Affoué Tindo

Sylvie Konan

Romain Richard
email: romain.richard@iut-tlse3.fr

Caroline Andriantsiferana
email: caroline.andriantsiferana@iut-tlse3.fr

Kouassi Benjamin Yao

Marie-Hélène Manero
email: marie-helene.manero@iut-tlse3.fr

Ouverte

Keywords: Ozonation, Low-cost activated carbon, Adsorption, 2,4-Dimethylphenol, Wastewater treatment

This work is aimed at elaborating an innovative material from palm tree waste that can be used in industrial applications for adsorption and catalytic oxidation. This low-cost activated carbon, elaborated from Borassus palm tree waste (bpAC) showed textural and chemical properties comparable with common activated carbon. Moreover, it demonstrated interesting behaviour when it was coupled with ozone to remove a phenolic compound, 2,4-dimethylphenol (2,4-DMP). This refractory model molecule was removed two times faster in the presence of bpAC and was achieved in only 7 min. The total organic carbon (TOC) removal by only adsorption on bpAC was satisfactory with a value of 93.8% after 8 h. Nevertheless, the most interesting result concerned the TOC removal during catalytic ozonation which increased from 26% (single ozonation, i.e., without bpAC) to 91% in the presence of bpAC. The addition of a radical scavenger (tert-butanol) showed that hydroxyl radicals were involved during ozonation with bpAC. Moreover, a kinetics study highlighted that both radical and molecular mechanisms were acting in heterogeneous reaction. All the results were finally validated by treating real wastewater spiked with 2,4-DMP, confirming the ability of this bpAC to enhance the treatment of phenolic wastewaters.

Introduction

The global market for activated carbon was estimated at around USD 3 billion in 2015, corresponding to a production of nearly 10 million tons, of which about 40% of the production is used in water treatment plants to produce either high quality drinking water or to treat wastewater. Around the world, particularly in Asia, increasingly restrictive rejection standards involve significant development of wastewater treatment plants and consequently a growing demand of activated carbon. The perspectives are equivalent for the other activated carbon applications: air treatment, purification or storage of gases (30% of the activated carbon produced), car industry, pharmaceutical/medical and food processing. At medium-term, the demand is expected to dramatically increase since the consumption may triple by 2024 [START_REF]Activated Carbon Market Size, Share & Trends Analysis Report By Product (Powdered Activated Carbon, Granular Activated Carbon), By Application, By End-use, And Segment Forecasts[END_REF][START_REF] Wong | Recent advances in applications of activated carbon from biowaste for wastewater treatment: a short review[END_REF]. Coal, lignite and peat were initially the preferential precursor materials for the production of activated carbons. Nowadays, carbon-rich renewable resources such as stems, shells, husks, seeds or wood are also used. Since activated carbon must be regularly changed due to its loss of efficiency (saturation), low cost materials have been focusing interest for several years. Numerous research activities have aimed to find new low-cost precursors, accessible and available in abundant quantities such as rice husk, cashew nut shells, vine shoots, apple pulp and potato waste [START_REF] Wong | Recent advances in applications of activated carbon from biowaste for wastewater treatment: a short review[END_REF][START_REF] Menya | Production and performance of activated carbon from rice husks for removal of natural organic matter from water: a review[END_REF][START_REF] Matos | Influence of activated carbon upon titania on aqueous photocatalytic consecutive runs of phenol photodegradation[END_REF][START_REF] González-García | Activated carbon from lignocellulosics precursors: a review of the synthesis methods, characterization techniques and applications[END_REF]. Many studies have shown comparable efficiency of activated carbon derived from such precursors to commercial adsorbents for wastewater treatment applications: elimination of dyes, metal ions, pharmaceutical products, organic pollutants or natural organic matter [START_REF] Wong | Recent advances in applications of activated carbon from biowaste for wastewater treatment: a short review[END_REF][START_REF] Menya | Production and performance of activated carbon from rice husks for removal of natural organic matter from water: a review[END_REF].

Activated carbon is not only used for adsorption applications. Indeed, it can be used as a catalyst support for precious metal or metal oxide to synthesize value-added chemical products (hydrogenation or oxidation for example). Activated carbon may also have catalytic properties for many reactions. Its notable performances are attributed to its large specific surface area, high porosity and excellent electron conductivity [START_REF] Coughlin | Carbon as Adsorbent and Catalyst[END_REF]. In the field of water treatment, this material is often used to catalyse oxidation reactions. Indeed, Fortuny et al. [START_REF] Fortuny | Wet air oxidation of phenol using active carbon as catalyst[END_REF] showed that catalytic effects of activated carbon are comparable with conventional catalysts for degrading phenols using catalytic wet air oxidation (Cu 2+ , Fe 2+ , metal oxide, metal supported). This effect has also been demonstrated in photocatalysis where a synergistic effect between activated carbon and TiO 2 has been reported by several researchers [START_REF] Matos | Influence of activated carbon upon titania on aqueous photocatalytic consecutive runs of phenol photodegradation[END_REF][START_REF] Andriantsiferana | Photocatalytic degradation of an azo-dye on TiO 2 /activated carbon composite material[END_REF].

Oxidation with ozone is one of the most powerful operations for degrading biorefractory compounds but effective purification is sometimes difficult to achieve. Nervertheless, oxidation processes with ozone can be improved by adding an adsorbent in the process. Several studies have dealt with the interest of catalytic ozonation in presence of zeolite or activated carbon (AC) for the last two decades [START_REF] Khamparia | Adsorption in combination with ozonation for the treatment of textile waste water: a critical review[END_REF][START_REF] Aboussaoud | Combined ozonation using alumino-silica materials for the removal of 2,4-dimethylphenol from water[END_REF][START_REF] Ikhlaq | Catalytic ozonation of chlorinated VOCs on ZSM-5 zeolites and alumina: formation of chlorides[END_REF][START_REF] Chen | Effective mineralization of diclofenac by catalytic ozonation using Fe-MCM-41 catalyst[END_REF][START_REF] Wang | Fe-based catalysts for heterogeneous catalytic ozonation of emerging contaminants in water and wastewater[END_REF][START_REF] Merle | Comparison of activated carbon and hydrophobic zeolite efficiencies in 2,4-dichlorophenol advanced ozonation[END_REF][START_REF] Gümüs | A comparative study of ozonation, iron coated zeolite catalyzed ozonation and granular activated carbon catalyzed ozonation of humic acid[END_REF][START_REF] Rozas | Organic micropollutants (OMPs) oxidation by ozone: effect of activated carbon on toxicity abatement[END_REF][START_REF] Valdés | Heterogeneous and homogeneous catalytic ozonation of benzothiazole promoted by activated carbon: kinetic approach[END_REF][START_REF] González-Labrada | Enhancement of ciprofloxacin degradation in aqueous system by heterogeneous catalytic ozonation[END_REF]. This coupling generates the production of non-selective radicals such as hydroxyl radicals which have a higher oxidation potential than molecular ozone. The heterogeneous catalytic ozonation in the presence of activated carbon was reported to be more efficient than molecular ozonation particularly for the treatment of effluents containing compounds with low biodegradability [START_REF] Khamparia | Adsorption in combination with ozonation for the treatment of textile waste water: a critical review[END_REF][START_REF] Merle | Comparison of activated carbon and hydrophobic zeolite efficiencies in 2,4-dichlorophenol advanced ozonation[END_REF][START_REF] Gümüs | A comparative study of ozonation, iron coated zeolite catalyzed ozonation and granular activated carbon catalyzed ozonation of humic acid[END_REF][START_REF] Rozas | Organic micropollutants (OMPs) oxidation by ozone: effect of activated carbon on toxicity abatement[END_REF][START_REF] Valdés | Heterogeneous and homogeneous catalytic ozonation of benzothiazole promoted by activated carbon: kinetic approach[END_REF][START_REF] Beltrán | Kinetics of heterogeneous catalytic ozone decomposition in water on an activated carbon[END_REF]. In all these papers, the reaction kinetics was enhanced due to the decomposition of ozone on the surface of the activated carbon into powerful non-selective free radicals: HO • and O 2 -• . All these authors showed that coupling AC and O 3 involves a better efficiency than the addition of the two techniques. The removal process of the contaminant can take place in the bulk phase (homogeneous oxidation) and/or at the AC surface. In so doing, AC may act as an adsorbent and/or as an initiator of free radicals [START_REF] Kasprzyk-Hordern | Catalytic ozonation of gasoline compounds in model and natural water in the presence of perfluorinated alumina bonded phases[END_REF][START_REF] Flouret | Advanced treatment of phenol by H 2 O 2 /UV/activated carbon coupling: influence of homogeneous and heterogeneous phase[END_REF]. The AC physico-chemical properties were found to influence the decomposition of O 3 on AC: Sánchez-Polo et al. [START_REF] Sánchez-Polo | Efficiency of activated carbon to transform ozone into • OH radicals: Influence of operational parameters[END_REF] and Alvárez et al. [START_REF] Alvárez | The influence of various factors on aqueous ozone decomposition by granular activated carbons and the development of a mechanistic approach[END_REF] evidenced the importance of the specific surface, the mesoporous volume and the presence of basic groups at the surface of the AC. Other researchers [START_REF] Valdés | Heterogeneous and homogeneous catalytic ozonation of benzothiazole promoted by activated carbon: kinetic approach[END_REF] suggest that acidic sites can also play a key role in the decomposition of O 3 if pH > pH PZC .

Many types of wastewaters were efficiently treated by ozonation in presence of AC, with improvements in the mineralization, in the removal of total organic compounds and in the biodegradability of the water: wastewater from the textile industry, water containing humic acids, phtalates, atrazine or dyes [START_REF] Khamparia | Adsorption in combination with ozonation for the treatment of textile waste water: a critical review[END_REF][START_REF] Gümüs | A comparative study of ozonation, iron coated zeolite catalyzed ozonation and granular activated carbon catalyzed ozonation of humic acid[END_REF][START_REF] De Oliveira | Use of ozone/activated carbon coupling to remove diethyl phthalate from water: influence of activated carbon textural and chemical properties[END_REF][START_REF] Guzman-Perez | Kinetics of catalytic ozonation of atrazine in the presence of activated carbon[END_REF]. So catalytic ozonation on activated carbon appears a very suitable process for water treatment for a variety of industries. Our objective is to show that a low cost and waste-based activated carbon could be used not only as an adsorbent but also as a catalyst for ozone treatments.

