, 127.7 (d, JCP = 1.2 Hz), 127.6 (d, JCP = 1.2 Hz), 127.3 (d, JCP = 5.3 Hz), 127.2, 127.0, 126.7, 126.5, 126.2, 126.1, 125.8, 125.7 (d, JCP = 6.0 Hz), 125.7, 121.9 (d, JCP = 7.9 Hz), 121.3 (d, JCP = 2.3 Hz), 121.0 (d, JCP = 4.4 Hz), 120.8, 120.7, 120.5 (d, JCP = 2.9 Hz), 120.4 (d, JCP = 2.3 Hz), 119.5, 119.2 (d, JCP = 3.2 Hz), 118.4 ppm; 31 P NMR (202 MHz, CDCl3) δ = -3.46, -5.28 ppm.

Compound 24: from 4 (80 mg, 1 eq), purified by column chromatography on silica gel using a mixture of petroleum ether/ethyl acetate 7/3 as eluent, to give a white solid, 230 mg, 56% yield. M.p. 171-173 °C; [α] 20 D +112 (c = 1.2, CHCl3); IR (neat, cm -1 ) 3059, 1614, 1590, 1506, 1463, 1321, 1296, 1260, 1208, 1188, 966, 950, 899, 884, 811, 747; ESI-HRMS calculated for C50H31O8P2 [M+H] + 821.1488, found 821.1496; 1 H NMR (300 MHz, CDCl3) δ = 8.00-7.89 (m, 3H), 7.75 (d, J = 8.1 Hz, 1H), 7.65-7.53 (m, 6H), 7.48-7.40 (m, 3H), 7.36-7.22 (m, 9H), 7.13 (d, J = 8.4 Hz, 1H), 7.05-6.89 (m, 4H), 6.19-6.15 (m, 3H) ppm; [START_REF] Legrand | Compound 6 has already been synthesized[END_REF] C NMR (75 MHz, CDCl3) δ = 147.0 (d, JCP = 11.5 Hz), 146.3 (d, JCP = 8.7 Hz), 145.9, 145.8, 145.7, 145.6 (d, JCP = 6.5 Hz), 134.5, 133.1, 132.7, 132.2, 132.0, 131.9 (d, JCP = 1.0 Hz), 131.6 (d, JCP = 1.0 Hz), 131.4, 131.0, 130.7, 130.2 (d, JCP = 4.0 Hz), 128.8, 128.6, 128.2, 127.8, 127.3, 127.2, 127.0, 127.0, 126.9, 126.8 (d, JCP = 2.6 Hz), 126.1, 125.9, 125.8, 125.6, 125.4, 123.3 (d, JCP = 3.4 Hz), 121.4 (d, JCP = 2.2 Hz), 120.9 (d, JCP = 4.4 Hz), 120.8, 120.7 (d, JCP = 3.1 Hz), 120.6 (d, JCP = 2.2 Hz), 119.5 (d, JCP = 3.3 Hz), 118.8, 118.6, 112.7, 112.5, 112.4, 112.0 (d, JCP = 9.3 Hz) ppm; 31 P NMR (202 MHz, CDCl3) δ = -3.55, -

Figure 1

 1 Figure 1 New chiral BINOL-derived phosphotriester general structures.

Scheme 1

 1 Scheme 1 Synthesis of monophosphates 6-9 from phenyl dichlorophosphate (5).

Figure 3

 3 Figure 3 ORTEP representations of compounds 8 (left) and 9 (right) at 50% thermal ellipsoids. Hydrogen atoms are omitted for clarity.

