Ana Sofia Rodrigues

Marie-Thérèse Charreyre
email: marie-therese.charreyre@univ-lyon1.fr

Arnaud Favier

José Carlos Baleizão

Paulo S Farinha
email: farinha@tecnico.ulisboa.pt

Sofia Rodrigues

José Paulo

S Farinha

Temperature-Responsive Copolymers without Compositional Drift by RAFT Copolymerization of 2-(Acryloyloxy)ethyl trimethylammonium chloride and 2-(Diethylamino)ethyl acrylate

    Temperature-responsive copolymers without compositional drift by RAFT copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride and 2-(diethylamino)ethyl acrylate

INTRODUCTION

Stimuli-responsive polymers have been studied for the last thirty years in order to mimic biological systems that continuously adapt themselves to changing conditions. 2 , 3 Such polymers can undergo significant reversible changes in their chemical or physical structure in response to a small external stimulus (temperature, pH, ionic strength, light, electric or magnetic field). Among stimuli-responsive polymers, those that respond to changes in temperature are especially attractive, finding applications in many fields such as drug delivery, tissue engineering, cell culture and separation devices. 4,5 Most thermoresponsive polymers are homopolymers, such as poly(N-isopropyl acrylamide) and its various derivatives, 6,7,8 which are characterized by a LCST (Lower Critical Solution Temperature) 9 , 10 and/or an UCST (Upper Critical Solution Temperature), 11 above/below which the polymer chains undergo a volume phase transition with a drastic change of conformation, collapsing from a coil to a globule. 6 The use of copolymers instead of homopolymers, offers important design advantages such as the modulation of the volume phase transition temperature (T VPT ) by finely tuning the molar composition. 12 Moreover, a structure based on a copolymer can be used to improve the mechanical properties of a thermoresponsive material, as demonstrated in the case of poly(N-isopropyl acrylamide-cobutyl methacrylate) hydrogels. 13 A study by the group of Mark Bradley reported the free radical copolymerization of [2-(acryloyloxy)ethyl] trimethylammonium chloride (AEtMACl) and 2-(diethylamino)ethyl acrylate (DEAEA) monomers in the presence of a cross-linker, in order to prepare thermoresponsive hydrogels adequate for stem cell culture. 1,14 These hydrogels were selected among more than 600 different monomer combinations by a combinatorial synthesis process, for their excellent performance in releasing cultured stem cells using only a mild temperature stimulus. These results indicate that copolymers of AEtMACl and DEAEA are very good camdidates for other controlled release applications. However, to the best of our knowledge, there are no other studies about this comonomer pair and no copolymerization kinetics was yet reported. In addition, no study about the temperature-responsiveness of those copolymers has been carried out, except for the effect of the hydrogels on stem cell culture.