In the present work, an innovative low-cost precursor has been used to produce activated carbon, the Borassus (aethiopum specie) palm, available in abundant quantities in tropical regions of Africa and Asia. To produce this activated carbon (bpAC), phosphoric acid has been chosen for chemical activation due to its high efficiency in activation of wood, its low cost and its non-toxicity [START_REF] Danish | A review on utilization of wood biomass as a sustainable precursor for activated carbon production and application[END_REF]. For a wastewater treatment application, the 2,4-dimethylphenol (2,4-DMP) was selected as a model molecule as it is representative of toxic biorefractory molecules detected in industrial rejections (disinfectants, solvents, insecticides, pharmaceuticals). After determining the physical and chemical properties of the bpAC, as well as its adsorption properties for 2,4-DMP elimination, this material was used as a catalyst in a lab-scale ozonation process to degrade 2,4-DMP. Experiments were conducted on two aqueous solutions, a synthetic one and a real wastewater spiked with the phenolic pollutant in order to demonstrate the feasibility of this new AC for adsorption and, above all, for catalytic ozonation.

Materials and methods

Reagents

Analytical grade 2,4-dimethylphenol C 8 H 10 O (2,4-DMP, 98% purity) and tert-butanol C 4 H 10 O (t-BuOH) were purchased from Sigma-Aldrich (Saint-Louis, Missouri, USA). 2,4-DMP properties are as follows: a molar mass of 122.17 g mol -1 , a boiling temperature of 212°C, a pK a of 10.6 and a kinetic diameter of 7.3 Å. Solutions were prepared with water purified by a Millipore Milli-Q UV. Ethanol (EtOH) and methanol (MeOH) used for high-performance liquid chromatography (HPLC) analyses are HPLC grade and were also purchased from Sigma-Aldrich (Saint-Louis, Missouri, USA). Phosphoric acid (H 3 PO 4 ) was purchased from Merck (Darmstadt, Germany) and the other reagents (HCl, NaOH, NaHCO 3 , Na 2 CO 3 ) used in this study were supplied by Sigma-Aldrich (Saint-Louis, Missouri, USA). Dioxygen (>99.99% purity) was supplied by Linde (Saint-Priest, France).

Preparation of 2,4 DMP synthetic solutions and spiked samples

This study was carried out on two aqueous solutions:

• A synthetic solution of 2,4-DMP (50 ± 2 mg L -1 ) prepared with water purified by a Millipore Milli-Q UV unit.

It was used to study the ozonation of 2,4-DMP in a simple medium without interaction with other organic substances. The concentration of 50 mg L -1 selected in this study is in the concentration range of phenolic compounds in industrial wastes (50 to 2,000 mg L -1 ) reported by many researchers [START_REF] Hussain | Kinetic study for aerobic treatment of phenolic wastewater[END_REF][START_REF] Garcia | Biodegradation of phenol compounds in vinasse using Aspergillus terreus and Geotrichum candidum[END_REF]. Moreover, at this concentration, the adsorption does not mask the effects of catalytic ozonation and offers better monitoring of elimination kinetics. • A wastewater spiked with 2,4-DMP (50 ± 2 mg L -1 ) was prepared using 1 L of filtered (with a sieve of 0.25 mm mesh) effluent sampled from the domestic sewage treatment plant of Nailloux village (Occitanie, France) in which 50 mg of 2,4-DMP was added. It was used to simulate a real medium and to study the performance of the ozonation of 2,4-DMP in complex conditions such as in industrial wastewaters. Its characteristics are detailed in Table 1.

Analytical methods

Liquid phase analysis

The 2,4-DMP concentration was determined by HPLC equipped with a UV detector (280 nm) and a C18 grafted silica column (Zorbax Eclipse XDB-C18, 5 µm average, 150 mm length, 4.6 mm diameter). Isocratic elution with a solvent mixture of 60% EtOH and 40% ultrapure water was applied at a flow rate of 0.7 mL min -1 . The liquid samples were filtered through a 0.45 µm membrane before being analysed using HPLC.

Degradation of 2,4-DMP leads to many by-products. All the substances contained in the water (intermediates and others) are globally followed by TOC analysis, using a TOC-LCPN Shimadzu (Noisiel, France) apparatus equipped with an infrared detector. The injection volume was 50 µL. combustion reactions were carried out in a quartz pipe at 680°C with a platinum catalyst. Oxygen was used as a carrier gas at a flow rate of 150 mL min -1 .

Solid-phase analysis

Concerning the bpAC, its textural characterization was obtained from nitrogen adsorption at -193°C using an ASAP 2010M apparatus (Micromeritics, Norcross, GA). The specific surface area was determined with the Brunauer, Emmett and Teller method (BET method) for a relative pressure range (p = p 0 ) of 0.01-0.2 [START_REF] Brunauer | Adsorption of gases in multimolecular layers[END_REF]. Methods from Horvath and Kawazoe [START_REF] Horváth | Method for the calculation of effective pore size distribution in molecular sieve carbon[END_REF] and Barrett et al. [START_REF] Barrett | The determination of pore volume and area distributions in porous substances. I. Computations from nitrogen isotherms[END_REF] have been used to assess the micropore and mesopore volumes.

Moreover, the identification and the quantification of the surface functions of bpAC were performed using Boehm titration [START_REF] Boehm | Some aspects of the surface chemistry of carbon blacks and other carbons[END_REF]. For the determination of acid groups, 1 g of activated carbon was stirred during 72 h with 50 mL of a solution (NaHCO 3 , Na 2 CO 3 or NaOH) at 0.1 mol L -1 . To determine the basic groups, the same operations were led with a solution of HCl at 0.1 mol L -1 . The basic or acid reactional volume was determined by titration with a solution of 0.1 mol L -1 of HCl or NaOH, respectively, for the acid and basic surface functions.

Finally, the pH of the point of zero charge (pH PZC ) was determined according to the so-called drift method [START_REF] Lopez-Ramon | On the characterization of acidic and basic surface sites on carbons by various techniques[END_REF], which consists in treating, during 48 h, 50 mL of NaCl solutions (0.01 mol L -1 ) with 0.15 g of bpAC at different pHs. The pH PZC is obtained when pH final is equal to pH initial .

Production and characterization of activated carbon

The innovative activated carbon (bpAC) was prepared using Borassus palm collected in the locality of Yamoussoukro (Côte d'Ivoire). The petioles of these palms were cut-up, washed and sun-dried during three days (Fig. 1a).

The dried samples were impregnated in a solution of orthophosphoric acid (H 3 PO 4 ) at 3 mol L -1 for 24 h and then carbonized at 600°C for 3 h under non-controlled atmosphere. The residues of carbonization were eliminated by an abundant washing with distilled water. In order to know if the activated carbon produced contained phosphorus, the orthophosphate content was regularly checked during washing. The final content was 0.01 mg L -1 . Then, activated carbon was dried at 105°C for 24 h and crushed in order to obtain a material with a granulometry of less than 500 µm. The final granular bpAC can be seen in Fig. 1b.

Experimental setup

The experiments were carried out in a semi-batch stirred slurry glass reactor (height: 0.26 m; diameter: 0.10 m; Fig. 2) with a jacket allowing to maintain a constant temperature (25°C). A volume of 1 L of the aqueous solution of 2,4-DMP was introduced into the reactor containing 5 g of bpAC.

The reactor was provided with a syringe dipping into the reactor for liquid sampling. Ozone (O 3 ) was produced by an ozone generator (WEDECO type modular 4 HC) fed with pure dioxygen. Ozonated stream was continuously added to the reactor at a flow rate of 30 L h -1 and a 10 g m -3 (NTP) ozone concentration. Thanks to this flow rate, a maximum amount of ozone in the liquid phase is obtained, which guarantees that ozone is not the limiting reagent. As a result, the kinetics being independent of ozone concentration, it can be considered of pseudo-first order. It was introduced through a tube provided with a porous diffuser placed under the turbine, to ensure a satisfactory mixing of liquid and gaseous phases. UV ozone analyser (BMT 964) was used to measure ozone gas concentrations at the inlet and outlet of the reactor. It was connected to a computer for continuous data acquisition. The off-gas was driven to an ozone thermal destructor before being released to the atmosphere.

To study the adsorption kinetics, 5 g of bpAC were introduced into the same reactor without any gas supply. The adsorption isotherm experiments were performed at 25°C in 125 mL stirred flasks, 0.1 g of bpAC with 100 mL of 2,4-DMP at different concentrations (in the range 0-1 g L -1 ). 2 COD (mg O2 L -1 ) 30 TOC (mg C L -1 ) 4 Ammonium (mg N L -1 ) 0.9 Nitrogen Kjeldahl (mg N L -1 ) 2.1 Nitrates (mg L -1 ) 1.5 Nitrites (mg L -1 ) 0.5 Total phosphorus (mg L -1 ) 0.3 IC (mg L -1 ) 79 BOD: biochemical oxygen demand; COD: Chemical oxygen demand.

Kinetic model

Organic compounds removal by the O 3 /bpAC coupling results from a complex combination of homogeneous (liquid phase) and heterogeneous reactions (on the surface of bpAC). Therefore, to better understand the mechanisms involved and in order to compare ozonation processes and adsorption under the same conditions, the kinetic study was performed on the first 5 min. As in the other studies in the literature [START_REF] Valdés | Heterogeneous and homogeneous catalytic ozonation of benzothiazole promoted by activated carbon: kinetic approach[END_REF][START_REF] Flouret | Advanced treatment of phenol by H 2 O 2 /UV/activated carbon coupling: influence of homogeneous and heterogeneous phase[END_REF][START_REF] Sánchez-Polo | Efficiency of activated carbon to transform ozone into • OH radicals: Influence of operational parameters[END_REF][START_REF] De Oliveira | Use of ozone/activated carbon coupling to remove diethyl phthalate from water: influence of activated carbon textural and chemical properties[END_REF], it was figured that the catalytic ozonation could be the result of five mechanisms. Each one was identified with its kinetics law and the simplified global kinetic law can be obtained by summing all the kinetic laws. For ozonation reactions (directly with O 3 or indirectly with HO • ), it is generally admitted that the kinetics is correctly described by a reaction of global order 2 [START_REF] Guzman-Perez | Kinetics of catalytic ozonation of atrazine in the presence of activated carbon[END_REF][START_REF] Acero | Degradation kinetics of atrazine and its degradation products with ozone and OH radicals: a predictive tool for drinking water treatment[END_REF]. Several hypotheses have been made to determine the kinetics law governing the phenomena in coupling O 3 /bpAC: (i) dissolved O 3 concentration in solution is assumed to be constant over time (as explained in the previous paragraph); (ii) hydroxyl radical (HO • ) concentration can also be assumed to be constant. In order to simplify the global model, it was assumed that the adsorption kinetics could be depicted by a pseudo-first order model [START_REF] De Oliveira | Use of ozone/activated carbon coupling to remove diethyl phthalate from water: influence of activated carbon textural and chemical properties[END_REF][START_REF] De Oliveira | Degradation kinetics of DEP in water by ozone/activated carbon process: influence of pH[END_REF].