Scheme 3

 3 Scheme 3 Synthesis of phosphates 13 and 15 in the presence of diethyl chlorophosphate (12).

Figure 4

 4 Figure 4 ORTEP representation of compound 14 at 50% thermal ellipsoids. Hydrogen atoms are omitted for clarity.

Figure 5

 5 Figure 5 Estimated scale of acidity of the different substrates.

Figure 6

 6 Figure 6 ORTEP representation of compound 18 at 50% thermal ellipsoids. Hydrogen atoms are omitted for clarity.

  These results corroborate the above hypothesis of a two-step reaction mechanism (Scheme 4) involving a transient species of type D which, owing to the strong acidity of 4 in conjunction with the nucleophilicity of the BINOL naphtholate, rearranges into an intermediate of type E. The latter then reacts with the excess of 17 to generate the final product. Template for SYNTHESIS © Thieme Stuttgart • New York 2020-11-25 page

Figure 7

 7 Figure 7 ORTEP representation of compound 24 at 50% thermal ellipsoids. Hydrogen atoms and one molecule of pentane are omitted for clarity.

Scheme 7 .

 7 Scheme 7 . Straightforward preparation of chiral bisphosphate 26 via in situ oxidation of bisphosphite 25.

Figure 8

 8 Figure 8 ORTEP representation of compound 26 at 50% thermal ellipsoids. Hydrogen atoms are omitted clarity.

  ; ESI-HRMS calculated for C26H26O4P [M+H] + 433.1563, found 433.1564;1 H NMR (300 MHz, 2H), 7.26 (d, J = 7.2 Hz, 2H), 3H), 7.10 (d, J = 8.4 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H), 2.83-2.79 (m, 4H), 2.71-2.64 (m, 2H), 2.31-2.25 (m, 2H), 1.80-1.78 (m, 6H), 1.61-1.51 (m, 2H) ppm; 13 C NMR (75 MHz, CDCl3) δ = 150.6 (d, JCP = 6.2 Hz), 147.0 (d, JCP = 11.0 Hz), 145.4 (d, JCP = 7.7 Hz), 138.8 (d, JCP = 1.5 Hz), 138.7 (d, JCP = 1.5 Hz), 136.4 (d, JCP = 2.2 Hz), 135.9 (d, JCP = 2.1 Hz), 130.6 (d, JCP = 1.4 Hz), 130.1 (d, JCP = 1.3 Hz), 129.9, 126.0 (d, JCP = 1.7 Hz), 125.9 (d, JCP = 1.9 Hz), 125.5, 120.1 (d, JCP = 5.0 Hz), 118.7 (d, JCP = 3.5 Hz), 117.9 (d, JCP = 3.9 Hz), 29.3, 29.2, 28.1, 28.0, 22.6, 22.5, 22.4, 22.3 ppm; 31 P NMR (202 MHz, CDCl3) δ = -5.24 ppm.

  [α][20][21][22][23][START_REF] Furuno | For the synthesis of H8-BINOL phosphorochloridate 19[END_REF][START_REF]For the synthesis of the bisphosphite intermediate 25[END_REF] CHCl3); IR (neat, cm-1 ) 2925, 1588, 1484, 1297, 1184, 1159, 1008, 938, 752, 686; ESI-HRMS calculated for C44H40O8P2Na [M+Na] + 781.2090, found 781.2088; 1 H NMR (300 MHz, CDCl3) δ = 7.32 (d, J = 8.4 Hz, 2H), 7.21-7.19 (m, 8H), 7.14-7.04 (m, 6H), 7.02-6.97 (m, 4H), 6.84-6.81 (m, 4H), 2.78-2.57 (m, 4H), 2.30 (td, J = 17.1, 6.0 Hz, 2H), 2.05 (dt, J = 17.1, 5.7 Hz, 2H), 1.63-1.50 (m, 8H) ppm; 13 C NMR (75 MHz, CDCl3) δ = 150.5 (d, JCP = 4.1 Hz), 150.4 (d, JCP = 4.1 Hz), 146.0 (d, JCP = 6.5 Hz), 137.9,

  18 (d, J = 9.0 Hz, 1H), 6.12 (d, J = 8.1 Hz, 1H) ppm; 13 C NMR (75 MHz, CDCl3) δ = 147.3 (d, JCP = 9.0 Hz), 147.2 (d, JCP = 9.0 Hz), 146.9 (d, JCP = 11.2 Hz), 146.2 (d, JCP = 6.0 Hz), 146.2, 146.0 (d, JCP = 6.0 Hz), 145.9, 133.3 (d, JCP = 9.8 Hz), 132.2 (d, JCP = 0.9 Hz), 131.9 (d, JCP = 0.9 Hz), 131.8, 131.5 (d, JCP = 1.0 Hz), 131.2, 131.