The AEtMACl and DEAEA monomers (Scheme 1) have already been homopolymerized and copolymerized with other comonomers but never together, except in the hydrogels of Bradley et. al.. AEtMACl has been copolymerized by free radical polymerization with several (meth)acrylate or acrylamide derivatives, such as acrylamide, [START_REF] Wandrey | Advances in Polymer Science[END_REF][START_REF] Pan | [END_REF] acrylic acid 17 (for polaroid instant photographic films), polyethylene glycol diacrylate 18 (for stationary phases in chromatography columns), and with a cationic methacrylate. 19 In most cases, the free radical (co)polymerization was carried out in water. AEtMACl has also been polymerized by controlled Reversible Deactivation Radical Polymerization (RDRP), in particular by the RAFT process in aqueous solution, either to prepare homopolymers 20 or block copolymers with ethylene glycol acrylate. 21 Concerning DEAEA, very few studies report its polymerization contrary to its methacrylate counterpart, diethylaminoethyl methacrylate (DEAEMA). DEAEA has been copolymerized by free radical polymerization with hydroxyethyl methacrylate (in bulk) to prepare hydrogels. 22 More recently, ATRP has been used to copolymerize DEAEA with n-butyl acrylate (in anisole) to synthesize polymer brushes on silica nanoparticles. 23 The RAFT process has also been used to polymerize DEAEA (in toluene) in order to prepare pH-sensors based on silicon films coated with pHresponsive polymer. 24 In most of the studies with AEtMACl or DEAEA, very little information is given about homopolymerization kinetics, except in a very recent study where the kinetic parameters of AEtMACl free radical polymerization were determined in aqueous solution. 25 Moreover, there are very few studies investigating the copolymerization kinetics of AEtMACl with other comonomers including the determination of reactivity ratios. [START_REF] Wandrey | Advances in Polymer Science[END_REF]19 Considering the high potential of thermoresponsive materials based on AEtMACl and DEAEA for controlled release applications, in particular for cell culture, and the absence of kinetic data on this comonomer pair, we carried out a thorough investigation of their RAFT homo-and copolymerization kinetics. We propose different methods to follow the conversion by 1 H NMR and suggested the determination of the molecular weights by static light scattering using a Debye plot. Finally, the temperature response of the copolymer was determined by dynamic light scattering, revealing a broad volume phase transition temperature that explains the excellent performance of this copolymer in stem cell culture. pentanoic acid (CPADB, >97%, Sigma-Aldrich) was used as received as chain transfer agent (CTA). Trioxane (>99%, Sigma-Aldrich), tetramethylsilane (TMS, >99.9%, Sigma-Aldrich), trimethyl silyl-3 propionic acid-d 4 (TMSP, S.d.S), chloroform-d (CDCl 3 , 99,8%, 0,03% TMS, Sigma-Aldrich), deuterium oxide (D 2 O, 99.9%, Sigma-Aldrich ), trifluoroacetic acid (TFA, 99%, Fisher), acetone (>99%, Fisher) and diethyl ether (>99.5%, puriss, Sigma-Aldrich) were used as received. MilliQ deionized water (Millipore system) was used.

General polymerization procedure

Solubility tests

The solubility of each monomer and CTA was tested in several solvents. A few drops (or mg) of monomer (or CTA) were added to ca. 1 mL of solvent in a glass vial. The results (full/partial/non-soluble, presence of an emulsion or of two phases) are reported in Table S1 in ESI.

Reversible Addition-Fragmentation chain Transfer (RAFT) Polymerization

In the experiments where DEAEA was protonated (DEAEA + ), a given molar equivalent of trifluoroacetic acid (TFA) was added to the monomer solution in ethanol, as indicated (Table 1). Monomer(s), AIBN, CPADB, ethanol (and trioxane if used) were introduced in a Schlenk tube, equipped with a magnetic stirrer (Table 1). The mixture was degassed by five freezeevacuate-thaw cycles. Before heating the mixture in a thermostated oil bath under a slight nitrogen overpressure (0.3 bar), a sample was withdrawn as t 0 . Periodically, samples were withdrawn from the polymerization medium via a cannula for analyses.

For each experiment, [AEtMACl] 0 , [DEAEA] 0 , and [CTA] 0 were chosen such as to give the desired M n value at 100% conversion (Eq. 1, neglecting AIBN-initiated chains) 26 :

[ ] [ ] [ ] CTA DEAEA DEAEA AEtMACl AEtMACl n M CTA p M DEAEA p M AEtMACl M + + = 0 0 0 (Eq. 1)
where [AEtMACl] Method 1 is based on the addition of an internal standard (trioxane, 5.1 ppm in CDCl 3 ) to the polymerization medium 27 to quantify the decrease of DEAEA or AEtMACl vinyl proton resonances (Figure S1 and calculation method in ESI). This method provided the best accuracy (below 2%) and reproducibility for experiments carried out below 80°C. When temperature was higher than the solvent boiling point (78°C for ethanol under 760 mmHg), we noticed that the accuracy decreased probably because of the co-evaporation of some trioxane.

Method 2 is based on the comparison between the 1 H NMR resonances of residual

AEtMACl monomer 21 and growing PAEtMACl homopolymer chain (backbone part) (Figure S2 and calculation method in ESI). A very good accuracy (around 2%) was obtained for conversions above 20%, with a sufficiently high signal/noise ratio for the polymer backbone resonances.

