Saida Leboukh

Hicham Gouzi
email: gouzi_h@yahoo.fr

Thibaud Coradin
email: thibaud.coradin@upmc.fr

Harek Yahia

An optical catechol biosensor based on a desert truffle tyrosinase extract immobilized into a sol-gel silica layered matrix

Keywords: Optical Biosensor, Tyrosinase, Bioencapsulation, Silica, Catechol

An optical biosensor for the determination of catechol, a widely used yet toxic and carcinogenic molecule, is proposed using a crude extract of desert truffle (Terfezia leonis Tul.) as an enzymatic source of tyrosinase. The biosensor is constructed by the immobilization of tyrosinase crude extract in a bi-layered silica gel film prepared by dip-coating of an alkoxide/colloidal silica solution containing the enzyme on glass slide. Encapsulation has a moderate effect of the enzyme optimal pH stability but largely increases its thermal stability.

Immobilized enzymes have a higher substrate affinity towards catechol but smaller maximum conversion velocity. The optical biosensor provides a linear response for catechol in the concentration range of 50-400 µM and a limit of detection was 52 µM. AFM studies show that the enzymes impact on the silica gel structure, preventing further deposition of additional layers. Comparison with similar dopamine biosensors points out that the impact of encapsulation on enzymatic activity may depend on the considered substrate.

Introduction

Catechol is an important phenolic compound which is widely used in tanning, plastics, pesticides, dyes, cosmetics, and pharmaceutical industry, and is released into the environment as an industrial waste and a source of environmental pollution [1]. Moreover, it is toxic to humans and animals even at very low concentrations [START_REF]Toxics Release Inventory Basis of OSHA Carcinogens[END_REF]. In consequence, it is crucial to establish a rapid and effective method to titrate catechol in contaminated waters.

Current methods for catechol detection include spectrophotometry, capillary electrophoresis, voltammetry and chromatography [START_REF] Tsai | [END_REF][4][5][6]. Catechol biosensors have also received considerable attention in recent years, using laccase and/or tyrosinase enzymes [7][8][9][10][11].

Tyrosinase is a copper-containing monooxygenase enzyme responsible for the first step of melanin pathway, starting from catechol as substrate leading to the formation of obenzoquinone, a strongly yellow colored product that can be used in simple optical bioassays [12,13].

For the fabrication of enzymes biosensors, it is very important to choose the appropriate preparation method and film matrix for immobilizing enzymes; these factors can directly affect the activity, stability as well as other performances of the biosensor [14][15][16][17]. Among available technologies, the sol-gel bioencapsulation process, mainly involving silica-based materials, appears as a suitable approach to obtain chemically and physically-stable immobilization hosts, with tailorable porosity and good optical transparency [18][19][20][21][22]. In this context, the encapsulation of tyrosinase in silica-based materials for sensing or degradation purposes has already been reported [23][24][25][26].

In particular we have previously shown that a crude enzymatic extract of a desert truffle exhibiting polyphenol oxidase activity could be immobilized in a bilayered silica film and used for the detection of dopamine [27]. The advantage of this strategy is that it avoids the need for enzyme purification that is both time-and cost-consuming.

In this work, we demonstrate that this approach can be extended to the design of a catechol biosensor but also show that the impact of encapsulation on the enzymatic activity depends on the considered substrate.

Experimental

Extraction of tyrosinase

The fresh black wild truffles (Terfezia leonis Tul.) for tyrosinase extraction were obtained from Algerian Southern. The protocol for extraction was described by Gouzi et al. [28]. First, 50 g of peridium from truffles were homogenized in 100 mL of 0.05 M phosphate buffer (pH 7.0), using a Waring blender for 2 min. The resulting homogenate was filtered through four layers of cheesecloth, and the filtrate was centrifuged for 10 min at 4,500 rpm. The supernatant (40 mL) constituted the tyrosinase crude extract and was kept frozen at -10°C until use.

Preparation of tyrosinase-based biosensor

The immobilization of the tyrosinase within silica layers was performed using a previously described method [27]. Briefly, 1.5 mL of a pre-hydrolyzed solution of tetraethoxysilane (TEOS) was mixed with 1.5 mL of colloidal silica LUDOX-HS 40 in acidic conditions (pH 2.0). The mixture was neutralized by addition of 1 mL of Tris-HCl (0.05 M-pH 8.6) containing 2 % glycerol. Finally, 1 mL of the desert truffle tyrosinase crude extract was added. The resulting solution was placed in transparent cuvettes (1-cm width). After 30 min of ageing at room temperature, glass slides (5 mm×45 mm), which had been soaked in concentrated nitric acid for 24 h and washed with acetone, ethanol and distilled water, were immersed in this solution for 10 s and then slowly withdrawn (rate = 0.25 cm/s). The films were dried for 30 min under ambient conditions and another dip-coating step was performed. The resulting two-layer films were place in an eppendorf tube containing sodium phosphate buffer (0.05 M-pH 7.0) and stored in the fridge (4°C).

Free and immobilized tyrosinase activity assays

The activity of free and immobilized tyrosinase was determined by using UV-vis Spectrophotometer (Amersham Biosciences UV/visible spectrophotometer Ultrospec™ 1100 pro), by measuring of the initial rate of o-benzoquinone formation at 410 nm. The reaction was initiated by adding appropriate enzyme amount or two layers of silica containing enzyme to catechol solution prepared in sodium phosphate buffer (0.05 M-pH 7.0).

Optimum pH and thermal stability

Thermal stability experiments were carried out with free and immobilized tyrosinase which were incubated in the absence of substrate at various temperatures (30-60°C). The temperature stability was determined by assaying the residual activity after incubating enzymes at a given temperature for 10 min. The heat-treated enzymes were rapidly cooled in an ice bath prior to assay for residual activity with catechol as substrate.

The optimum pH of free and immobilized enzyme was determined by carrying out the enzyme assay at different pH values using the following buffers: 0.05 M sodium acetate/acetic acid (pH 4.0-5.6); 0.05 M monosodium dihydrogen phosphate-disodium hydrogen phosphate (pH 6.0-8.0). All measurements and pre-equilibration were carried out at 30°C. The results for both pH and temperature were normalized, considering as 100% enzymatic activity the highest value obtained in each of the series of measurements made. All activity tests were conducted in triplicate.