  , JCP = 8.1 Hz), 121.1 (d, JCP = 2.2 Hz), 120.4 (d, JCP = 2.2 Hz), 120.2 (d, JCP = 2.8 Hz), 119.3 (d, JCP = 3.2 Hz), 119.2 ppm; 31 P NMR (202 MHz, CDCl3) δ = -3.58 ppm. Crystals suitable for X-ray diffraction study were grown by slow diffusion of a CH2Cl2 solution layered with pentane.

	Compound 23: from 3 (85 mg, 1 eq), purified by column
	chromatography on silica gel using a mixture of petroleum ether/ethyl
	acetate 7/3 as eluent, to give a white solid, 160 mg, 73% yield. M.p. 165-
	167 °C; [α] 20 D +141 (c = 1.1, CHCl3); IR (neat, cm -1 ) 3059, 1589, 1505,
	1475, 1433, 1304, 1212, 1187, 1000, 965, 951, 886, 747; ESI-HRMS
	calculated for C52H32O8P2Na [M+Na] + 869.1464, found 869.1464; 1 H NMR
	(300 MHz, CDCl3) δ = 7.95 (d, J = 9.3 Hz, 1H), 7.87-7.77 (m, 6H), 7.54-7.39
	(m, 7H), 7.35-7.30 (m, 3H), 7.23-7.06 (m, 11H), 7.01 (td, J = 8.1, 0.9 Hz,
	1H), 6.55 (dt, J = 8.1, 1.2 Hz, 1H), 6.

Scheme 6 Synthesis of unsymmetrical chiral bisphosphates 23 and 24 and plausible reaction pathway.

Acknowledgment

The authors thank Université de Rennes 1, CNRS, Rennes Métropole and Région Bretagne for financial support. The CRMPO of the Institut des Sciences Chimiques de Rennes for mass measurement, the Centre de Diffractométrie X for X-ray analyses and FEDER funds for acquisition of single crystal equipment are gratefully acknowledged. Part of this work has been performed using the PRISM core facility (Biogenouest©, UMS Biosit, Université de Rennes 35000 RENNES, FRANCE). Solenn Ferron is finally acknowledged for running 31 P NMR experiments.

Template for SYNTHESIS © Thieme Stuttgart • New York 2020-11-25 page

24.47 ppm. X-ray single quality crystals were grown by slow diffusion of Et2O in CDCl3 solution.

Synthesis of bisphosphate 20: Under an argon atmosphere, to a solution of 2 (150 mg, 1 eq) in dry THF (0.3 M), NaH (2.5 eq) was added at 0 °C and the mixture was stirred at this temperature for 30 min before the addition of (S)-H8-BINOL phosphorochloridate 19 (2.1 eq). The mixture was stirred at room temperature for an additional 30 min, then hydrolyzed with H2O and extracted with CH2Cl2. The organic phase was washed with an aqueous 1N HCl solution and with H2O, then dried over anhydrous MgSO4. The solvent was removed under vacuum and the residue was purified by column chromatography on silica gel using a mixture of petroleum ether/ethyl acetate 7/3 to 1/1 as eluent, to give a white solid, 438 mg, 88% yield. M.