In homogeneous phase ([2,4-DMP] represents the concentration of 2,4-DMP in the bulk phase):

• Direct ozonation due to a molecular type reaction between O 3 and the pollutant.

- ⎡ ⎣ ⎤ ⎦ = ⎡ ⎣ ⎤ ⎦ ⎡ ⎣ ⎤ ⎦ = ⎡ ⎣ ⎤ ⎦ d dt k k 2 4 2 4 2 4 1 3 1 , , , -DMP -DMP O -DMP obs (1)
• Indirect ozonation, due to a radical-type reaction (the radicals resulting from the decomposition of O 3 in water).

- ⎡ ⎣ ⎤ ⎦ = ⎡ ⎣ ⎤ ⎦ ⎡ ⎣ ⎤ ⎦ = ⎡ ⎣ ⎤ ⎦ • d dt k k 2 4 2 4 2 4 2 2 , , , -DMP -DMP HO -DMP obs (2)
In heterogeneous phase ([2,4-DMP] represents the concentration of adsorbed 2,4-DMP in equilibrium with the 2,4-DMP in the bulk phase through the Freundlich type equilibrium isotherm):

• Adsorption of pollutant on the bpAC.

- ⎡ ⎣ ⎤ ⎦ = ⎡ ⎣ ⎤ ⎦ d dt k 2 4 2 4 3 , , -DMP -DMP (3)
• Direct ozonation of adsorbed products on the bpAC (molecular oxidation).

- ⎡ ⎣ ⎤ ⎦ = ⎡ ⎣ ⎤ ⎦ ⎡ ⎣ ⎤ ⎦ = ⎡ ⎣ ⎤ ⎦ d dt k k 2 4 2 4 2 4 4 3 , , , -DMP -DMP O -DMP 4obs (4)
• Indirect ozonation of products adsorbed on the bpAC (radical oxidation).

- ⎡ ⎣ ⎤ ⎦ = ⎡ ⎣ ⎤ ⎦ ⎡ ⎣ ⎤ ⎦ = ⎡ ⎣ ⎤ ⎦ • d dt k k 2 4 2 4 2 4 5 5 , , , -DMP -DMP HO -DMP obs (5)
Thus, the elimination kinetics of 2,4-DMP by single ozonation can be modelled by:

- ⎡ ⎣ ⎤ ⎦ = ⎡ ⎣ ⎤ ⎦ + ⎡ ⎣ ⎤ ⎦ d dt k k 2 4 2 4 2 4 1 2 , , , -DMP -DMP -DMP obs o bs (6)
- ⎡ ⎣ ⎤ ⎦ = ⎡ ⎣ ⎤ ⎦ d dt k 2 4 2 4 , , -DMP -DMP homo (7)
With:

k k k homo obs o bs = + 1 2 (8)
k homo represents the kinetic constant of reactions (direct or indirect) occurring in liquid phase in absence of bpAC (min -1 ). k homo can be deducted with the slope of the curve ln[2,4-DMP] = f(t) for single ozonation.

The elimination kinetics of 2,4-DMP by catalytic ozonation has been simplified as follows:

- ⎡ ⎣ ⎤ ⎦ = ⎡ ⎣ ⎤ ⎦ d dt k 2 4 2 4 , , -DMP -DMP global (9)
With: The contribution of heterogeneous mechanisms (δ hetero ) in the O 3 /bpAC coupling is given by Eq. ( 12):

k k k global homo hetero = + (
δ hetero hetero global homo = × = - ( ) × k k k k k global global 100 100 (12)
Eqs. ( 13) and ( 14) allow to model 2,4-DMP removal kinetics for experiments performed on the synthetic water with O 3 , bpAC and the radical scavenger t-BuOH [START_REF] De Oliveira | Use of ozone/activated carbon coupling to remove diethyl phthalate from water: influence of activated carbon textural and chemical properties[END_REF]:

d dt k k k 2 4 2 4 2 4 2 4 1 3 4 , , , , -DMP -
DMP -DMP -DMP obs obs ⎡ ⎣ ⎤ ⎦ = ⎡ ⎣ ⎤ ⎦ + ⎡ ⎣ ⎤ ⎦ + ⎡ ⎡ ⎣ ⎤ ⎦ (13)
- ⎡ ⎣ ⎤ ⎦ = ⎡ ⎣ ⎤ ⎦ d dt k 2 4 2 4 , , -DMP -DMP mol ( 14
)
k mol represents the kinetic constant of reactions due to molecular ozone and also adsorption onto the bpAC. The estimation of the kinetic contribution of radical reactions (δ HO • to 2,4-DMP removal is given by Eq. ( 15):

δ HO global mol global • = - × k k k 100 (15)

Results and discussion

Properties of the bpAC and adsorption capacities

The prepared bpAC from Borassus palm was characterized in terms of textural properties, chemical attributes and adsorption capacities thanks to the techniques previously described. These properties are compared with those of commercial activated carbons prepared from natural raw materials (Table 2).

The BET surface area is quite high (888 m 2 g -1 ), giving to this adsorbent an interesting retention capacity. Indeed, the value is consistent with usual commercial activated carbon BET values, from 980 to 1,860 m 2 g -1 for ACs selected. Equivalent values were found for mesoporous (0.32 cm 3 g -1 )

and microporous (0.35 cm 3 g -1 ) volumes, as expected for AC prepared from agricultural residues [START_REF] Yahya | Agricultural bio-waste materials as potential sustainable precursors used for activated carbon production: a review[END_REF][START_REF] Ioannidou | Agricultural residues as precursors for activated carbon production-A review[END_REF]. Because of the activating treatment used, many acidic functions were generated at the surface of bpAC. That's why carboxylic and lactonic groups are predominant at its surface. The low pH PZC value of our activated carbon (2.4) is due to the activating chemical agent we used: H 3 PO 4 . Because of its high phosphorus and oxygen content, the H 3 PO 4 chemical activation process generates acidic ACs. The other ACs in Table 2 are commercial and have been activated by a physical process which leads to higher pH PZC . In the literature, researchers found pH PZC values (of activated carbons prepared by chemical activation with H 3 PO 4 ) ranging from 2.4 to 1.9, depending on the level of impregnation [START_REF] Sych | Porous structure and surface chemistry of phosphoric acid activated carbon from corncob[END_REF][START_REF] Danish | Effect of acidic activating agents on surface area and surface functional groups of activated carbons produced from Acacia mangium wood[END_REF][START_REF] Ould-Idriss | Preparation of activated carbons from olive-tree wood revisited. I. Chemical activation with H 3 PO 4[END_REF], which is comparable with ours. Other researchers have obtained slightly higher pH PZC values of 3.2 [START_REF] Ahmedna | The use of nutshell carbons in drinking water filters for removal of trace metals[END_REF] or 3.5 [START_REF] Ao | Microwave assisted preparation of activated carbon from biomass: a review[END_REF].

Concerning adsorption properties, isotherm experiments were carried out with 2,4-DMP in contact with bpAC at 25°C. Compared with commercial adsorbents, bpAC showed good retention capacities of 2,4-DMP totally consistent with other activated carbon results (Table 3). Equilibrium adsorption capacities of bpAC are lower than commercial ACs (ratio between 0.25 and 0.5) but they are significantly higher than the values obtained for different adsorbents such as ZSM-R and Mordenite and rather the same than Faujasite-γ.

Even if its smallest specific surface area impacts its adsorption capacity, the lower adsorption performance of bpAC compared with commercial AC is mostly due to the presence of many acidic functions on its surface. These functions are generally unfavourable for the adsorption of phenolic compounds. They are used to retain water molecules by H-bonds and therefore compete with the targeted molecules. This phenomenon is also accentuated by the absence of basic groups. Indeed, as widely accepted, the adsorption mechanism is based on π-π dispersion interactions between the π-electrons of aromatic ring and the π-electrons of the activated carbon: the presence of basic functional groups has been considered to favour these interactions (π-electron rich functional groups). The absence of basic oxygen functions at the surface of the bpAC therefore tends to be a disadvantage for these interactions [START_REF] Moreno-Castilla | Adsorption of organic molecules from aqueous solutions on carbon materials[END_REF]. Finally, the Freundlich model, typical of multilayer adsorptions, correctly represents the adsorption isotherm at 25°C, which can be explained by the micro-mesoporous properties of bpAC. Concerning Freundlich constants, the high value of n (0.1 < 1/n < 1) indicates the stronger adsorption interactions between adsorbate and adsorbent [START_REF] Kamaraj | Removal of lead from aqueous solutions by electrocoagulation: isotherm, kinetics and thermodynamic studies[END_REF]; the low value of K F is in agreement with the weakest adsorption capacities of bpAC.