Method 3 is based on a given proton of DEAEA for which the corresponding 1 H NMR resonances in residual monomer and homopolymer are close but clearly resolved (Figure S3 and calculation method in ESI). A very good accuracy (around 2%) was obtained for conversions as low as 10%, since the considered resonance at 4.2-4.4 ppm is much narrower than the polymer backbone resonances (between 1.4 and 2.5 ppm) and thus exhibits a better signal/noise ratio. For DEAEA, Method 3 gave better reproducibility than Method 2. For AEtMACl, Method 3 could not be applied since there was no zone where monomer and polymer resonances did not overlap.

Method 4 is based on the comparison between the 1 H NMR resonances of residual monomers and growing copolymer backbone (Figure S4 and calculation method in ESI). A very good accuracy (around 2%) was obtained for conversions above 20%, with a sufficiently high signal/noise ratio for the copolymer backbone resonances.

Purification of the (co)polymers

Several tests were carried out in order to find the best non-solvents to precipitate the (co)polymers for purification (Table S2 in ESI). The PAEtMACl homopolymer as well as the various copolymers were precipitated first in acetone, centrifuged (Eppendorf centrifuge, model 5810R, 10 minutes at 7232 g), re-solubilized in ethanol and precipitated twice in diethyl ether (centrifuged for 10 min at 12,857 g). The PDEAEA + homopolymer was precipitated three times in diethyl ether and centrifuged (10 min at 12,857 g). The final polymers were dried under vacuum for several days. The complete elimination of residual monomers was confirmed by 1 H NMR analysis.

Characterization of the (co)polymers

H NMR spectroscopy

Each purified (co)polymer was analysed by 1 H NMR spectroscopy (Bruker spectrometer Avance 300 MHz) and all the resonances were identified in order to determine the molar composition of the various copolymers.

Static Light Scattering (SLS)

Molecular weights were determined by SLS, performed on Zetasizer Nano ZS (Malvern)

operating at an angle of 173º, using SUPRASIL quartz cells 10x10 mm (the same cell was used for all the measurements on each experiment). All the material used was cleaned to remove dust, using compressed air. Ethanol (filtered on PTFE filter 0.45 µm) was used to wash the cell between measurements and toluene was used as scattering standard. The polymer samples were found to be hygroscopic, and thus were weighted in a glovebox.

Deionized water from a Milipore Milli-Q system (with a Millipak membrane filter 0.22 µm)

was used for the preparation of polymer solutions with a concentration of 1 mg mL -1 . These solutions were left stirring until used. The dilutions (80%, 60%, 40%, 20% and 10% from original solution) of each polymer sample were prepared on the day of the measurements, using fresh Milli-Q water. The molecular weight measurement sequence requires a series of intensity measurements: first of a standard (toluene) to establish the reference scattering intensity, then of the solvent (Milli-Q water) and then of each of the prepared solutions. [START_REF]Zetasizer Nano Series User Manual[END_REF] Ten measurements were made for each concentration (1, 0.8, 0.6, 0.4, 0.2 and 0.1 mg mL -1 ).

The SLS measurements where analyzed with the Rayleigh equation (Eq. 2):

) ( 2 1 2 θ θ P C A M R KC w         + = (Eq. 2)
where is the Rayleigh ratio, which characterize the scattered intensity at the scattering angle , M w the sample molecular weight; A 2 the 2 nd virial coefficient; C the concentration.

( ) is the form factor, which is related to the shape and size of the dispersing object and the angle at which the scattering is determined. For measurements at very large angle (as in our case), the form factor is approximately 1, and therefore a plot of KC/ versus C is expected to be linear (Debye Plot) with an intercept equal to 1/M w (Eq. 3)

        + = C A M R KC w 2 2 1 θ (Eq. 3)
with the optical constant, K, defined as

2 2 0 4 0 2 n A c d K n N d π λ   =     (Eq. 4)
where is the solvent refractive index (1.330 for water), the laser wavelength (633 nm, in the used apparatus), the Avogadro's constant and dn/dc the differential refractive index increment (depends on the polymer sample).