Determination of kinetic constants

The kinetic constants, Km and Vmax values of the free and the immobilized tyrosinase were determined by measuring initial rates of the reaction with catechol at different concentrations (0.5-20 mM) in phosphate buffer (0.05 M, pH 7.0) at 30°C.

Km and Vmax values of the free and immobilized enzyme were calculated from Lineweaver-Burk plot from the initial rate of the reaction data obtained during enzymatic oxidation of catechol by using the Kaleidagraph software.

Calibration curve of the tyrosinase-based optical biosensor

To assess the biosensor performance, the optical density versus catechol concentration was plotted and the linearity domain determined via a linear regression model using the Kaleidagraph software.

Characterization of surface morphology

The surface morphology of silica sol-gel tyrosinase-based biosensor was investigated by atomic force microscopy, AFM (DI CPII, Veeco) using tapping mode, with commercially ultrasharpened Si3N4 tips in a 3 µM×3 µM scanning area. The measurements were performed at air-ambient (25°C and 35% relative humidity). A tyrosinase-free film was also imaged for comparison. Sol-gel thin films were also characterized for refractive index and thickness using a Woollam M200-U variable-angle spectroscopic ellipsometer. The ellipsometric spectra recorded ranged from 250 nm to 900 nm with incident angle of 45°. Reflectance spectrum at an incident angle of 75° was collected. A simple Cauchy's equation was used for modeling the optical properties of the porous silica films.

Results and discussion

Optimal pH and thermal stability of free and immobilized tyrosinase

pH is one of the most important factors influencing not only the side groups of the amino acid dissociations in the protein structure but also the solution chemistry of the insoluble support. pH therefore plays an important role in the sensing efficiency of the biosensor. The effect of pH on the activity of free and immobilized desert truffle tyrosinase towards was studied in the pH range 3.5-8.0 (Figure 1). The optimum pH for both the free and immobilized enzymes was observed at pH 7.0, in agreement with previous studies [23].

The immobilized tyrosinase also displayed significantly improved stability at lower and higher pHs compared to that of the free enzyme, as already widely reported for sol-gel encapsulated enzymes [29].

The thermal stability of an immobilized enzyme is one of the most important criteria of their application. In general, the activity of the immobilized enzyme is more resistant than that of the soluble form against heat and denaturing agents [30]. Below 30°C, free and immobilized tyrosinase maintained their activity. After 10 min incubation at 50°C, 53% and 30% activity losses occurred for free and immobilized enzyme respectively (Figure 2). At 60 •C, free enzyme was completely inactivated while immobilized enzyme maintained 40% of its initial activity. Therefore, immobilized tyrosinase showed better thermal stability than free enzyme at high temperatures. This may be attributed to the fact that the confinement of the enzyme within the silica matrix can prevent its unfolding [31].

Kinetics parameters

Maximum reaction rates, Vmax, and Michaelis-Menten constants, Km, were calculated using Lineweaver-Burk plot. Vmax and Km values for free and immobilized tyrosinase were determined at constant pH (pH = 7.0) and temperature (T = 30°C) while varying catechol concentrations. The typical hyperbolic growth and saturation upon increasing the catechol concentration is evident in the Figure 3a. The kinetic parameters for encapsulated tyrosinase in silica matrix were obtained from a Lineweaver-Burk double reciprocal plot (Figure 3b), where the apparent Michaelis-Menten constant (Kmapp) and maximum velocity (Vmaxapp) were 0.35 mM and 6 µM.min -1 , respectively. These values are significantly lower than those obtained for free tyrosinase (Km = 2.5 mM; Vmax = 102 µM.min -1 ) using the same substrate.

The Kmapp value reflects the affinity of the enzyme for the substrate; the smaller the Kmapp value, the greater the affinity. Therefore, the affinity of immobilized tyrosinase for catechol is stronger than that of the free tyrosinase, in agreement with the literature [32]. In general, the KM of an immobilized enzyme is different from that of the free enzyme due to diffusional limitations, steric effects and ionic strength. The change in affinity of the enzyme to its substrate is probably caused by structural changes in the enzyme introduced by immobilization procedure. There is also partitioning of substrate between the solution and support so that the substrate concentration in the neighborhood of the enzyme may be significantly different from that in the bulk solution, leading to a decrease of the Vmax value upon immobilization.

Biosensor performances

Under the optimal conditions (50 mM phosphate buffer solution (pH 7.0, 30°C), we determined catechol concentration by spectrophotometry. The absorbance increased linearly and proportionally with the catechol concentration in the range 50-400 µM, following the equation Abs410 nm = 2.6×10 -5 [Catechol] (µM) + 3.6×10 -3 (r 2 = 0.9924) (Figure 4), indicating that the enzyme catalytic reaction of catechol is a first-order reaction. A deviation from a linear relationship between concentration catechol and absorbance was observed at high concentrations that can be explained by strong molecular interactions [33].

The data have been fitted to a straight line and the limit of detection (LOD) was calculated according to Miller and Miller [START_REF] Miller | Statistics and Chemometrics for Analytical Chemistry[END_REF]:

𝐿𝑂𝐷 = &3 × 𝑆 */, - 𝑎
where a is the sensitivity of the method (the slope of the calibration curve) and Sy/x is the standard deviation of the fit. The LOD was calculated to be 52 µM, in the upper limit of the acceptable concentration of phenolic compounds in treated wastewater [START_REF] Mandal | [END_REF].

Reproducibility and stability are important parameters to be considered for a biosensor. They were examined in the solution containing 2 mM catechol. In terms of reproducibility, five different films were used and measurements were achieved with a variation coefficient of 4.7%.

In terms of operational stability, as seen in Figure 5, 30% of initial tyrosinase activity was retained up to three cycles of reutilization. The decrease in the tyrosinase activity may be due to enzyme denaturation, leaching of enzyme from the carrier and due to inactivation of enzyme by catechol. In particular tyrosinases are prone to suicide inactivation resulting in the release of one of the two copper ions from the enzyme active site [36].