Oxidation of 2,4-DMP: single and catalytic ozonation

As expected, 2,4-DMP can be easily removed by single ozonation, without any catalyst (Fig. 3a). Indeed, 2,4-DMP has a high electron density thanks to its aromatic structure and its double bonds, and the electron-donating group -OH. Ozone, which is an electrophilic agent, strongly reacts with this type of molecule: its electrophilic attack opens the aromatic rings, leading to the formation of carboxylic acid compounds. Other works found the same fast 2,4 DMP removal by single ozonation, even with other concentrations [START_REF] Aboussaoud | Combined ozonation using alumino-silica materials for the removal of 2,4-dimethylphenol from water[END_REF][START_REF] Moreno-Castilla | Adsorption of organic molecules from aqueous solutions on carbon materials[END_REF][START_REF] Vittenet | Removal of 2,4-dimethylphenol pollutant in water by ozonation catalyzed by SOD, LTA, FAU-X zeolites particles obtained by pseudomorphic transformation (binderless)[END_REF][START_REF] Trapido | Advanced oxidation processes for degradation of 2,4-dichlo-and 2,4-dimethylphenol[END_REF][START_REF] Vittenet | Catalytic ozonation with γ-Al 2 O 3 to enhance the degradation of refractory organics in water[END_REF]. Thanks to the addition of bpAC, the kinetics of reaction is highly increased, from 0.31 to 0.69 min -1 (Table 4). It is difficult to draw comparison with the results of other researchers due to different experimental conditions, however, activated carbon always increases the reaction rate of the contaminant if the pH is not too acidic and if the water does not contain radical scavengers [START_REF] Kasprzyk-Hordern | Catalytic ozonation of gasoline compounds in model and natural water in the presence of perfluorinated alumina bonded phases[END_REF]. Moreover, the contribution of activated carbon is interesting when considering the by-products of the oxidation reaction. Indeed, it can be seen in Fig. 3b that only 26.0% of the TOC is removed during single ozonation whereas 91.4% of the TOC is eliminated during catalytic ozonation (in the presence of 5 g L -1 of bpAC). Other works found almost the same increase (300%) with 2 g L -1 of Al 2 O 3 and with an O 3 dose of 2 g m -3 [START_REF] Vittenet | Catalytic ozonation with γ-Al 2 O 3 to enhance the degradation of refractory organics in water[END_REF]. In addition, an acidification of the medium is also noted, the pH decreasing from 5.3 to 3.1 and from 5.3 to 1.7 for single ozonation and catalytic ozonation, respectively, confirming the generation of acidic species. With bpAC, this pH decrease could be (i) due to the degradation of bpAC surface by ozone attack [START_REF] Merle | Comparison of activated carbon and hydrophobic zeolite efficiencies in 2,4-dichlorophenol advanced ozonation[END_REF][START_REF] Valdés | Effect of ozone treatment on surface properties of activated carbon[END_REF] and also (ii) due to the consequence of the leaching of residual phosphoric acid contained inside the pores even though it has been carefully rinsed.

Interesting results in terms of TOC adsorption (Fig. 3b) were obtained with 93.8% of TOC elimination after 8 h. Nevertheless, in this adsorption experiment, there is no oxidation and only the 2,4-DMP molecule was considered among the TOC. In the context of this study, the objective was not only to adsorb the 2,4-DMP molecule but also to completely degrade it (and reach a total mineralisation).

The presence of the bpAC, therefore, has a very significant impact on the overall yield of the reaction and proceeds to further acidification of the treated water. This more effective degradation can be explained by several phenomena that add up and/or combine. On the one hand, the decrease of organic specie concentrations can be explained by the retention of the molecules (2,4-DMP and some by-products) by adsorption. Indeed, as shown in Fig. 3a,2,4-DMP compounds can be removed by adsorption in this reactor with a good efficiency (90%). However, it is unlikely that all the oxidation by-products of carboxylic acid type are adsorbed. Indeed, previous studies have shown that the by-products of 2,4-DMP ozonation are mainly acetic acid, formic acid and oxalic acid [START_REF] Vittenet | Catalytic ozonation with γ-Al 2 O 3 to enhance the degradation of refractory organics in water[END_REF] and it is known that these polar molecules are hardly adsorbed on activated carbon [START_REF] Vittenet | Removal of 2,4-dimethylphenol pollutant in water by ozonation catalyzed by SOD, LTA, FAU-X zeolites particles obtained by pseudomorphic transformation (binderless)[END_REF][START_REF] Yousuf | Recovery of carboxylic acids produced during dark fermentation of food waste by adsorption on Amberlite IRA-67 and activated carbon[END_REF][START_REF] López-Garzón | Recovery of carboxylic acids produced by fermentation[END_REF]. So even if adsorption plays a role in decreasing TOC, other phenomena also exist.

The second explanation for the stronger decrease in TOC when bpAC is added is an oxidation of the molecules by the radical route. Indeed, it is known that molecular ozone can generate free radicals in contact with solids such as activated carbon [START_REF] Merle | Comparison of activated carbon and hydrophobic zeolite efficiencies in 2,4-dichlorophenol advanced ozonation[END_REF][START_REF] Gümüs | A comparative study of ozonation, iron coated zeolite catalyzed ozonation and granular activated carbon catalyzed ozonation of humic acid[END_REF][START_REF] Rozas | Organic micropollutants (OMPs) oxidation by ozone: effect of activated carbon on toxicity abatement[END_REF]. As these species are much more reactive than ozone and non-selective, they are able to attack compounds refractory to molecular ozone. Other works reported an increase of the ozone decomposition into hydroxyl radicals in the presence of adsorbent, even at low pH: Valdés and Zaror [START_REF] Valdés | Heterogeneous and homogeneous catalytic ozonation of benzothiazole promoted by activated carbon: kinetic approach[END_REF] confirmed in 2006 that aqueous ozone decomposes into free radicals in the presence of AC at pH 2, Guzman-Perez et al. [START_REF] Guzman-Perez | Kinetics of catalytic ozonation of atrazine in the presence of activated carbon[END_REF] for atrazine removal at pH 3 and González-Labrada et al. [START_REF] González-Labrada | Enhancement of ciprofloxacin degradation in aqueous system by heterogeneous catalytic ozonation[END_REF] for ciprofloxacin removal at pH 3. This higher degradation rate in the presence of free radicals comes from the higher value of the kinetic rate constant: k O3-DMP = 9.9 10 4 L mol -1 s -1 [START_REF] Gurol | Kinetic behavior of ozone in aqueous solutions of substituted phenols[END_REF] and k HO•-alkyl phenol = 10 9 -10 10 L mol -1 s -1 [START_REF] Ross | Selective specific rates of reactions of transients in water and aqueous solutions. Part III. Hydroxyl radical and perhydroxyl radical and their radical ions[END_REF][START_REF]Reaction rate constants of hydroxyl radicals with micropollutants and their significance in advanced oxidation processes[END_REF].

Mechanisms

A series of catalytic ozonation experiments were carried out with a radical scavenger in order to highlight the existence of a radical pathway. Tert-butyl alcohol (t-BuOH) is commonly used as a radical scavenger as it reacts preferentially with radicals: its reaction rate is much higher with hydroxyl radicals (6 × 10 8 L mol -1 s -1 [START_REF] Buxton | Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals ( • OH/ • O-in aqueous solution[END_REF]) than with ozone (3 × 10 -3 L mol -1 s -1 [START_REF] Hoigné | Rate constants of reactions of ozone with organic and inorganic compounds in water-I: Non-dissociating organic compounds[END_REF]). The adsorption of t-BuOH onto AC is supposed to be negligible and does not compete with 2,4-DMP: it cannot be easily adsorbed by AC because of its high polarity and its very low adsorption rate in comparison with the decomposition rate [START_REF] Alvárez | The influence of various factors on aqueous ozone decomposition by granular activated carbons and the development of a mechanistic approach[END_REF]. Nevertheless, introduced Table 3 Adsorption capacities for various 2,4-DMP/adsorbent systems [START_REF] Aboussaoud | Combined ozonation using alumino-silica materials for the removal of 2,4-dimethylphenol from water[END_REF][START_REF] Bourneuf | Desorption experiments and modeling of micropollutants on activated carbon in water phase: application to transient concentrations mitigation[END_REF][START_REF] Lesage | Etude d'un procédé hybride Adsorption / Bioréacteur à membranes pour le traitement des effluents industriels[END_REF] Adsorbent (reference) S BET (m 2 g -1 ) Operating conditions q e (mg g -1 ) Appropriated model (R 2 ; 1/n; K) bpAC (our work) 888

C adsorbent = 1 g L -1 ; T = 25°C 0 < C 0 < 1,000 mg L -1 47 a 130 b
Freundlich R 2 = 0.99 1/n = 0.34 in large excess, there is enough t-BuOH near the AC surface to react with the radicals formed in the heterogeneous phase. Fig. 4 shows the evolution of 2,4-DMP concentration during catalytic ozonation with and without t-BuOH. In presence of t-BuOH, a complete elimination of 2,4-DMP was obtained after 14 min of catalytic ozonation whereas it only needs 7 min for catalytic ozonation without t-BuOH. This shows that a molecular mechanism exists at this acidic pH since all the 2,4-DMP is eliminated (pH i = 5.3). Nevertheless, its kinetics is much slower than the radical mechanism one, especially at the beginning of the kinetics: for example, after 3 min, only 63% of 2,4-DMP is removed, compared with 85% without t-BuOH (see the two highlighted points in Fig. 4). The half reaction times (t 1/2 = 1 min without t-BuOH and 2.5 min with t-BuOH) evidence this statement and confirm that the presence of the scavenger changes the kinetics of oxidation by reacting with the radicals HO • instead of 2,4-DMP.

K F = 27 Activated carbon 1240 Ceca [46] 980 0.2 < C adsorbent < 8 g L -1 C 0 = 2 g L -1 ; T = 25°C
These radical reactions can take place in the bulk liquid phase or on the surface of the activated carbon. In the first case, this can occur if the radicals generated on the surface of the solid have sufficient lifetimes to be able to diffuse in the bulk liquid phase [START_REF] Guzman-Perez | Kinetics of catalytic ozonation of atrazine in the presence of activated carbon[END_REF]. In the second case, the reactions occur in the adsorbed phase, near the sites of decomposition of ozone. There are major controversies in the literature about the nature of ozone decomposition sites. Some researchers indicate that it is the basic oxygenated functional groups and the delocalized π electrons that are responsible for the decomposition of ozone into free radicals [START_REF] Alvárez | The influence of various factors on aqueous ozone decomposition by granular activated carbons and the development of a mechanistic approach[END_REF][START_REF] Xing | Activated carbon-enhanced ozonation of oxalate attributed to HO • oxidation in bulk solution and surface oxidation: effects of the type and number of basic sites[END_REF]. Other researchers show that acidic sites can also decompose ozone [START_REF] Valdés | Heterogeneous and homogeneous catalytic ozonation of benzothiazole promoted by activated carbon: kinetic approach[END_REF][START_REF] Dehouli | Influences of pH, temperature and activated carbon properties on the interaction ozone/activated carbon for a wastewater treatment process[END_REF]. Our study reinforces this second point of view. Indeed, although our activated carbon bpAC has a large quantity of acidic groups (2.75 meq g -1 , Table 2) and no basic group, it seems to decompose ozone into free radicals since radical oxidation takes place. It is very likely that the acidic groups of the surface of the material are at the origin of this decomposition. Indeed, the pH PZC of our activated carbon (2.4) being lower than the pH of water (5.3), the acidic functional groups are present in dissociated form and this deprotonated form can react with ozone. Several researchers have shown that the interaction between ozone and surface acids in deprotonated form can interact with ozone to lead to the formation of highly reactive free radicals such as O 2 -• and O 3 -•

, which are promoters of the chain reaction on the carbon surface [START_REF] Valdés | Heterogeneous and homogeneous catalytic ozonation of benzothiazole promoted by activated carbon: kinetic approach[END_REF][START_REF] Nawrocki | Catalytic ozonation in water: controversies and questions[END_REF].