The specific refractive index increment (dn/dc) in acetate buffer (acetic acid 0.2 M + ammonium acetate 0.15 M, pH 4.5, 40°C) was determined for PDEAEA + (dn/dc = 0.15 ± 0.01), for PAEtMACl (dn/dc = 0.19 ± 0.03) and for the P(AEtMACl-co-DEAEA + ) copolymer with a 75/25 molar ratio (dn/dc = 0.21 ± 0.01) using a manual differential refractometer (C.

N. Wood RF-600) operating at 658 nm. The dn/dc of the other copolymers was determined by interpolation of the values obtained for PDEAEA + and PAEtMACl.

Dynamic Light Scattering (DLS)

To determine the temperature response of the copolymers, dynamic light scattering experiments were performed using a Zetasizer Nano ZS (Malvern), for temperatures between 10 and 40 ºC. The auto-correlated functions were analyzed by the CONTIN method, to determine the hydrodynamic diameter. Polymer solutions (2 mg mL -1 ) of P(AEtMACl-co-DEAEA + ) copolymer with a 75/25 molar ratio (Exp. 17) were prepared in phosphate buffer (pH 7.5, 1 M), in Milli-Q water at pH 7 with 0.15 M NaCl, and in Milli-Q water at pH 9 (using a 0.1 M NaOH solution to adjust the pH).

RESULTS AND DISCUSSION

The excellent performance of AEtMACl/DEAEA based thermoresponsive hydrogels in cell culture 1 and the lack of information on the use of the RAFT process to produce wellcontrolled copolymer chains from those monomers, motivated us to perform a systematic study on the different parameters controlling their RAFT (co)polymerization. In addition to the kinetics experiments, our objective was to produce copolymer chains of at least 50000 g mol -1 , in order to favor a strong thermoresponsive behavior.

As chain transfer agent (CTA), we selected 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid (CPADB, Scheme 1), successfully used with acrylate monomers. [START_REF] Favier | [END_REF] Additionally, it has a carboxylic group that can be further used to graft the chains on the surface of solid supports. 30 The first experimental step was to select an appropriate polymerization solvent for both monomers and the CTA. After several tests ( 1).

To study the (co)polymerization kinetics, we developed convenient 1 H NMR methods adapted to AEtMACl and DEAEA to follow monomer conversion (see experimental part and ESI); up to now, the few papers reporting kinetics studies with AEtMACl used HPLC, 19 gravimetry, 20,21 or near-infrared spectroscopy 25 methods.

Kinetics of AEtMACl RAFT homopolymerization

The RAFT homopolymerization of AEtMACl in ethanol was performed under similar conditions to its FRP, except for the presence of CPADB as CTA. Since temperature is a key parameter in RAFT polymerization, 31 several experiments were carried out at different temperatures (oil bath), 70°C (Exp. 5), 80°C (Exp. 6) and 85°C (Exp. 7) (Table 1 and Figure 1), with a CTA/initiator ratio of 5. Exp. 8 ( ) is similar to Exp. 7 but carried out at a larger scale (5.6 g of monomer instead of 0.8 g). Conversions were determined by 1 H NMR using Method 2 (except for Exp. 5 where Method 1 was used).

At 70°C (Exp. 5), the induction period (initial step with very low monomer consumption) 32 was around 2 hours and the conversion plateau reached 40-50%. When temperature was increased to 80°C, the conversion plateau was significantly higher (75%) as expected, and the induction period reduced to 20 min as better evidenced by the ln([M] 0 /[M] t ) versus time plots (Figure S7 in ESI). Moreover, the linear increase of these plots indicated that the concentration of radical species remained constant until rather high conversions (for instance, 67% for Exp. 6). After that (here, ca. 3 hours), the new radicals produced by AIBN decomposition did not compensate any more the loss of propagating radicals via irreversible termination reactions, therefore conversion reached a plateau. Finally, at 85°C (Exp. 7), the induction period was just a few minutes (7 min) and it was possible to reach 81 % conversion in 4 hours. Taking into account the initial amounts of AEtMACl and CTA, that conversion corresponded to a calculated M n value of 61000 g mol -1 , in agreement with our objective.