Effect of the number of silica layers

The optical response of tyrosinase biosensor depends on the amount of enzymes, which is a function of the number of assembled desert truffle tyrosinase silica layers. As seen on Figure 6, the initial rate of catechol oxidation by the double-layer system studied above was twice as high as for a single silica-tyrosinase layer. However, when a third layer was deposited, this rate only increased moderately and in a less reproducible manner, suggesting that the coating process was only partially successful [37].

However, the possibility to obtain three successive silica layers following the same protocol, except for the absence of the tyrosinase, was previously reported [38]. We therefore hypothesized that the enzyme could impact on the structure of the deposited layers. AFM imaging of the surface of a two-layer silica-tyrosinase film showed a porous structure formed by the aggregation of well-defined nanoparticles 10-30 nm in diameter, with an average roughness Ra of 17 nm (Figure 7a). The thickness and refractive index of one and two layers of silica films have been calculated from the ellipsometry data. A single layer had a thickness of 222 ± 4 nm while the two layers were 362 ± 5 nm thick. Refractive index under ambient conditions was 1.505 ± 0.005 for both conditions, confirming that the layers were porous.

As a comparison, the surface of an enzyme-free film showed a much denser network of aggregated nanoparticles with Ra of 12 nm (Figure 7b). It indicates that the presence of the enzyme extract interferes with the process of gel formation. The resulting porosity may lead to the non homogenous deposition of the silica sol during the third coating step and therefore contribute to its poor reproducibility"

Comparison with related dopamine biosensors

It is interesting to compare these results with a previous study describing dopamine biosensors prepared using the same enzymatic catalysts and the same sol-gel process [27]. In this previous work, the pH domain of maximum activity was extended over 2 pH units compared to the enzyme alone and the optimal temperature was shifted by ca. 10°C after encapsulation. Since the enzyme and the matrices are the same, such a difference should mainly reflect modification of the substrate reactivity. It is possible to suggest that this is related to the presence of an additional alkylamine chain on the aromatic ring of dopamine compared to catechol, making the former more sensitive to the pH and the silica surface compared to the later. Noticeably, Kmapp and Vmapp variations resulting from encapsulation were of the same order of magnitude for both substrates. However they were determined in different optimal conditions for dopamine (pH 5.6, T = 20°C) and catechol (pH 7.0, T=30°C), making a strict comparison difficult.

Conclusions

An optical biosensor allowing for catechol detection was obtained by encapsulation of a tyrosinase extract within a bi-layered silica film. It exhibits a linear response range over one concentration decade. However its limit of detection was in the upper range of recommended value for phenolic molecules content in treated wastewaters indicating that further efforts must be made, especially to increase the enzyme content of the device.

Considering the diversity of catalytic reactions that can be performed by tyrosinases, the hereproposed strategy may be extended to the detection of a wide range of other analytes, although our data suggest that the biosensor performance may significantly depend on the nature of the substrate.

Figure captions

Figure 1 .

 1 Figure 1. Optimum pH of free (•) and immobilized tyrosinase (○).

Figure 2 .

 2 Figure 2. Temperature stability of free (•) and immobilized tyrosinase (○). The samples were

Figure 3 .

 3 Figure 3. Michaelis-Menten (a) and Lineweaver-Burk (b) graphics of free (•) and immobilized

Figure 4 .

 4 Figure 4. Biosensor response as optical density at 410 nm after 3 min of contact with tyrosinase

Figure 5 .

 5 Figure 5. Repeated uses of immobilized tyrosinase.

Figure 6 .

 6 Figure 6. Increase of the rate of benzoquinone formation as a function of the number of silica

Figure 7 .

 7 Figure 7. AFM images of bilayered silica films (a) with and (b) without tyrosinase

Acknowledgments

The authors thank Dr Marco Faustini and Dr Christophe Depagne (LCMCP) for AFM experiments and Dr. Cédric Boissière (LCMCP) for ellipsometry analysis. We would also like to thank Pr. Aziz Amine for her careful proofreading and copy-editing for grammar.

Conflict of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.


Somasoudrame Rassou

Nicolas Illy

Ozgul Tezgel

Philippe Guégan
email: philippe.guegan@upmc.fr

Anionic ring opening polymerization of N-glycidylphthalimide: combination of phosphazene base and activated monomer mechanism

Anionic ring-opening polymerization of N -glycidylphthalimide: Combination of phosphazene base and activated monomer mechanism