It is, therefore, likely that four mechanisms are responsible for the good performance of the ozonation mineralization 4, based on the kinetic constants, the contributions of both heterogeneous and homogeneous mechanisms are of the same order of magnitude, with a slight predominance of the heterogeneous pathway: 55% for δ hetero against 45% for δ homo .

Application of the catalytic ozonation process to the wastewater spiked with 2,4-DMP

This study was carried out with the wastewater spiked with 2,4-DMP to evaluate the efficiency of this coupling process in a complex medium. The operating conditions were the same as those applied for the study of the synthetic water. The removal of pollution is followed during 8 h and results are presented according to 2,4-DMP removal (Fig. 5a) and TOC removal (Fig. 5b).

For these experiments, the initial TOC value includes 2,4-DMP and a low contribution of the wastewater (around 10%). The same conclusion as for the synthetic solution can be found for 2,4-DMP oxidation (Fig. 5a): the kinetics of 2,4-DMP removals was improved by the presence of bpAC, with a 2,4-DMP complete elimination time divided by 2. The presence of HCO 3 -ions does not have much influence on 2,4-DMP degradation kinetics while these ions are known to be radical scavengers. As reported by Sanchez-Polo et al. [START_REF] Sanchez-Polo | Combination of ozone with activated carbon as an alternative to conventional advanced oxidation processes[END_REF], acidic functions on the AC surface may react with HCO 3 -to form H 2 CO 3 and then limit their inhibitor effect. Surprisingly, concerning the TOC evolution (Fig. 5b), single ozonation process applied to the wastewater spiked with 2,4-DMP showed a real decrease of the TOC and a very high elimination of the TOC after 8 h. This better result can be attributed to different pH conditions. Indeed, the initial pH of this real wastewater was 8, with a slight decrease to 6.7 during single ozonation. As it is well known, basic conditions are favourable to the ozone decomposition into hydroxyl radicals. Thus, a better intermediates removal can only be obtained in these experimental conditions rather than with a synthetic solution (acidic conditions).

As it is the case in the synthetic solution, the addition of bpAC improves the efficiency of the TOC removal, but only during 3 h. After this time, the single ozonation process is more efficient than the catalytic one. The presence of natural organic matter (NOM) in wastewater can explain this phenomenon with alternative by-products formation. The presence of NOM can also create unfavourable conditions for the catalyst, being settled on its surface by the NOM. Nevertheless, the residence time in continuous processes being less than 3 h, the interest of the addition of bpAC is clearly shown: it enhanced the kinetics in a dramatic way.

In order to distinguish the homogeneous and heterogeneous reactions share, the modelling of the kinetics of 2,4-DMP removal during the first 5 min was done and the values k homo = 0.38 min -1 and k global = 0.62 min -1 were determined (Table 4). Here, the homogeneous reactions are predominant. Unlike the synthetic solution, the contribution of heterogeneous mechanism (δ hetero = 39%) is less important in this case. This may be due to the presence of NOM in effluent sampled from domestic sewage treatment. The TOC removal is then mainly due to the direct and indirect action of ozone.

Conclusion

This work shows that, for the first time, it is possible to develop a valuable activated carbon from Borassus palm waste (bpAC). This bpAC presents a high specific surface, with a good adsorption capacity of 2,4-DMP, a model phenolic compound. Furthermore, the presence of bpAC in the ozonation process showed a quantitative and quick removal of 2,4-DMP and the TOC in the synthetic solution and the wastewater spiked with 2,4-DMP. The study conducted with t-BuOH, a radical scavenger, showed that hydroxyl radicals were strongly responsible to the 2,4-DMP degradation during catalytic ozonation with bpAC. The presence of both molecular ozone and radicals, in solution and also at the surface of bpAC, may be responsible for the fast kinetics obtained. A global model of the first order based on adding the contributions of the different mechanisms of the catalytic ozonation made it possible on the one hand, to correctly represent the kinetics of degradation of 2,4-DMP, and on the other hand to estimate the kinetics contribution of heterogeneous reactions and homogeneous reactions to eliminate this pollutant. The contribution of heterogeneous mechanisms being more important in the case of the synthetic solution (δ hetero = 55%) showed that the reactions occurred mainly on the surface of bpAC. However, in the case of the wastewater spiked with 2,4-DMP, δ homo was more important (61%) because of the initial pH (8.0), which confirms the hypothesis that ozone decomposition is favourable in basic conditions.

Fig. 1 .

 1 Fig. 1. Raw Borassus palm (a) and activated carbon produced (b).

Fig. 2 .

 2 Fig. 2. Schematic diagram of experimental setup.

k

  hetero represents the mechanisms occurring on bpAC surface involving the elimination of 2,4-DMP at the bpAC surface by adsorption, degradation by molecular ozone and also by radical mechanisms. Likewise, the representation of ln[2,4-DMP] = f(t) enables the determination of k global and the deduction of the value of k hetero .

  C adsorbent : NC c C 0 : NC c ;

Fig. 3 .Fig. 4 .

 34 Fig. 3. Dimensionless evolution of the concentration of 2,4-DMP (a) and TOC (b). [bpAC] = 5 g L -1 ; [2,4-DMP] 0 = 50 mg L -1 , TOC 0 = 39 mg L -1 , pH i = 5.3.

Fig. 5 .

 5 Fig. 5. Dimensionless evolution of 2,4-DMP concentration (a) and TOC (b) spiked wastewater: [2,4-DMP] 0 = 50 mg L -1 , TOC 0 = 42 mg L -1 , pH i = 8.

Table 1

 1

	Characteristics of the effluent sampled from the domestic
	sewage treatment plant of Nailloux
	pH	8
	BOD (mg O2 L -1 )

Table 2

 2 Characterization of bpAC and commercial activated carbons[START_REF] Andriantsiferana | Competitive adsorption of p-hydroxybenzoic acid and phenol on activated carbon: experimental study and modeling[END_REF][START_REF] Quesada-Peñate | Comparative adsorption of levodopa from aqueous solution on different activated carbons[END_REF][START_REF] Mohamed | Competitive adsorption of phenolic compounds from aqueous solution using sludge-based activated carbon[END_REF]

	Activated	S BET	V porous (cm 3 g -1 )	Acidic group	Total acidic/basic	pH PZC
	carbons	(m 2 g -1 )	mi/me	Car/Lac/Ph (meq g -1 )	groups (meq g -1 )
	bpAC	888	0.35/0.32	1.02/1.07/0.66	2.75/0	2.4
	PICA S23	1,230	0.49/0.04	0.00/0.09/0.21	0.30/0.98	9.7
	PICA L27	1,860	0.77/0.48	NC	1.85/0.59	6.2
	MERCK 2514	980	0.37/0.20	0.035/0.015/0.16	0.22/0.46	8.9
	1240 Ceca	980	0.39 a	0.00/0.06/0.00	0.06/0.54	9.3
	mi: microporous, me: mesoporous; Car: carboxylic, Lac: lactonic, Ph: phenolic; NC: not communicated.

a Total porous volume.

Table 4

 4 Kinetic constants of 2,4-DMP degradation in the synthetic solution and the wastewater spiked with 2,4-DMP

		k global (min -1 )	k homo (min -1 )	R 2	k hetero (min -1 )	δ homo	% ( )=	homo global k k	×	100	δ hetero	% ( )=	hetero global k k	×	100
	Synthetic solution	0.69	0.31	0.987	0.38	45					55
	Wastewater spiked	0.62	0.38	0.986	0.24	61
	with 2,4-DMP

operation in the presence of bpAC: two mechanisms in liquid homogeneous phase, molecular oxidation and radical oxidation, and two mechanisms in heterogeneous phase, radical oxidation in adsorbed phase and adsorption. Considering the results given in Table

Acknowledgement

This work was supported by the project Appui à la Modernisation et à la Réforme des Universités et Grandes Ecoles de Côte D'Ivoire (AMRUGE-CI).


Stefanie Wagner

Dirk Hauck

Michael Hoffmann

Roman Sommer

Ines Joachim

Rolf Müller

Anne Imberty

Annabelle Varrot

Alexander Titz
email: alexander.titz@helmholtz-hzi.de

Covalent lectin inhibition and its application in bacterial biofilm imaging

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

Lectins are carbohydrate-binding proteins with very diverse functions that are found in all domains of life. [1,[START_REF] Varki | Essentials of Glycobiology[END_REF] These proteins play crucial roles in numerous processes such as cell-cell recognition, infection processes and immune defense. They are generally characterized by an intermediate to low affinity towards their carbohydrate ligands that is often overcome by Nature through multivalency of both the lectin receptors and their carbohydrate ligands resulting in avidity with an increase in apparent affinity.