Synthesis of bisphosphates 26:

Under an argon atmosphere, to a solution of PCl3 (1 eq) in toluene (0.1 M), phenol (1,07 g, 2 eq) and Et3N

(5 eq) were added at 0 °C. The mixture was stirred at 0 °C for 30 min before the addition of 1 (820 mg, 0.5 eq). The reaction was stirred at room temperature for 1 h, then the mixture was filtered over a pad of celite and the solvent was removed under vacuum. The crude was dissolved in EtOAc, washed with H2O, an aqueous 5% NaOCl solution and brine, then dried over anhydrous MgSO4. The solvent was removed under vacuum and the residue was purified by column chromatography on silica gel using a mixture of petroleum ether/ethyl acetate 3/1 as eluent to give a white solid, 1. 6, 131.2, 130.5, 129.7, 129.5, 128.1, 127.3, 126.2, 125.8, 125.4

Supporting Information

YES


Maya Marinova

Antoine Bonnefont

Thierry Achard

Aline Maisse-François

Stéphane Bellemin-Laponnaz

Chiral stimuli-responsive metallo-supramolecular assembly induced by Cu II

We investigated the selective formation of homoleptic and heteroleptic metal complexes controlled by means of the chiral molecular instruction of the ligand and the coordination geometry of the metal. Our results showed that chiral self-recognition or self-discrimination may be induced by Cu I /Cu II redox transition using cyclic voltammetry. The further use of chiral ditopic ligands led to metallo-supramolecular copolymers with stimuli-responsive controlled arrangement.

Chirality is the molecular instruction encoded in the chemical structure itself, which allows nature to perform most biological functions essential for life. [START_REF] Campbell | Biology: Concepts and Connections[END_REF] Chirality enables high recognition processes, through intermolecular noncovalent interactions, that are crucial for example to create protein functions or to store genetic information. Understanding of chiral recognition encouraged synthetic chemists to develop simplified molecular systems with the purpose of mimicking biology and developing smart molecular materials. Consequently, supramolecular chiral self-assembly is now considered as the most attractive and promising area of supramolecular chemistry. 2 A property of a complex biological system may be the result of the interplay of simpler integrated parts subjected to dynamic reversible interactions and that can respond to a stimulus. 3 Therefore, the mimic of chirality-induced biological selfassembly should also incorporate the dynamic nature inherent in non-covalent interactions that can evolve to generate a particular function, especially under the action of an external stimulus. Supramolecular chiral recognition between enantiomeric molecules can lead to self-discrimination or self-recognition to generate pairs of enantiomers that are homochiral or heterochiral entities, respectively. The recognition will govern the outcome of the self-assembling process. This assembly route that links molecular and supramolecular chirality is unidirectional and occurs under thermodynamic control. 4 Contrary to molecular chirality, supramolecular chirality can easily be influenced by external parameters (solvents, temperature, light, electrical bias, etc.) that could provide the opportunity to modulate the self-assembly and thus modify the equilibrium state of the system. 5 We show here that the self-assembly of two chiral C2-symmetric ligands around a metal may be electrochemically controlled. This strategy can lead to the generation of metallopolymers in which the arrangement of the structural unit of the polymer may be controlled by the oxidation state of the metal. The switching from alternating metallopolymers to block metallopolymers is electrochemically possible in both directions (Figure 1). The heteroformation or homoformation of ML2-type complexes may be controlled by the chiral molecular 'instruction' of the ligand in combination with the prevalent coordination geometry of the metal. 6,7 Chiral neutral C2symmetric bisoxazoline 1 in its racemic form spontaneously generates heterochiral complexes with tetrahedral Zn II 2 (Figure 2 We anticipated that self-recognition or self-discrimination in this system could be induced under the action of electrochemical signal akin to molecular machines induced by Cu I /Cu II redox transition, as described by Sauvage et al. 9,10 Indeed, Cu I , d 10 , is a metal ion that adopts geometry only determined by sterics and should be isomorph to what observed here with zinc. [START_REF]The preferential formation of heterotopic heterochiral Cu I complexes was confirmed by mass spectrometry experiments using the bisoxazoline ligand (R-1) and the S-bisoxazoline functionalized with a methyl-pyrene moiety (S-1pyr, see ESI for details)[END_REF] Thus, the homochiral/heterochiral interconversion could be triggered electrochemically by changing the oxidation state of Cu atom. 12 The cyclic voltammogram (CV) of the homochiral enantiopure complex [Cu(R-1)2](BF4)2 is displayed in Figure 3 (blue curve).