Somasoudrame Rassou, Nicolas Illy, Ozgul Tezgel, Philippe

INTRODUCTION

The field of anionic ring-opening polymerization (AROP) of epoxides experiences a recent revival. 1 Polyethers are a very important class of materials that are industrially produced in million tons scale per year. They are used in high diffusion applications (e.g. surfactants, cosmetics) or in more advanced applications (e.g. biomedical field). 2 The polymerization of functional epoxide monomers that possess reactive groups either directly accessible or protected are of particular interest. However, even if the demand for functional polyethers increases steadily, the number of functional epoxide monomers that can be polymerized by AROP is still limited. 1,3 Amino functional polyethers are of particular interest due to the number of potential applications, such as heavy metal recovery, 4 gene delivery, 5,6 wastewater treatment, 7 surface engineering. 8 Polyfunctional primary amine-containing polyethers can be prepared by two different strategies. The first one is the postmodification of a precursor polymer using, for example, thiol-ene addition of poly(allyl glycidyl ether), 9 azidation-reduction of polyepichorhydrine 10 or reduction of poly(epicyanohydrin). 11 However, postmodification reactions often result in nonquantitative conversion or require multi-step syntheses. Therefore, the second strategy consisting in the direct polymerization of protected amine groups is an interesting but under-explored solution. There are a few epoxide monomers functionalized with protected amino groups reported to be polymerized by AROP: N,Ndibenzylglycidylamine (DBGA), 12,13 N,Ndiallyglycidylamine (DAGA), 14 Boc-protected butanolamine glycidyl ether (BBAG). 15 Moreover, glycidyl phthalimide is an interesting protected-amine-containing monomer which has been used to functionalize hyperbranched polyglycerols with amine groups. 16,17 However, ABSTRACT : Anionic ring-opening polymerization of glycidyl phthalimide, initiated with alcoholphosphazene base systems and based on monomer activation with a Lewis acid (iBu3Al), has been studied. No propagation occurred for initiator : iBu3Al ratios less or equal to 1 : 3. For larger Lewis acid amounts, the first anionic ring-opening polymerizations of glycidyl phthalimide were observed. Polymers were carefully characterized by NMR, MALDI-TOF mass spectrometry and size exclusion chromatography and particular attention was given to the detection of eventual transfer or sidereactions. However, polymer precipitation and transfer reaction to aluminum derivative were detrimental to monomer conversion, polymerization control and limited polymer chain molar masses. The influence of reaction temperature and solvent on polymer precipitation and transfer reactions were studied and reaction conditions have been optimized leading to afford end-capped poly(glycidyl phthalimide) with narrow molar mass distributions. Keywords : Anionic polymerization; Ring Opening Polymerization; Phosphazene bases; epoxy; living polymerization. the homopolymerization of the monomer by AROP has never been successfully achieved and only single additions of epoxides were observed. Möller and coworkers reacted glycidyl phthalimide with potassium tert-butoxide in dioxane and DMSO but no polymerization took place. 10 The use of Oct4NBr in combination with triisobutylaluminum (iBu3Al) Lewis acid according to an activated monomer mechanism in toluene initiated the polymerization, but it did not enable further growth of polymer chains. 10 Protic species -phosphazene base systems have emerged as efficient catalyst systems for the living polymerization of various functional monomers. 13,[18][19][20] Protic groups are deprotonated by phosphazene bases and initiated the anionic polymerization. The protonated bulky cations formed by the deprotonation process generate highly reactive ion pairs which results in an increase of the propagation rate. Satoh and coworkers reported that the combination of tBuP4 and alcohol is an excellent catalytic system for the living ringopening polymerization of several N,Ndisubstituted glycidyl amine derivatives, such as N,N-dibenzylglycidylamine, N-benzyl-Nmethylglycidyl amine or N-glycidylmorpholine. 13 Nevertheless, attempts to apply the previous initiating system to N-glycidylphtalimide failed to obtain corresponding polymers. 13 This lack of polymerizability has been attributed to the high steric hindrance of the phthalimide group. 3 To the best of our knowledge, poly(glycidylphthalimide) was only synthesized once according to a cationic ring-opening polymerization mechanism with Lewis acid and methylaluminium bis(2,6-di-tert-butyl-4methylphenoxide) (MAD) as initiator by Kanoh et al. 21 The synthesis was performed in toluene at 100 °C and a polymer with a 1700 g.mol -1 number average molar mass (Mn) and a large dispersity (Ð = 1.63) was reported. Harsh polymerization conditions are required to obtain a polyether backbone with phthalimide pendant groups. Only one reaction was carried out and the polymer was not thoroughly characterized, e.g. end-chains were not identified. In addition, the difference between theoretical and experimental molar masses has not been explained.

In this paper, polymerization of the very challenging glycidyl phthalimide monomer by AROP is reported for the first time. For this purpose, we used a Lewis acid which activates the monomer by increasing its electrophilicity and a phosphazene base resulting in highly nucleophilic propagating centers due to a high degree of ion-pair separation with phosphazenium counterion. 22 In addition, we have also studied the effect of solvent in polymerization, since the solubility of polyglycidylphthalimide is a limiting factor in chain growth. The resulting polymers were fully characterized by NMR, SEC and MALDI-TOF. A particular attention was given to the determination of molar masses, potential transfer reactions as well as end-groups.

EXPERIMENTAL

Materials.

Phosphazene base tBuP4 solution (0.8 mol L -1 in hexane, Aldrich), 2-methyltetrahydrofuran (98%, Aldrich), benzyl alcohol (99%, Aldrich) and triisobutylaluminium iBu3Al solution (1.1 mol L -1 in toluene, Acros) were used without further purification and were stored in a glove box. N-(2,3-Epoxypropyl)phthalimide (95%, Acros, glycidyl phthalimide) was sublimated at 90°C under reduce pressure. Toluene, dichloromethane and tetrahydrofuran were dried with an MBRAUN MB SPS-800 solvent purification system under nitrogen. Ndimethylformamide (anhydrous, 99.8%, Aldrich) was distilled over CaH2 two times prior to use. Instruments 1 H and 13 C NMR analyses were performed on a Bruker Avance 300 MHz and a Bruker Avance 400 MHz spectrometers. All spectra are internally referenced to residual proton signals of the deuterated solvent: CDCl3 or CD2Cl2. Polymer molar masses were determined by size exclusion chromatography (SEC) using DMF (containing 1.0 g L -1 lithium bromide as an additive) or THF as eluent. In DMF, the analyses were performed at 60°C, and at a flow rate of 0.8 mL.min -1 , at a polymer concentration of 5 mg.mL -1 after filtration through 0.22 μm. The steric exclusion was carried out on two PSS GRAM 1000 Å columns (8 × 300 mm; separation limits: 1 to 1000 kg.mol -1 ) and one PSS GRAM 30 Å (8 × 300 mm; separation limits: 0.1 to 10 kg.mol -1 ) coupled with three detectors (Viscotek, TDA 305) : a differential refractive index (RI) detector and an UV detector. In THF measurements were carried out on three PL Gel Mixte C 5µm columns (7,5 × 300 mm; separation limits: 0,2 to 2000 kg.mol -1 ) maintained at 40°C coupled with 2 modular detectors: a differential refractive index (RI) detector Viscotek 3580 and a Diode Array UV Detector Shimadzu SPD20-AV. THF was used as the mobile phase at a flow rate of 1 mL.min -1 , toluene was used as a flow rate marker. All polymers were injected (50 μL) at a concentration of 5 mg.mL -1 after filtration through a 0.45 μm pore-size membrane. In both cases, the OmniSEC 4.6.2 software was used for data acquisition and data analysis. The numberaverage molar masses (Mn), the weight-average molar masses (M� w), and the molar mass distributions (Đ = Mw/Mn) were determined by SEC with a calibration curve based on narrow poly(methyl methacrylate) (PMMA) standards (from Polymer Standard Services), using the RI detector.

Flight (MALDI-TOF) mass spectrometry (MS).