Because these carbohydrate-binding proteins play essential roles in a number of pathological processes, they have become attractive targets for therapy. However, the fact that lectins display moderate affinities to their ligands renders this class of proteins as difficult targets for drugs. [START_REF] Ernst | [END_REF] Despite this drawback, a number of recent success stories impressively demonstrated their potential for therapy: the selectin antagonist GMI-1070 is currently in phase III clinical trials and various FimH inhibitors are in the late preclinical stage. [START_REF] Ernst | [END_REF][4][5][6][7] Lectins are involved in infections with the Gram-negative bacterium P. aeruginosa, one important member of the often highly drug resistant ESKAPE pathogens which are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, P. aeruginosa, and Enterobacter species and currently cause most of the severe hospital infections in western countries. [8,9] The two bacterial lectins, [10] LecA and LecB, are virulence factors and important for bacterial adhesion and biofilm formation. [11,12] The latter is especially problematic as resistance against antibiotics inside a biofilm is increased by a factor of 10-1000. [13] Thus, the inhibition of these lectins provides a promising way to dismantle the bacterium from the protective biofilm environment and restore immune defense and activity of antibiotics. [14] Current approaches to inhibit both lectins range from small molecules to multivalent structures and are summarized in recent reviews. [15][16][START_REF] Titz | Carbohydrate-Based Anti-Virulence Compounds Against Chronic Pseudomonas aeruginosa Infections with a Focus on Small Molecules[END_REF][START_REF] Sommer | [END_REF][19] We focus on the development of small molecules and recently published various potent glycomimetic inhibitors for the high affinity lectin LecB as inhibitors of P. aeruginosa adhesion. [20][21][22][23][24][25] In contrast, LecA only has an intermediate affinity for its monovalent Dgalactose-derived ligands in the 50-100 µM range. [10,26,27] Phenyl β-D-galactosides and derivatives showed an increased affinity of approx. 10 µM, e.g. compound 1 (Fig. 1), but despite a high number of derivatives analyzed, no further significant increase in potency could be achieved. [28][29][30][31] Covalent inhibition is one strategy to avoid dissociation of the inhibitor from the target and thus to persistently inactivate proteins. To date, a specific covalent inhibition of the carbohydrate binding site in a lectin has not been achieved despite attempts using squaric acid to target FimH [32] or photoactivatable substituents for the targeting of galectins. [33][34][35] The latter probes covalently bind to unspecific residues of the protein in proximity (3 Å) to the photoactivated center. The crystal structure of LecA [26,36] reveals the presence of one cysteine residue (Cys62) in the carbohydrate binding domain (Figure 1). The specific targeting of cysteine residues with electrophilic warheads is a general strategy in the search for cysteine protease inhibitors, [37] but has never been addressed in carbohydrate recognition domains. In order to target Cys62, we designed the two diastereoisomeric galactose-derived epoxides 2 and 3 (Figure 1) as potential covalent active site inhibitors of LecA. A.

B.

Figure 1: A. Galactoside recognition by the bacterial lectin LecA (pdb: 3ZYF [36] ); B. Electrophilic epoxide derivatives The 8 step synthesis of epoxides 2 and 3 started from D-galactose (4) with epoxidation as the last step (Scheme 1). 4 was protected as diacetonide (→5) and the free primary hydroxyl group was oxidized under Swern conditions (→6) in good yields. Then, heptose 7 was established in a Wittig reaction followed by a change in protecting groups from acetonides to acetates (→8). Lewis acid catalyzed glycosylation of phenol with 8 gave phenyl β-glycoside 9 in good yield. 9 was deprotected in a Zemplén transesterification reaction to give olefin 10 in 97% yield. Late stage oxidation with mCPBA yielded the two diasteroisomeric epoxides 2 and 3 in 9% and 19%, respectively. The stereochemistry of 3 was established by X-ray crystallography in complex with LecA (see Figure 3).

(vii) (viii) (i) (ii) (iii) (iv)-(v)
Both epoxides 2 and 3 and olefin 10 were then tested in a recently developed competitive binding assay [31] for inhibition of LecA. No inhibition was observed for olefin 10 up to 3 mM. In contrast, the epoxides showed a strong diastereoselectivity for inhibition of LecA: 3 was a good inhibitor with an IC50 = 64 µM, whereas its diastereomer 2 was not recognized (IC50 > 3 mM), indicating a specific binding of epoxide 3 to LecA.

To assess the binding mode of 3 with LecA, we analyzed LecA in presence and absence of 3 by mass spectrometry (Figure 2). LC-MS measurements on intact protein level showed a mass shift of 268.1 Da when incubated with 3 and thus prove a covalent binding of the epoxide to LecA (Figure 2A,B). In order to localize the binding site of the epoxide, an MS based sequencing using MALDI in source decay (ISD) was performed for both samples (Figure 2C). MALDI-ISD experiments with c-ion series annotated in a range from 5000 to 8000 m/z were instrumental to identify the peptide sequence ranging from Arg48 to Thr74 and Asn71, respectively. For the LecA sample co-incubated with 3, a mass increase of 268 Da to the c62 ion indicated the binding of compound 3 to Cys62 of LecA. Thus, 3 showed covalent binding to LecA and the nucleophile for the epoxide ring opening was the sulfhydryl group of Cys62 in the carbohydrate recognition domain. We then crystallized LecA in complex with epoxide 3 and solved the structure by X-ray crystallography (Figure 3). In this complex, 3 adopts a coordination to the calcium ion bound to LecA as it had been reported for other galactosides before. [26,36] Surprisingly, despite its orientation towards Cys62 the epoxide moiety in 3 is still intact and the covalent adduct could not be observed in this structure. These differences to the covalent adduct observed by mass spectrometry are likely a result of the different pH values of the buffers employed: lectin binding assays and mass spectrometry were performed at a physiologically buffered pH (7.4), whereas LecA is crystallized at In order to exploit this unique covalent lectin ligand for biological applications such as lectin specific staining, we synthesized alkyne-bearing derivatives that were then coupled to a fluorescent azide in a Huisgen dipolar cycloaddition (Scheme 2). Glycosyl donor 8 was reacted under Lewis acid catalysis with the acceptor monopropargyl hydroquinone to give the glycoside 11. Here, we first oxidized the peracetylated olefin 11 using mCPBA and the two diasteromeric epoxides 12 and 13 were obtained after chromatographic separation in 21% and 46% yield, respectively. Subsequently, the acetates were removed in a Zemplén type reaction to individually give the 14 or 15. Both were then tested for inhibition of LecA showing a comparable diastereoselectivity as observed for the unsubstituted phenyl derivatives 2 and 3 before: 6D epoxide 15 inhibited LecA with an IC50 of 109 µM, whereas the diastereomeric 6L epoxide 14 was inactive (IC50 > 3 mM). The stereochemistry of 14 and 15 was unambiguously assigned by combining the activity data and NMR chemical shift and coupling constant analysis and comparison with analogs 2 and 3. The active diasteromer 15 was then coupled in a copper(II)-catalyzed click reaction to the azide 16 [38] to give fluorescent probe 17 in good yields. Tools to visualize the presence of carbapenem-resistant bacterial pathogens in a test tube by specific activity-based probes have recently been reported. [39] The visualization of bacterial biofilm structures is of outstanding current interest, both, in vitro and in vivo. [40][41][42] LecA is involved in the biofilm formation of P. aeruginosa and LecA-deficient strains were shown to have thinner biofilms with a reduced biomass. [12,43] Because the expression of LecA is upregulated in biofilms and it is located extracellularly, [12,44,45] this protein is a promising target for the imaging of biofilms. We therefore explored whether the LecA-directed epoxides reported here can specifically stain biofilms of P. aeruginosa. Bacterial biofilms were grown using mCherry-expressing PAO1 wildtype bacteria and the corresponding LecA-deficient strain PAO1 ∆lecA and then analyzed by confocal fluorescence microscopy (Figures 4,5). Under shaking growth conditions, bacterial aggregates [46] of the biofilm were observed in the PAO1 wildtype strain, whereas the ∆lecA strain generally showed a heavily reduced number of aggregates with smaller sizes and therefore also a higher number of planktonic bacteria since bacterial growth is comparable (Figures 4, 5, S21). After addition of the LecA-directed dye 17 to the bacterial cultures, a specific staining of the wildtype biofilm aggregates was observed and no staining was visible in case of the aggregates formed by the ∆lecA strain. The green fluorescence originating from 17 was observed on the entire structure of the wildtype bacterial aggregates, whereas no or only a very faint color on the surface of the ∆lecA strain aggregates was observed without any detectable fluorophore inside these aggregates. The largest aggregate found for the ∆lecA strain was also analyzed (Figures S22-S24) and staining was LecAspecific and independent of aggregate size. Thus, the bacterial lectin LecA can be exploited as a target to visualize biofilms of P. aeruginosa using conjugates of LecA-ligands, such as the galactose-derived epoxide 17. In summary, we have developed the first covalent inhibitor of carbohydrate binding sites by rational structure-based design. Both diastereomers of the epoxygalactoheptoside 2 and 3 were synthesized and biologically evaluated. LecA displayed a strong diastereoselectivity for the 6D epimer 3 over its 6L isomer 2. The binding site and its covalent nature at physiological pH was established using mass spectrometry-based sequencing and the non-covalent crystal structure of 3 in complex with

LecA was solved at pH 4.6. Finally, we used the fluoresceine-derivative 17 for the LecA-specific staining of P. aeruginosa biofilms. Such conjugates may lead to the development of pathogenspecific imaging agents to localize bacterial biofilm-associated infections inside an infected host enabling pathogen-and tissue-directed therapy.

Experimental

General Experimental Details

Commercial chemicals and solvents were used without further purification. C). Chemical shifts are given in ppm and were calibrated on residual solvent peaks. [47] Multiplicities were specified as s (singlet), m (multiplet) or interpreted according to 1 st order and higher order where possible. The signals were assigned with the help of 1 H, 1 H-COSY, DEPT-135edited 1 H, 13 C-HSQC and 1 H, 13 C-HMBC experiments. Preparative HPLC-MS was performed on a Thermo Dionex Ultimate 3000 HPLC with UV detection. Analytical HPLC-MS was performed on a Thermo Dionex Ultimate 3000 HPLC coupled to a Bruker amaZon SL for low resolution mass spectra or on a Bruker maxis 4G hr-QqToF spectrometer for high resolution, and the data were analyzed using DataAnalysis (Bruker Daltonics, Bremen, Germany).

1,2:3,4-Di-O-isopropylidene-α-D-galacto-hexodialdo-1,5-pyranose (6). 6 was synthesized from 1,2:3,4-Di-O-isopropylidene-α-D-galactopyranose [48] (5, 500 mg, 1.92 mmol) by Swern oxidation following the procedure by Streicher and Wünsch. [49] After chromatography, 6 (348 mg, 1.35 mmol)

was obtained as colorless amorphous solid. The NMR of 6 corresponded to the one given in the literature. [50] 6,7-Dideoxy-1,2:3,4-di-O-isopropylidene-α-D-galacto-hept-6-enopyranose (7). 7 was synthesized by treating 6 with a Wittig reagent in analogy to Lehmann and Schäfer. [51] In contrast to the literature, we used methyltriphenylphosphonium iodide and sodium hydride in DMSO to generate the ylid. Crude olefin 7 was obtained after extraction and was used without purification in the next step. The NMR of the crude product corresponded to literature values. [50] 6,7-Dideoxy-1,2,3,4-tetra-O-acetyl-α/β-D-galacto-hept-6-enopyranose (8). α-8 was first described by Lee et al. and the NMR corresponded to literature values. [50] Phenyl 2,3,4-tri-O-acetyl-6,7-dideoxy-β-D-galacto-hept-6-enopyranoside (9).