Starting from positive potential, the reduction wave of Cu II (R-1)2 into Cu I (R-1)2., denoted c1, is first observed at -0.05 V.

Upon the reverse scan, the generated Cu I (R-1)2 species is oxidized at ca. 0.01 V (oxidation wave denoted a1 in Figure 3). The Cu II /Cu I redox transition is reversible, suggesting that no reorganization of the coordination Cu(I) complex occurs on time scale of the experiment. Similar features are obtained for the CVs of the homochiral enantiopure complex [Cu(S-1)2](BF4)2 (see figure S7 in the ESI).

Next, the reduction wave of the racemic mixture of 1 mM homochiral Cu( 1)2(BF4)2 3 (red curve in Figure 3) occurs at the same potential and with approximately the same intensity as for the homochiral enantiopure Cu II complexes. However, during the backward scan, two oxidation waves, separated by 350 mV are observed. The first oxidation wave at 0.01 V, denoted a1', corresponds to the oxidation of the metastable homochiral Cu I (R-1)2 and Cu I (S-1)2 complexes and exhibits much weaker intensity compared to the a1 peak of the homochiral enantiopure complexes. The second oxidation wave, denoted a2, appears at higher potential (i.e. +0.360 V) and is attributed to the presence of more stable heterochiral [Cu (S-1)(R-1)] species resulting from the homochiral/heterochiral interconversion triggered by the reduction of Cu II into Cu I . The rate of the reorganization of the coordination shell of the Cu I species can be estimated from the evolution of the shape of the CV as a function of the potential sweep rate (Fig. 4a). 13 Above 1000 mV s -1 only the peak a1 was observed in the CV of the racemic mixture of homochiral Cu II complex. This indicates that the characteristic time of the Cu II /Cu I reduction/oxidation process at this sweep rate (less than 1 s) is too short to allow the reorganization of coordination shell and to generate detectable amount of heterochiral Cu I species. The peak a2 is clearly visible on CVs recorded at 500 mV s -1 . When the sweep rate was decreased from 500 to 50 mV s -1 , the intensity of the a1 peak decreased concomitantly with the increase of the a2 peak. At 20 mV s -1 , the time scale of the Cu II /Cu I reduction/oxidation experiment was of ca. 40 s and the peak a1 was not observed anymore (cf. Fig. S8). Thus, the homochiral/heterochiral interconversion occurs on a time scale of a few seconds. This order of magnitude is confirmed by the fit of the CVs using digital simulations and assuming an EC mechanism. The homochiral/heterochiral interconversion rate was found to be kc = 0.20±0.05 s -1 (see Fig. S9). Note however that the rate of the homochiral/heterochiral interconversion is expected to depend on the complex and/or ligand concentration. It should be also mentioned that the wave corresponding to the possible reduction of the heterochiral Cu II complex was never observed even at high scan rate (0.5 V s -1 ) confirming its instability and the rapid conversion to homochiral Cu II complex. The full-square scheme is summarized in Fig. 4b. Stimuli-responsive polymers are promising materials because of their potential applications ranging from drug delivery to sensors. 14 Among them, metallo-supramolecular polymers are ideal candidates to access new stimuli-responsive materials because they offer properties modifiable by various external stimuli including electric stimulus. 15 We previously established that chiral ditopic ligand such as ligand 4 (Figure 5, a) is suitable to generate metallo-supramolecular assemblies and thus evaluated for the possibility to implement the chiral self-sorting assembly of the metallo-polymer by Cu II /Cu I change. [START_REF] Miller | For an example of redox-induced polymerization / depolymerisation of metallo-supramolecular polymer[END_REF] The potential axis is referred to the Fc + /Fc redox potential.