Mass spectra were recorded by matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) using a Bruker autoflex III smartbeam mass spectrometer, equipped with the laser that produces pulses at 337 nm using dithranol as a matrix and NaI as cationizing agent. Spectra were recorded in reflector or in linear mode at an accelerating potential of 20 kV. Samples were prepared by dissolving the polymer in THF at a concentration of 5 mg.mL -1 . A 10 μL aliquot of this solution was mixed with 20 μL of matrix solution and 10 μL of NaI solution (both at 20 mg.mL -1 in THF). Standards (poly(ethylene oxide) of known structures, Mn = 1120, 3500 and 5500 g.mol -1 purchased from Polymer Standards Service) were used to calibrate the mass scale. In all cases, to determine m/z, the molar mass of the sodium cation was added.

Anionic ring opening polymerization of glycidylphthalimide

Polymerizations

were carried out in polymerization tubes equipped with a magnetic stirrer according to the following procedure. All the mixtures were performed in a glovebox under a nitrogen atmosphere. In the polymerization tube, benzyl alcohol (1 equiv, 3 μL, 0.03 mmol) was deprotoned by the phosphazene base (1 equiv, 37 μL, 0.03 mmol) in dried toluene (2,5 mL) into polymerization tube for 10 min. In a separate vial, triisobutylaluminium (7 equiv, 190 μL, 0.21 mmol) was added on sublimated monomer (50 eq, 0.3 g, 1.50 mmol) in 2.5 mL of dried toluene. The monomer mixture was added dropwise in the polymerization tube. The polymerization was stirred at 25°C, left to react for 3h and quenched with acetic acid. The solvent was evaporated yielding a brown product. This product was solubilized in methylene chloride and precipitated into 150 mL diethyl ether to remove the phosphazene base (tBuP 4), monomer and triisobutylaluminium. A white solid was obtained. The polymer conversion was determined by 1 H NMR spectroscopy and molar masses were determined by SEC.

RESULTS AND DISCUSSION

Initially, polymerization reactions were carried out in toluene (Table 1, run 1-12). 21 For all the experiments, targeted degrees of polymerization (Xn), as calculated from [M]0/[ROH]0 ratio (i.e. assuming quantitative initiation), were deliberately set between 20 and 100 so that chain ends could be easily characterized by 1 H NMR and MALDI-TOF mass spectrometry. In our first trial, 1 equiv of the phosphazene base with respect to the primary alcohol initiator (benzyl alcohol) was used and this condition did not lead to polymerization (Table 1, run 1), which is Table 1. Experimental conditions and molecular characteristics of Poly(glycidylphthalimide) samples initiated benzyl alcohol in the presence of tBuP4 and iBuAl3 in toluene.

Run

[M] (mol.L -1 )

[M]0/[ROH]0/[tBuP4 ]/[iBu3Al] T°C Time (h) Conv a (%)
Mn,th b (g.mol -1 )

Mn,sec c (g.mol -1 ) supporting the earlier results published by Isono et al. 13 Further, we included iBu3Al to our system in addition to tBuP4 and benzyl alcohol. Therefore, for the rest of the polymerization experiments, 1 equiv of the phosphazene base with respect to benzyl alcohol and an increasing amount of iBu3Al, were used. 23,24 Experiments performed with 2.5 equiv (Table 1, run 2) and 3.0 equivalents of iBu3Al (Table 2, run 3) did not generate polymers. Several authors used glycidyl phthalimide monomer to end-cap polyglycidol by ring-opening reaction, 10,16 and they interpreted this lack of homopolymerization to high steric hindrance or aggregation issue caused by the phthalimide group rather than the monomer reactivity.

Ð % ISO 1 0.3 50/1/
The anionic ring-opening polymerization of glycidyl phthalimide has been observed for the first time when [iBu3Al]/[ROH] ratio ≥ 3.6 (Table 1, runs 4-12). A minimum amount of Lewis acid is required for the propagation step to occur. This ratio is still unexplained but we assume this might result from the establishment of coordination complexes between propagating center, Lewis acid and phosphazenium cation and by the necessity to activate monomers by coordination of an excess of Lewis acid with glycidylphthalimide molecules (Scheme 1). A complexation equilibrium is established between free monomer, monomer complexed with iBu3Al either on carbonyl groups, on nitrogen atom or on ether linkage. When iBu3Al interacts with ether linkage, electrons are transferred from the oxygen atom to the aluminum, generating a strong electronwithdrawing effect on the α-carbon. 23 The αcarbon electrophilicity is increased resulting in monomer activation followed by an attack of the nucleophilic alcoholate complex and the ringopening of the monomer.

FIGURE 1 SEC trace of poly(glycidylphthalimide) initiated by benzyl alcohol in the presence of 1equiv of tBuP4 and 3.6 equiv of iBu3Al (Table 1, run

For experiments carried out with 3.6 equiv iBu3Al (Table 1, run 4-5) at [M]0 = 0.3 mol.L -1 , monomer conversion and molar masses are limited by the precipitation of polymer chains during propagation step and the resulting polymers exhibit quite large molar mass distributions (Ð = 1.5-1.6) (Figure 1). Polyglycidylphthalimide (PGht) polymers were purified by precipitation in diethyl ether. Molar masses were determined by SEC using PMMA standards. PGht was obtained with molecular weights, Mn up to 1900 g mol -1 (Table 1, run 4-7, Xn= 9, figure 1) and molar mass distributions ranged from 1.5 to 1.6. Molar masses given by the MALDI-TOF spectra are higher than Mn obtained by SEC (approx. 3000 vs 1900). We assume that molar masses determined by SEC are strongly underestimated due to interaction between phthalimide lateral substituents and the column stationary phase. Molecular weight is limited by transfer reactions with triisobutylaluminium (Figure S2). One iBu3Al molecule forms four-center aluminatetype complex with alkoxide and phosphazenium cation according to a similar mechanism to the one proposed by Rejsek et al. in the case of ethylene oxide polymerization initiated by butyl lithium in the presence of iBu3Al. 24 Inside the complex, a ligand exchange can be observed followed by a new complex formation (iBu2AlOR) and isobutyl-phosphazenium moieties. Then, the isobutyl group attacks activated glycidylphthalimide and initiates a new macromolecular chain.