8 (1.24 g, 3.6 mmol) and phenol (678 mg, 7.2 mmol) were dissolved in dry CH2Cl2 (24 mL) and added to a round bottom flask with powdered activated molecular sieves (3Å, 600 mg) under argon.

After cooling to 0 °C, BF3•Et2O (1.77 mL, 14.4 mmol) was added in four portions over 1 h. Then, the reaction was stirred for 50 min at 0 °C after which the reaction was allowed to warm to r.t.

during 20 min. The reaction was stopped by pouring on cold aqueous satd. NaHCO3, after addition of CH2Cl2 (60 mL) the phases were separated and the organic layer was washed with cold aqueous satd. NaHCO3 (2 x 60 mL). The combined aqueous layers were extracted with CH2Cl2 (3 x 60 mL).

The combined organic layers were dried over anhydrous Na2SO4, filtered and the volatiles were removed in vacuo. The residue was purified by MPLC (SiO2, petrol ether/EtOAc gradient of 5-50%) to elute first the anomer α-9 (55 mg, 4%), then the title compound 9 (760 mg, 56%) and then recovered starting material 8 (358 mg, 26%). Analytical data for 9: 1 After a reaction time of 10 d, the reaction was diluted with EtOAc (25 mL) and washed with brine

(2 x 3 mL). The organic layer was dried over Na2SO4, filtered and the volatiles were removed in vacuo. The aqueous phase contained the desired products and was therefore lyophilized and the 3.27 (dd, J = 6.7, 1.2 Hz, 1H, H5), 2.83 (dd, J = 4.9, 4.2 Hz, 1H, H7a), 2.71 (dd, J = 5.0, 2.7 Hz, 1H, H7b). 13 was prepared from 8 (800 mg, 2.32 mmol) and hydroquinone monopropargyl ether [52] (688 mg, 4.64 mmol) as described for 9. Purification of the crude product yielded 11 (627 mg, 62%) as an oil and small amounts of its α-anomer and recovered α-8. Analytical data for 11: p-Propargyloxyphenyl 6,7-dideoxy-6,7-epoxy-β-D-galacto-heptopyranoside (6L)-14.

Acetylated (6L)-12 (34 mg, 76 µmol) was dissolved in dry MeOH (380 µL) and cooled to 0 °C.

Freshly prepared NaOMe (60 mM in MeOH, 570 µL) was added dropwise and the reaction was stirred for 1.5 h at 0 °C. Then, Amberlite IR120/H + was added, the reaction was filtered and the volatiles were removed in vacuo. Analytically pure (6L)-14 (21 mg, 86%) was obtained. 1 Acetylated (6L)-13 (70 mg, 156 µmol) was was treated with NaOMe as described before for its diastereomer. Analytically pure (6D)-15 (47 mg, 94%) was obtained. Propargylated epoxide (6D)-15 (1.28 mg, 4 µmol) and azide modified FITC 16 [38] (2.8 mg, 4 µmol)

were dissolved in DMF (120 µL). After the addition of CuSO4*5H2O (0.

Competitive binding to LecA using fluorescence polarization

The protein LecA was expressed and purified as described previously. [27,31] The competitive binding assay was performed as described previously. [31] In brief, to 20 GmbH, Germany) and fitted according to the four parameter variable slope model. Bottom and top plateaus were defined by the standard compounds included as controls in each assay (methyl α-Dgalactoside and phenyl β-D-galactoside, respectively) and the data was reanalyzed with these values fixed. A minimum of three independent measurements on three plates was performed for each inhibitor.

Mass spectrometry

All intact protein ESI-MS-measurements were performed on a Dionex Ultimate 3000 RSLC system using an Aeris Widepore XB-C8, 150 x 2.1 mm, 3.6 μm dp column (Phenomenex, USA).

Separation of 0.5 μL sample was achieved by a linear gradient from (A) H2O + 0.1% HCOOH to (B) MeCN + 0.1% HCOOH at a flow rate of 300 μL/min and 45 °C. The gradient was initiated by a 1.0 min isocratic step at 2% B, followed by an increase to 75% B in 10 min to end with a 3 min step at 75% B before reequilibration with initial conditions. UV spectra were recorded by a DAD in the range from 200 to 600 nm. The LC flow was split to 75 μL/min before entering the maXis 4G hr-ToF mass spectrometer (Bruker Daltonics, Bremen, Germany) using the standard Bruker ESI source. In the source region, the temperature was set to 180 °C, the capillary voltage was 4000 V, the dry-gas flow was 6.0 L/min and the nebulizer was set to 1.1 bar. Mass spectra were acquired in positive ionization mode ranging from 600-1800 m/z at 2.5 Hz scan rate. Protein masses were deconvoluted by using the Maximum Entropy algorithm (Spectrum Square Associates, Inc.).

Crystallization and structure determination

LecA dissolved in water to 10 mg/mL was co-crystallized with 2 mM ligand 3 after incubation during one day at room temperature. Crystallization screening was performed using the vapor diffusion method. Sitting drops of 200 nl drops containing a 1/1 (v/v) mix of protein and reservoir solution at 20 °C were made using the robot of the HTXlab, Grenoble, France. Crystal clusters were obtained in five days from solution 6 from the PEGs-I (Qiagen): 25% peg 2KMME 0.1M sodium acetate pH 4.6. A broken part of the cluster was directly mounted in a cryoloop and flash-freezed in liquid nitrogen. Diffraction data were collected at 100 K at the European Synchrotron Radiation Facility (Grenoble, France) on BM30A-FIP using an ADSC Q315r detector. The data were processed using XDS. [53] All further computing was performed using the CCP4 suite. [54] Data quality statistics are summarized in Table S1. The structure was solved by molecular replacement using PHASER and the tetramer coordinates of PDB-ID 1OKO as search model. [55] This model was initially rebuild using ARP/WARP [56] and then the structure was refined with restrained maximum likelihood refinement using REFMAC 5.8 and local NCS restrains [57] iterated with manual rebuilding in Coot. [58] Five percent of the observations were set aside for cross-validation analysis, and hydrogen atoms were added in their riding positions and used for geometry and structure-factor calculations. Incorporation of the ligand was performed after inspection of the ARP/WARP 2Fo-DFc weighted maps. Water molecules, introduced first with ARP/WARP and then automatically using Coot, were inspected manually. The model was validated with the wwPDB Validation server: http://wwpdb-validation.wwpdb.org. The coordinates were deposited in the Protein Data Bank under code 5MIH.

Microbiology

Generation of fluorescent Pseudomonas aeruginosa strains

Bacterial cultures were grown in LB medium at 37 °C and 180 rpm. P. aeruginosa PAO1 wt (DSM19880) and P. aeruginosa PAO1 ∆lecA (source Bodo Philipp, University of Münster) were transformed with mCherry expressing plasmid pMP7605 [59] by three parental mating using E. coli DH5α (source Rolf Müller lab) as donor strain and E. coli HB101 [RK 600] (source Bodo Philipp, University of Münster) as helper strain. Bacterial pre-cultures were inoculated from single colonies in 5 mL LB with appropriate antibiotics (30 µg/mL chloramphenicol for bacteria containing pRK600; 15 µg/mL gentamicin for for bacteria containing pMP7605) and grown at 37 °C and 180 rpm overnight to stationary phase. For each mating, 100 µL pre-culture was combined and washed twice with pre-warmed LB medium. Strain mixtures were resuspended in 30 µL pre-warmed LB medium and spotted onto pre-warmed LB agarose plates and incubated overnight at 37 °C. After 24 hours from each plate all growth was resuspended in 1 mL LB per plate. 100 µL of the bacterial suspension and 100 µL of a 1:10 dilution were plated on LB-gentamicin (120 µg/mL) agar plates and incubated at 37 °C for 24 hours. Successfully transformed bacteria were gentamicin-resistant and the colonies were identified by their pink color and restreaked.

Biofilm experiments

Bacterial pre-cultures of P. aeruginosa PAO1 w.t. pMP7605 or ∆lecA pMP7605 were inoculated from single colonies in 5 mL LB and grown at 37 °C and 180 rpm overnight to stationary phase.

For the biofilm assay the bacterial pre-cultures were diluted to an OD600nm of 0.02 in fresh LB medium. 400 µL bacterial culture were transferred to each well of a 24-well imaging plate (cat no 3231, zell-kontakt GmbH, Germany). Plates were incubated at 37 °C and shaking at 180 rpm for 20

2 and 3

 3 for the targeting of Cys62. The distances from Cys62-S to C6 and O6 of 1 are between 4.1 and 4.3 Å. (sc = side chain, bb = back bone)

Figure 2 :

 2 Figure 2: Covalent binding mode of 3 with LecA established by mass spectrometry. Deconvoluted intact protein MS spectra of LecA without inhibitor (A) and with inhibitor 3 (B). (C) MALDI-ISD experiments with c-ion series annotated in a range from 5000 to 8000 m/z.

pH 4 . 6 .Figure 3 :

 463 Figure 3: Crystal structure of epoxide 3 in complex with LecA at 1.80 Å resolution in the non-covalent binding mode obtained at pH 4.6 (pdb code 5MIH). A. Electron density displayed at 1σ for ligand and Cys62 side chain. B. Interaction of the ligand with LecA: the epoxy-oxygen atom accepts hydrogen bonds from His50 and one protein-bound water molecule. In addition, His50 established a CH-π interaction with the phenyl agylycon. In the crystal, the sulfur atom of Cys62 is 3.3 Å away from C7 of ligand 3.

Scheme 2 :

 2 Scheme 2: Synthesis of LecA-directed propargylated epoxides with LecA inhibition data and synthesis of fluorescent derivative 17. (i) hydoquinone monopropargyl ether, BF3*Et2O, CH2Cl2, -20 °C -r.t.; (ii) mCPBA, NaHCO3, CH2Cl2, 0 °C -r.t.; (iii) NaOMe, MeOH, 0 °C; (iv) CuSO4, sodium ascorbate, H2O, DMF, r.t.;

Figure 4 :Figure 5 :

 45 Figure 4: Galleries of LecA-dependent staining of P. aeruginosa biofilms with 17. P. aeruginosa PAO1 wt (A) or the lecA knockout (∆lecA) mutant (B) expressing mCherry from pMP7605 were incubated at 37 °C for 24 hours with agitation (180 rpm). Biofilms were stained with the covalent LecA ligand fused to fluorescein (17) for 10 -30 min. Zstacks (232 x 232 µm) were recorded every 2 µm at 561 nm for mCherry (red, A and B, upper panels) and 488 nm for fluorescein (green, A and B middle panels). The galleries show every 4th z-stack recorded. Lower panels show merged images of both channels (488 nm and 561 nm).