Kinetics of DEAEA RAFT homopolymerization

The RAFT homopolymerization of DEAEA was performed under similar conditions to those for AEtMACl. However, it exhibited an unexpected behavior. First, an experiment conducted at 70°C led to very low conversions (results not shown). An increase in temperature to 80°C (Exp. 9, Table 1 and Figure 2) did not improve significantly the conversion that reached a plateau at approximately 10%. Second, the color of the polymerization medium turned from pink (color of the CPADB dithiobenzoate in ethanol at t 0 ) to pale yellow after 10 min (instead of the expected salmon color usually generated in the presence of dithiobenzoate, Figure S8 in ESI). Such discoloration of the medium generally indicates a degradation of the CTA. 33 Third, during the polymerization reaction, we noticed some intriguing changes in the 1 H NMR spectrum (already observable at t 1 , i.e. less than 5% conversion), with new resonances appearing just besides the vinyl proton resonances (see for instance t 4 compared to t 0 , respectively b and a in Figure 3; the parasite resonances are marked with stars). A possible explanation is the hydrolysis of the monomer, previously observed for a polymer based on dimethylaminoethyl acrylate (DMAEA), a monomer very similar to DEAEA.

PDMAEA suffers hydrolysis in aqueous solution leading to 2-dimethylamino ethanol and poly(acrylic acid). 34,35 Hydrolysis of DEAEA was confirmed here, with the observed new resonances assigned to acrylic acid vinyl protons. Those observations were related to the basicity of the medium (DEAEA solution in ethanol corresponded to pH≈10) that could explain both hydrolysis of the monomer and rapid degradation of the CTA (thiocarbonylthio compounds are sensitive to basic conditions). 36 To avoid such hydrolysis, DEAEA was protonated with trifluoroacetic acid (TFA). To investigate the influence of the protonation on the RAFT polymerization kinetics, two experiments (Exp. 10 and 11, Table 1 and Figure 2) were carried out in the same conditions as Exp. 9, but with 1 and 1.25 molar equivalent of TFA, respectively (with respect to monomer).

In Exp. 10 (1 eq. TFA), conversion reached almost 50% in 4 hours compared to 10% in the absence of TFA. Moreover, the color of the polymerization medium did not change to yellow after 10 min but to the usual salmon color (stable afterwards). A slight excess of TFA (Exp.

11, 1.25 eq. TFA) appeared favorable since conversion reached around 80% in 4 hours. A further excess (1.5 eq.) did not lead to better results (data not shown). On the 1 H NMR spectra, we noticed that in the case of 1 eq. TFA (pH≈7-8), the acrylic acid resonances related to monomer hydrolysis still appeared but with a lower intensity. However, with 1.25 eq. TFA (pH≈5-6), no parasite resonance appeared during the whole polymerization course (see for instance t 7 compared to t 0 , respectively d and c in Figure 3). These results clearly show that polymerization kinetics was drastically influenced by monomer protonation. In the presence of 1.25 eq. TFA, monomer hydrolysis was fully avoided and DEAEA + conversion reached 80% in a few hours.

Taking into account the initial amounts of DEAEA + and CTA, the conversion plateau of Exp. 11 corresponded to a calculated M n value of 30000 g mol -1 . In order to produce longer chains (of about 50000 g mol -1 ), the [DEAEA + ] 0 /[CTA] 0 initial molar ratio was increased from 217 (Exp. 11, calculated M n = 37400 g mol -1 at 100% conversion) to 388 (Exp. 13, Table 1, calculated M n = 66700 g mol -1 at 100% conversion), keeping the [CTA] 0 /[AIBN] 0 ratio equal to 5 (Figure S9 in ESI). However, the conversion plateau decreased from 80 to 65%, a behavior different from that observed with the N-acryloylmorpholine/tertbutyldithiobenzoate system, where the kinetics remained identical whatever the targeted chain length provided that the CTA/AIBN ratio was kept constant. 31 Therefore, to achieve higher conversion and the desired longer chains, a temperature of 85°C was preferred in the following experiments.