Experiments were performed at lower monomer concentrations in order to minimize the precipitation of the growing polymer chains and thus to increase monomer conversion (Table 1, runs 6-7). As expected, monomer conversion increases when decreasing initial monomer concentrations. But at the same time, undesirable transfer side reactions become more and more important, limit molar masses and negatively affect the control of polymer endgroups. For example, on the MALDI-TOF spectrum of run 7 Table 1, benzyl terminated chains (P1) are not the main distribution anymore which now consists of isobutylterminated chains (P2) (Figure 2.b). Another major peak series can be attributed to polyglycidylphthalimide chains with methyl and hydroxyl end groups (P4), which indicates the occurrence of a transfer reaction to the aluminum derivative resulting in hydride initiation. 25 Peaks corresponding to isobutyl endgroups are observed at 0.61, 0.77 and 1.15 ppm in 1 H NMR spectra. The molar fraction of macromolecular chains resulting from transfer reactions to aluminum derivative (%ISO, Table 1) was roughly calculated by 1 H NMR using the endgroup aromatic protons and the CH3 end-group protons resulting from transfer reactions. Chains initiated by moisture traces are neglected in the previous calculation. These values are in good agreement with the relative sizes of the different populations in the MALDI-TOF spectra. A kinetic study was carried out (Table 1, run 6). According to an activated monomer mechanism, 23 we assume an apparent zero monomer order with respect to the monomer. Plot of monomer conversion versus time is shown in Supporting Information (Figure S3). The monomer conversion rapidly increased during the first minutes up to 30 %. Then, the increase is greatly slowed down but the propagation is not fully stopped. This event was observed in parallel to the polymer precipitation. In the next step, the amount of aluminum derivative was increased, in order to shift the chemical equilibrium toward the activated form of the monomer, promoting the propagation rate versus the precipitation rate. The following experiments were carried out with a ratio [iBu 3Al]/[ROH] = 7,5 (Table 1, run 8-10) at 25°C. However, a higher ratio of iBu3Al did not help to prevent polymer precipitation and we did not observe a significant improvement on the monomer conversion rate or molar masses. In addition, these conditions favored transfer reaction as illustrated by the comparison between the MALDI-TOF spectra (Figures 2a and2c) and the 1 H NMR spectra (Figures S1a andS1c) of reactions performed with 3.6 and 7.5 equivalents of iBu3Al, respectively. On the other hand, decreasing the reaction temperature to 0°C led to an increase of the monomer conversion and of the Mn (3100 g.mol - 1 , Table 1, runs 11). However, we obtained the polymer with a high polydispersity (Ð = 2.4) and having a low molecular weight fraction (red line-Figure 3). MALDI TOF analysis confirmed the occurrence of transfer reactions to the aluminum derivative and that polymer endgroups are not controlled. For a targeted degree of polymerization of 100 (Table 1, run 12), the dispersity was higher and the molar mass distribution was clearly bimodal: one high molar mass population with a number-average molar mass equal to 18 000 g.mol -1 and a low molar mass distribution with Mn equal to 2200 g.mol -1 .

FIGURE 3 SEC profiles in DMF of poly(glycidylphthalimide)

synthesized at [M]0=0.3 mol.L -1 with 7.5 equiv iBu3Al.

A polymerization reaction was carried out a second time under the same conditions (Table 1, run 12). ) and after 1 h reaction time, precipitate and supernatant were analyzed separately. Both size exclusion chromatograms are superimposed in Figure 4. The precipitate essentially consisted of large macromolecules (Mn = 6700 g.mol -1 , Ð = 1.66) while the supernatant mostly contained macromolecules with lower molar masses. One possible explanation is that macromolecules grew until their precipitation when the solubility limit of the polymer is exceeded. This solubility limit is temperature dependant: increase of the solubility limit with lower temperatures. Smaller macromolecules in the supernatant are generated by transfer reactions mostly to the aluminum derivative. In conclusion, the limitation of the achievable molar masses is related to a physical issue rather than a chemical reactivity issue. Polymerization reactions were investigated in solvents of moderate polarity (i.e. dichloromethane, THF and MeTHF) and high polarity (i.e. DMF) in order to concomitantly increase the solvatation of the propagating anions and the polymer chain solubility. The stabilization of the propagating center by solvatation should also slightly decrease the interaction between the propagating center and the aluminum derivative resulting in a decrease of the transfer reaction to the aluminum derivative DMF has already been used as solvent for the anionic polymerization of oxirane monomers. 26 However, no glycidylphtalimide polymerization was observed after 94 h for a reaction performed at 25°C in this polar solvent (Table 2, run 1). The strong interaction between the highly polarized carbonyl and triisobutylaluminum is one plausible explanation for this result. Poly(glycidylphthaliminde) is soluble in dichloromethane, which has been used for NMR analyses. In addition, Carlotti and coworkers have shown that a living polymerization of ethylene or propylene oxide can be achieved in dichloromethane. 24 A reaction was carried out in this solvent (Table 2, run 2). The polymerization system stayed homogenous and no precipitation was observed. But the MALDI-TOF spectrum (Figure S4) shows a major distribution corresponds to polymer chains initiated by chloride anions resulted from transfer reactions to the solvent. Therefore, use of dichloromethane was ruled out. Run 3 Table 2 was performed in THF. The reaction mixture stayed homogenous during all the polymerization time. The MALDI-TOF spectrum (Figure S5) shows that the main distribution corresponds to polymer chains with the expected end-chains and the numberaverage molar mass value is in agreement with the theoretical one. Nevertheless, even after 4 days, monomer conversion was low compared to reaction performed in toluene (Table 1). THF behaves like a Lewis base and interact with trialkylaluminum decreasing the monomer activation efficiency. This result is in agreement with Carlotti and coworkers who showed that THF is not a suitable solvent for epoxide polymerization in the presence of triisobutylaluminum due to competitive complexation. 27 Later, we used MeTHF as a solvent. Compared to THF, the methyl group of MeTHF decreases its polarity and thus the aforementioned interaction should be reduced. 28 Moreover, MeTHF is a biomass-derived chemical, which is currently considered as a promising environmentally friendly alternative to petrobased solvents. For a [ROH]/[tBuP4]/[iBu3Al] ratio = 1/1/7.5 (Table 2, run 4) the polymerization system was homogenous for 1 hour and then a heteregeous system was observed. A MALDI-TOF spectrum of the final polymer (Figure 5) displays a major peak distribution corresponding to the expected polymer cationized by a sodium ion and endterminated by a benzyloxy group on one end and by an hydroxyl group on the other end. As expected the molecular weight difference between two successive peaks is strictly equal to the molecular weight of one repeat unit (203.06 mass units). Figure 5 shows this mass difference between two monoisotopic ions, m/z 1959.60 and 2162.68, corresponding to the Na +cationized oligomers containing 9 and 10 repeat units. Four very minor distributions with the same isotopic distribution and unit distribution as the main one could also be observed on the spectrum. Transfer reactions were suppressed in MeTHF for these molar masses.