  H NMR (400 MHz, CHCl3-d1) δ 7.34 -7.25 (m, 2H, ArH), 7.10 -6.99 (m, 3H, ArH), 5.76 (ddd, J = 17.3, 10.7, 4.8 Hz, 1H, H6), 5.51 (dd, J = 10.5, 8.0 Hz, 1H, H2), 5.46 -5.37 (m, 2H, H4, H7a), 5.27 (dt, J = 10.7, 1.4 Hz, 1H, H7b), 5.15 (dd, J = 10.4, 3.4 Hz, 1H, H3), 5.08 (d, J = 7.9 Hz, 1H, H1), 4.33 (dq, J = 4.7, 1.

3 and 17 mg of 2 .

 32 combined residues were purified by normal phase flash chromatography to give impure 19 mg of Both were then separately purified by reversed phase (C18) flash chromatography to give 2 (5.0 mg, 9%) and its epimer 3 (11.0 mg, 19%). The stereochemistry of 2 and 3 was assigned by co-crystallization of the active diastereomer 3 with LecA (vide infra). Analytical data for (6L)-2: (DH174-Fr1("Prod.B")):1 H NMR (500 MHz, MeOH-d4) δ 7.32 -7.25 (m, 2H, ArH), 7.13 -7.07 (m, 2H, ArH), 7.00 (tt, J = 7.5, 1.1 Hz, 1H, ArH), 4.83 (d, J = 7.8 Hz, 1H, H1, signal overlap with residual water), 3.93 (dd, J = 3.4, 1.1 Hz, 1H, H4), 3.82 (dd, J = 9.8, 7.7 Hz, 1H, H2), 3.56 (dd, J = 9.7, 3.4 Hz, 1H, H3), 3.35 -3.29 (m, 1H, H6, signal overlap with residual solvent),

p-Propargyloxyphenyl 2 , 3 , 4 -

 234 tri-O-acetyl-6,7-dideoxy-β-D-galacto-hept-6-enopyranoside 11. 11

p-Propargyloxyphenyl 2 , 3 , 4 - 13 .

 23413 tri-O-acetyl-6,7-dideoxy-6,7-epoxy-β-D-galacto-heptopyranoside (6L)-12 and (6D)-Olefin 11 (256 mg, 0.59 mmol) was dissolved in CH2Cl2 (12 mL) and cooled to 0 °C. mCPBA (511 mg, 2.96 mmol) was added portionwise under stirring and cooling. Then, the reaction was allowed to warm to r.t. and stirring was continued for 6 d when the reaction was diluted with CH2Cl2 (60 mL) and extracted with satd aq. Na2S2O3, NaHCO3, H2O. The organic layer was dried over Na2SO4, filtered and the volatiles were removed in vacuo. The residue was purified by MPLC (silica, PhMe/ EtOAc 5-15%) to give first 13 (119 mg, 46%) and then 12 (55 mg, 21%). Analytical data for (6D)-13: 1 H NMR (500 MHz, CHCl3-d1) δ 7.00 -6.83 (m, 4H, ArH), 5.55 (dd, 1H, J = 3.4, 1.0 Hz, H4), 5.46 (dd, J = 10.5, 7.9 Hz, 1H, H2), 5.09 (dd, J = 10.4, 3.4 Hz, 1H, H3), 4.90 (d, J = 7.9 Hz, 1H, H1), 4.64 (d, J = 2.4 Hz, 2H, HCCCH2-OR), 3.64 (dd, J = 4.7, 1.2 Hz, 1H, H5), 3.10 (ddd, J = 4.7, 3.

  H NMR (500 MHz, MeOH-d4) δ 7.06 (d, J = 9.2 Hz, 2H, ArH), 6.91 (d, J = 9.1 Hz, 2H, ArH), 4.72 (d, J = 7.8 Hz, 1H, H1), 4.67 (d, J = 2.4 Hz, 2H, HCCCH2-OR), 3.91 (dd, J = 3.5, 1.1 Hz, 1H, H4), 3.79 (dd, J = 9.8, 7.7 Hz, 1H, H2), 3.54 (dd, J = 9.8, 3.4 Hz, 1H, H3), 3.33 -3.29 (m, 1H, H6), 3.23 (dd, J = 6.7, 1.2 Hz, 1H, H5), 2.91 (t, J = 2.4 Hz, 1H, HCCCH2-OR), 2.83 (dd, J = 4.9, 4.2 Hz, 1H, H7a), 2.70 (dd, J = 4.9, 2.7 Hz, 1H, H7b); 13 C NMR (126 MHz, MeOH-d4) δ 154.58 (ArC), 153.83 (ArC), 119.12 (2C, ArCH), 116.86 (2C, ArCH), 103.79 (C1), 79.99 (HCCCH2-OR), 78.74 (C5), 76.54 (HCCCH2-OR), 74.47 (C3,) 72.08 (C2), 71.47 (C4), 57.19 (HCCCH2-OR), 52.92 (C6), 44.44 (C7); LC-HRMS: [C16H18O7+HCOO] -calcd: 367.1035, found: 367.1031. p-Propargyloxyphenyl 6,7-dideoxy-6,7-epoxy-β-D-galacto-heptopyranoside (6D)-15.

  All MALDI-ToF measurements were acquired on a Bruker ultrafleXtreme MALDI-ToF/ToF mass spectrometer (Bruker Daltonics, Germany) equipped with a smartbeam II solid state 1 kHz laser. Insource decay (ISD) experiments for top-down sequencing were performed in positive ion reflectron mode ranging from 4000-9000 m/z. The acceleration voltage was set to 25.00 kV, extraction voltage to 22.55 kV, lens voltage was held at 7.5 kV and reflector voltage was set to 26.45 kV. The ISD spectra comprised 80000 accumulated laser shots (arbitrary laser power of 20%) and were externally calibrated using c-type fragment ions generated from intact ubiquitin. All ISD samples were prepared by pre-mixing sample solution (250 μM protein in H2O) and matrix solution (1,5diaminonaphthalene saturated in MeCN: H2O + 0.1% TFA (50:50)) in a ratio of 1:2 (v:v) and spotted 0.5 μL onto a ground steel target.

-

  24 hours. FITC-labelled epoxide 17 was suspended in H2O/10% DMSO (0.57 mg in 680 µL), centrifuged and the supernatant was used in the staining experiments. Biofilms were stained for the presence of LecA by the addition of 44 µL FITC-labelled epoxide 17 to each well. After an incubation time of 10 -30 min at r.t., bacterial biofilms were directly visualized by measuring the fluorescence of the fluorescein-labeled LecA ligand and mCherry using a confocal laser scanning microscope (Leica TCS Sp8 CLSM). Fluorescein was excited with a 488-nm laser and mCherry at 561 nm. Focal planes were acquired starting from the bottom of the plate (position 0) with an interplane distance (z-step size) of 2 μm using a 25× numerical-aperture water objective. Images were batch-processed using the ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016.) for background correction and noise filter ("remove outliers").

3 mM IC 50 > 3 mM IC 50 = 64 ± 42 µM Scheme

	4	5, 66%	6, 70%	7		8, 33% (3 steps)
						HO
					HO	O
						O
					HO	O
	(vi)			HO		2, 9%
	8		HO	O
				O
			HO	CH 2		HO
		9, 56%		10, 97%	HO	O	O
				IC 50 >	HO	O
						3, 19%

1: Synthesis of LecA-directed epoxides and competitive binding IC50 values for LecA. (i) acetone, ZnCl2, H2SO4; (ii) (COCl)2, DMSO, NEt3, CH2Cl2, -78 °C to 0 °C; (iii) PPh3*MeI, NaH, DMSO; (iv) 70% HOAc aq.; (v) Ac2O, pyridine; (vi) PhOH, BF3*Et2O, CH2Cl2, -20 °C -r.t.; (vii) NaOMe, MeOH, r.t.; (viii) mCPBA, NaHCO3, CH2Cl2/MeOH;

  3 mg, 1.2 µmol) and sodium ascorbate (80 µL, 100 mM in H2O, 8 µmol), the reaction was stirred at r.t. for 3 h. After lyophilization, the residue was purified by preparative HPLC (C18, H2O/MeCN, gradient of 20-60%) to give pure (6D)-17 (2.8 mg, 76%). LC-MS: [C45H48N5O15S] + calcd: 930.3, found: 930.3.

  μL of a stock solution of LecA and fluorescent ligand (final assay concentration 10 nM) in TBS/Ca were added 10 µL serial dilutions of test compounds in TBS/Ca in triplicates in black 384-well microtiter plates (Greiner Bio-One, Germany, cat no 781900). After addition of the reagents, the microtiter plates were centrifuged at 800 rpm for 1 min at 23 °C and subsequently incubated for 4-6 h at r.t. in a humidity chamber on a rocking table. Fluorescence was measured on a PheraStar FS plate reader (BMG Labtech GmbH, Germany) with excitation filters at 485 nm and parallel and perpendicular emission filters at 535 nm. The measured intensities were reduced by the values of only LecA in buffer. The fluorescence polarization data were calculated and analyzed with MARS Data Analysis Software (BMG Labtech

Acknowledgements

The authors acknowledge the continuous support of Prof. Rolf W. Hartmann (HIPS). We are further grateful to Prof. Bodo Philipp (University of Münster) and Prof. Arthur F. J. Ram (Leiden University) for providing bacterial strains and plasmids used in this study, to MSc. Sarah Henrikus (Saarland University) for the synthesis of 16 and to MSc. Laura Becker (University of Konstanz) for initial synthetic experiments towards the galactose-derived epoxides and Dr. Josef Zapp (Saarland University) for acquisition of NMR spectra. Emilie Gillon prepared LecA for crystallization and performed some crystallization trials. A.V. acknowledges financial support from the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570). The authors thank the EMBL-Grenoble staff for assistance in using the High Throughput Crystallisation (HTX) facility and the European Synchrotron Radiation Facility, Grenoble, France for the access and technical support to beamline BM30A-FIP. A.T. acknowledges the following funding agencies for generous funding: the Helmholtz-Association (grant no. VH-NG-934), the Deutsche Forschungsgemeinschaft (grant no. Ti 756/2-1) and the European Research Council for an ERC Starting Grant (SWEETBULLETS).