Finally, the homochiral/heterochiral interconversion was investigated by cyclic voltammetry starting from the racemic mixture of 1 mM homochiral Cu II metallopolymer (red curve in Figure 5). The current-potential curve exhibits one reduction wave c1 and two oxidation waves a1 and a2 which are typical for the case where the homochiral/heterochiral conversion of the Cu I coordination shell occurs, consistent with reorganization scheme from Fig. 1. Again, the characteristic timescale for the reorganization can be extracted from the evolution of CVs with the scan rate. The peak a2 disappears for potential scan rate above 200 mV s -1 suggesting that the time scale for the reorganization is of approximately 10 s. It is interesting to note that the Cu II metallopolymer reorganization is only slightly slower than the racemic discrete Cu II complex, In summary, homoleptic homochiral copper complexes have been prepared and their ligand rearrangements have been investigated by Cu I /Cu II redox change using cyclic voltammetry. The chiral self-recognition and self-discrimination properties have been clearly established which can lead to a metallosupramolecular copolymer that can be stimuli-responsive. This feature will now be exploited to develop more complex metallo-supramolecular systems that are able to fulfil such functions.

Figure 1

 1 Figure 1 Principle of the electrochemically-induced control of the metallopolymer arrangement: generation of block or alternating metallopolymers through the selective formation of homoleptic (top) or heteroleptic complexes (down).

  , top). Homochiral complexes 3 are formed with Cu II reduction oxydation = M = ditopic ligandprecursor (Figure2, bottom), as deduced from the crystallisation of solutions of these species, and resolution via X-ray diffraction studies (data available in ESI). These observations were confirmed in solution by determining the log β complexation values. Addition of the ligand (enantiopure or racemic) to a solution of Zn(BF4)2 or Cu(BF4)2 was analyzed by UV-vis.8 With Cu salt, a fitting of the experimental data to an ML2 model allowed calculation of the overall stability log β being 6.0±0.4 and 5.8±0.4 for the enantiopure and racemic, respectively. A log β of 8.2±0.4 was determined with Zn salt and rac 1. However, the titration data for the addition of the enantiopure ligand to Zn(BF4)2 were fitted as a simple ML binding model with a log β = 3.7±0.4 suggesting that the homochiral Zn complex could be generated here.

Figure 2

 2 Figure 2 Selective formation of heterochiral or homochiral entities as the function of the metal precursor. Top: Crystal and molecular structure of the heterochiral complex [Zn(R-1)(S-1)](BF4)2 2 (tetrahedral geometry). Bottom: Crystal and molecular structure of the two enantiomeric forms of homochiral complex 3 [Cu(R-1)2](BF4)2 and [Cu(S-1)2] (BF4)2 (distorted square planar geometry). ‡

Figure 3

 3 Figure 3 Cyclic voltammograms of the homochiral enantiopure complex Cu(R-1)2(BF4)2 (blue curve) and of the racemic mixture of homochiral Cu(1)2(BF4)2 3 (red curve) on a glassy carbon electrode at a concentration of 1 mM in 0.1 M TBAPF6 CH2Cl2 solution. Sweep rate 200 mV s -1 . The potential axis is referred to the Fc + /Fc redox potential.

Figure 4

 4 Figure 4 a) Cyclic voltammograms at different sweep rates 50 mV s -1 (top), 500 mV s -1 (middle), 5000 mV s -1 (bottom); Left: with (R) enantiopure Cu complex; Right: with racemic Cu complex. b) Schematic representation of the electrochemically triggered rearrangement.

Figure 5

 5 Figure 5 a) molecular structure of ditopic ligand R-4. b) Cyclic voltammograms of the homochiral enantiopure metallopolymer [Cu(R-4)2(BF4)2]n (blue curve) and of the racemic mixture of [Cu(4)2(BF4)2]n (red curve) at a concentration of 1 mM in 0.1 M TBAPF6 CH2Cl2 solution on a glassy carbon electrode . Sweep rate 50 mV s -1 .

  of Cu II metallopolymer to reorganize upon electrochemical stimulation.

This work was funded by the French National Research Agency (ANR) through the Programme d'Investissement d'Avenir under contract ANR-11-LABX-0058_NIE. SBL would like to thank Jean-Claude Chambron (CNRS-Université de Strasbourg) for the fruitful discussion.

Conflicts of interest

There are no conflicts to declare.