A reaction was carried out with 3.6 equiv of iBu3Al in MeTHF. The polymerization kinetics was followed by 1 H NMR, SEC and MALDI-TOF (Figures S6 andS7). As previously, monomer conversion increased over time and reached a plateau corresponding to polymer precipitation. Mn increasing of the only peak series is clearly evidenced on the MALDI-TOF spectra. Mn, MALDI is equal to 2275 g.mol -1 and is in a very good agreement with the theoretical value. None of the side reactions described previously has been evidenced by NMR or by MALDI-TOF. A kinetic study in MeTHF (Table 2, run 6; Figure S6) shows that before polymer precipitation, the propagation rate is much higher than in THF but slightly lower than in toluene, which confirms that the interactions between MeTHF and iBu3Al are strongly reduced compared to THF, leading to more efficient monomer activation. At the beginning of the precipitation, the achieved monomer conversion in MeTHF was higher than in toluene (40% and 29%, respectively). In conclusion, the synthesis of heterotelechelic polymers with controlled end-groups and low dispersities is possible in MeTHF. To complete the polyglycidylphtalimide solubilization study, toluene-MeTHF (1/4 v/v) mixture was also used as reaction solvent (Table 3). The MALDI-TOF spectra of reactions with 7.5 or 3.6 equiv of iBu3Al (Table 3, run 1 and 2) are shown in Figure S8. In both cases, the major population corresponds to the expected structure and only very minor populations coming from side-reaction were visible. Sidereactions (initiation with water, transfer reaction to iBu3Al) were slightly more present for the reaction with 7.5 equiv of iBu3Al which is consistent with a slightly lower molar mass as measured by SEC, 1 H-NMR and MALDI-TOF. The overlay of the size exclusion chromatography elution profiles reveals a slightly higher dispersity as well (Figure S9). A 13 C NMR of run 2 Table 3 is shown in Supporting Information (Figure S11): signals associated with benzyl endgroups are observed around 128 ppm. A kinetic study was performed with 3.6 equiv of iBu3Al (Table 3, run 3) and experimental data were fitted according to a zero-order equation. Polymer precipitation was observed after 45 minutes of reaction and 45 % monomer conversion. Before precipitation, the degree of polymerization increases linearly with conversion and narrow molar mass distribution are observed for the various aliquots (Figure 6) with a fairly good control of the polymerization. On the whole, results obtained in toluene/MeTHF and in MeTHF are very similar.

CONCLUSIONS

In summary, this article has demonstrated the first anionic ring-opening polymerization of glycidyl phthalimide monomer using benzyl alcohol / phosphazene base as initiating system and iBu3Al as monomer activator. Optimal conditions for the polymerization have been studied. A minimal amount of iBu3Al is necessary for the polymerization to occur but the addition of a too large amount favors transfer reactions. MeTHF is particularly suitable solvent which allows a fairly good control of the polymerization. Heterotelechelic polymers with narrow molar mass distributions were prepared. However, the achievable molar masses are limited by polymer precipitation above a specific degree of polymerization. A decrease of the reaction temperature allowed an increase of the prepared molar masses but led cocomitently to an increase of transfer reaction frequency and is therefore detrimental to polymer dispersities. These results combined with the well-known deprotection reaction of the amine groups open a new synthetic pathway for the preparation by anionic ring-opening polymerization of homopolymer or copolymers of polyethers possessing pendant amine groups with potential applications in the field of drug delivery or nonviral vectors for gene transfection.

SCHEME 1

 1 SCHEME 1 Postulated activated mechanism for the ROP of glycidylphthalimide using phosphazene base and iBu3Al as catalytic system.

2

 2

FIGURE 2

 2 FIGURE 2 MALDI-TOF spectra (linear mode) of poly(glycidylphthalimide) initiated by benzyl alcohol/ tBuP4/iBu3Al (1/1/3.5) under different reaction conditions.

FIGURE 5 .

 5 FIGURE 5 . MALDI-TOF spectrum of a poly(glycidylphthalimide) synthesized in MeTHF with benzyl alcohol as initiator and in the presence of 1 equiv tBuP4 and 7.5 equiv of iBu3Al (Table 2, run 5).

FIGURE 6

 6 FIGURE 6 AROP of glycidylphalimide synthesized with benzyl alcohol / iBu 3Al / tBuP4 as initiating system at 25 °C in toluene-MeTHF (Table 3, run 3: [I]0 = 0.02 mol.L -1 , [M]0 = 0.3 mol.L -1 , M/benzyl alcohol/iBu3Al/tBuP4 = 20/1/3.6/1): (a) zeroorder kinetic plot ; (b) relationship observed between Mn (diamond; linear trend is indicated by the dashed line) or Mw/Mn (squares) and monomer conversion.

Figure S6 .

 S6 Figure S6. MALDI-ToF spectra of poly(glycidylphthalimide)s synthesized in MeTHF with benzyl alcohol as initiator and in the presence of 1 eq. tBuP4 and 3.6 eq. of iBu3Al at various reaction times (Table 2, run 6).

  Les paramètres requis sont manquants ou erronés.

Figure S7 .