Kinetics of RAFT copolymerization of the AEtMACl/DEAEA comonomer pair

Similarly to DEAEA homopolymerization, the AEtMACl/DEAEA copolymerization kinetics was also strongly influenced by the protonation of DEAEA. For example, for a 75/25

AEtMACl/DEAEA initial molar ratio, two RAFT copolymerization experiments were compared, without and with TFA (1.2 eq. with respect to DEAEA, Exp. 14 and 15 respectively, Table 1). In the presence of TFA, the conversion plateau reached 75% (instead of 25-30% without TFA, Figure S10 in ESI) and there was no degradation of CTA and no hydrolysis of DEAEA, unlike what was observed without TFA. Consequently, the following copolymerization experiments were all conducted in the presence of TFA.

In order to get polymer chains of at least 50000 g mol -1 while keeping a high final conversion, two parameters were simultaneously modified taking into account the above results for DEAEA + homopolymerization: the [M] 0 /[CTA] 0 molar ratio was increased from 217 to 388 and the temperature from 80 to 85 ºC (Exp. 16, Table 1, calculated M n = 73200 g mol -1 at 100% conversion). Comparison with Exp. 15 (Figure 4), indicated a shorter induction period at 85°C (8 min) than at 80°C (39 min) and a similar conversion plateau (75%), corresponding to a calculated M n value of 54900 g mol -1 in agreement with our objective. To obtain larger amounts of that copolymer sample, the total amount of comonomers was increased (from 1.35 to 6.7 g, Exp. 17, Table 1). This scale-up was a success and exhibited a similar copolymerization behavior (Exp. 17 compared to Exp. 16, Figure 4). ). Conversions were determined by 1 H NMR using Method 4.

In order to obtain copolymers with different compositions, we explored the RAFT copolymerization kinetics for different initial AEtMACl/DEAEA + molar ratios: 60/40, 25/75 and 90/10 (Exp. 18, 19 and 20, respectively, Table 1), in the same optimized conditions as Exp. 17 To go deeper in the copolymerization kinetics, we tried to access the individual monomer conversions, since they can usually yield the apparent reactivity ratios as well as the instantaneous and cumulative copolymer molar composition. 26,27 However, in the case of the AEtMACl/DEAEA + pair, 1 H NMR determination of the individual monomer conversions was complicated by the presence of TFA. Indeed, the vinyl proton resonances of DEAEA + were shifted to lower field than those of the non-protonated monomer (Figure 3, see Exp. The AEtMACl and DEAEA + individual conversion values were very close (often equal) not only for all polymerization times but also for all initial molar ratios (Figure 6, left), which is rarely observed in copolymerization. Generally, one monomer is consumed faster than the other and consequently, the molar composition of the generated copolymer varies with global conversion (known as compositional drift). [START_REF] Odian | Principles of Polymerization[END_REF] For a given comonomer pair, it may exist a particular initial molar ratio (the so-called azeotropic composition) for which the two comonomers copolymerize at the same rate, leading to a copolymer without compositional drift. 26,27,[START_REF] Odian | Principles of Polymerization[END_REF] However, in such a case, it usually takes place at a specific feed molar composition value or for a narrow composition range around that value. [START_REF] Mignard | [END_REF] Here, for the AEtMACl/DEAEA + comonomer pair, an azeotropic behavior is observed for a wide range of initial molar ratios (at least from 75/25 to 25/75). From the individual conversion values, p AEtMACl and p DEAEA , the cumulative copolymer composition (expressed as molar fractions, AEtMACl and DEAEA , respectively, calculated from Eq. 5 and 6) was plotted versus global conversion (Eq. 7) for the different copolymerization experiments (Figure 6, right). It is clearly visible that there is almost no compositional drift whatever the initial molar ratio, the cumulative copolymer composition being very close to the feed composition.