 S7 Figure S7. Anionic ring-opening polymerization of glycidylphalimide synthesized with benzyl alcohol / iBu3Al / tBuP4 as initiating system at 25°C in MeTHF (Table 2, run 6: [I]0 = 0.02 mol.L -1 , [M]0 =0.3 mol.L -1 , M/benzyl alcohol/iBu3Al/tBuP4 = 15/1/3.6/1): (a) zero-order kinetic plot ; (b) relationship observed between number-average molar mass (diamond; linear trend is indicated by the dashed line) or dispersity (squares) and monomer conversion.

Figure S8 .

 S8 Figure S8. MALDI-TOF spectra of a poly(glycidylphthalimide) synthesized in toluene-MeTHF (1/4 v/v) with benzyl alcohol as initiator and in the presence of 1 equiv tBuP4 and 7.5 equiv (a, Table 3 run 1) or 3.6 (b, Table3run 2) equiv of iBu3Al.

Figure S9 .

 S9 Figure S9. SEC profiles of poly(glycidylphthalimide) synthesized in toluene-MeTHF (1-4) under different reaction conditions.

Figure S10. 1 H

 1 Figure S10.1 H NMR spectra in CD2Cl2 at room temperature of poly(glycidylphthalimide) synthesized in Toluene-MeTHF (4-1) with benzyl alcohol-tBuP4 as initiating system in the presence of 7.5 eq. of iBu3Al with [M]0 = 0.3 mol.L -1 (Table2, run 4).

Figure

  Figure S11.13 C NMR spectra in CD2Cl2 at room temperature of poly(glycidylphthalimide) synthesized in Toluene-MeTHF (4-1) with benzyl alcohol-tBuP4 as initiating system in the presence of 7.5 eq. of iBu3Al with [M]0 = 0.3 mol.L -1 (Table3, run 2).

Table 2 .

 2 Experimental conditions and molecular characteristics of Poly(glycidylphthalimide) samples initiated benzyl alcohol in the presence of phosphazene base and iBu 3Al in polar solvent at 25°C. Determined by 1 H NMR spectra in CD2Cl2, b Mn,th(g.mol -1 ): theoretical number average molecular weight Mn=(Xn*conv*M0)+M(alcohol), c Mn,sec (g.mol -1 ): number average molecular weight estimated by SEC in DMF (60°C, PMMA standard), d This value is overestimated due to the presence of macromolecular chains initiated by moisture traces. FIGURE4SEC in THF profiles of the precipitate (red line) and the supernatant (blue line) of Table1run 12 after 1 h reaction time.

	Run Solvent ε	[M]0	[M]0/[ROH]0/	Times	Conv a Mn,th b	Mn,sec	Ð	Mn
				(mol.L -1 )	[tBuP4]/[iBu3Al]	(h)		(g.mol -1 )	(g.mol -1 )		(NMR)
	1	DMF	38.3 0.3	50/1/1/7.5	94	0	-	-	-	-
	2	CH2Cl2 8.93 0.3	50/1/1/7.5	48	20	2500	1100	1.22 -
	3	THF	7.58 0.3	50/1/1/7.5	93	20	2100	1100	1.45 -
	4	MeTHF 6.29 0.15	50/1/1/7.5	18	48	5000	2000	1.40 4100
	5	MeTHF 6.29 0.15	25/1/1/7.5	16	54	3000	1700	1.15 3900
	6	MeTHF 6.29 0.3	15/1/1/3.6	12	55	1800	1100	1.24 4100 d
	a

Table 3 .

 3 Ring Opening Polymerization of glycidylphthalimide using benzyl alcohol (ROH) as initiator, in the presence of tBuP4 and iBu3Al in toluene/MeTHF (1/4 v/v). Determined by1 H NMR spectra in CDCl3, b Mn,th(g.mol -1 ): theoretical number average molecular weight Mn=(Xn*conv*M0)+M(alcohol), c Mn,sec (g.mol -1 ) were determined by SEC in DMF (60°C, PMMA standards)

	Run [M]0	[M]0/[ROH]0/[tBuP4]	Time	Conv a Mn,th	Mn,NMR	Mn,SEC	Ð
		(mol.L -1 )	/[iBu3Al]	s (h)		(g.mol -1 ) b	(g.mol -1 )	(g.mol -1 ) c
	1	0.30	50/1/1/7.5	24	29	3000	3200	1900	1.32
	2	0.30	50/1/1/3.6	94	43	4500	3900	2000	1.18
	3	0.30	20/1/1/3.6	0.75	46	2000	3000	1700	1.12

a

Table 3 ,

 3 run 2).

ACKNOWLEDGEMENTS REFERENCES AND NOTES

Les paramètres requis sont manquants ou erronés.

Figure S1

. 1 H NMR spectra in CD2Cl2 at room temperature of poly(glycidylphthalimide) synthesized in toluene with benzyl alcohol-tBuP4 as initiating system in the presence of 3.6 eq. of iBu3Al with [M]0 = 0.3 mol.L -1 (a, Table 1 run 5), with [M]0 = 0.07 mol.L -1 (b, Table 1 run 6), or in the presence of 7.5 eq. of iBu3Al with [M]0 = 0.3 mol.L -1 (c, Table 1 run 9).

Proposed mechanisms for the transfer reactions during the anionic ring-opening polymerization of glycidylphthalimide in the presence of triisobutylaluminum.

Les paramètres requis sont manquants ou erronés.

Figure S3

. Anionic ring-opening polymerization of glycidylphalimide synthesized with benzyl alcohol / iBu3Al / tBuP4 as initiating system at 25 °C in toluene (Table 1, run 6: benzyl alcohol as initiator and in the presence of 1 eq. tBuP4 and 7.5 eq. of iBu3Al (Table 2, run 3).


Pierre Ouagne

Sullivan Renouard

Davina Michel

Eric Laine

Mechanical Properties of Flax and Hemp Yarns Designed for the Manufacturing of Geotextiles. Improvement of the Resistance to Soil born Microorganisms Introduction

Mechanical properties of flax and hemp yarns designed

for the manufacturing of geotextiles. Improvement of the resistance to soil born microorganisms.

Materials and Methods

Materials

Results

Tensile strength

Tensile strength of un-protected yarns:

Tensile strength of chitosan impregnated yarns:

  Figure 1:

  Figure 2:

Figure 3 :

 3 Figure 3:

Acknowledgements

Discussion

Conclusion