[ ] [ ] [ ] [ ] [ ] [ ] DEAEA AEtMACl AEtMACl AEtMACl p DEAEA p AEtMACl p AEtMACl DEAEA AEtMACl AEtMACl F × + × × = ∆ + ∆ ∆ = 0 0 0 (Eq. 5) [ ] [ ] [ ] [ ] [ ] [ ] DEAEA AEtMACl AEtMACl DEAEA p DEAEA p AEtMACl p DEAEA DEAEA AEtMACl DEAEA F × + × × = ∆ + ∆ ∆ = 0 0 0 (Eq. 6) [ ] [ ] [ ] [ ] 0 0 0 0 ) ( . DEAEA AEtMACl p DEAEA p AEtMACl t conv Global DEAEA AEtMACl + × + × = (Eq. 7)
It was also possible to follow the evolution of the feed molar composition versus global conversion (Figure S16 in ESI). From these plots, the values of the apparent reactivity ratios can be usually calculated using the Meyer-Lowry model. [START_REF] Odian | Principles of Polymerization[END_REF]39 However, in this case where the copolymerization proceeds with almost no changes in the feed molar composition, it was not possible to apply that model. This kind of copolymerization, where the copolymer composition is equal to the comonomer feed composition for a wide range of initial molar ratios, follows a Bernouillian behavior [START_REF] Odian | Principles of Polymerization[END_REF] and corresponds to the case with both apparent reactivity ratios, r AEtMACl and r DEAEA + , of about 1. Consequently, whatever the initial AEtMACl/DEAEA + molar ratio, the formed copolymer chains exhibit the same composition as the feed regardless of the conversion. Therefore, it is possible to prepare P(AEtMACl-co-DEAEA + ) copolymers with high composition and microstructure homogeneity for a large choice of molar ratios.

Characterization of the purified copolymers by 1 H NMR

After thorough purification of the (co)polymers by precipitation, the 1 H NMR spectra of PAEtMACl and PDEAEA + homopolymers and different P(AEtMACl-co-DEAEA + ) copolymers were recorded and assigned (Figure 7). They clearly show the variations of the For each purified copolymer, the average molar composition was quantified thanks to chosen zones of the 1 H NMR spectrum (shown for Exp. 17 as an example, Figure 7) and expressed as a molar fraction, P DEAEA /(P DEAEA + P AEtMACl ), where P DEAEA and P AEtMACl are the number of DEAEA + and AEtMACl units, respectively, in the copolymer. It could be determined either from zones A (1.25 -1.5 ppm) and C (2.9 -4 ppm) or from zones A and B (1.5 -2.9 ppm), according to Eq. 8 or 9, respectively. These average copolymer composition values were compared to those calculated from the corresponding individual monomer conversions according to Eq. 6 (Table 3). (Eq. 9) 3) (Figure S19 in ESI). For each polymer sample, the obtained value was consistent with the one calculated from the monomer conversion data (Table 4), which is an expected result for a well-controlled RAFT polymerization.

Characterization of the responsive behavior of the copolymers

As a preliminary test of the temperature response of the new copolymers, we measured the hydrodynamic diameter of the P(AEtMACl-co-DEAEA + ) copolymer in solution (75/25molar ratio, Exp. 17) by Dynamic Light Scattering. A copolymer concentration of 2 mg mL -1 was used, either in phosphate buffer at pH 7.5 (1 M) (A), or in Milli-Q water at pH 7 with 0.15 M NaCl (B), or in Milli-Q water at pH 9 (C) (Figure 8).

The most striking characteristic of these results is that the copolymer exhibits a broad volume phase transition below 30ºC, over which the chains collapse to ca. 50% of their low temperature (< 20 ºC) hydrodynamic diameter. At pH 7.5 (A), close to the pKa of the tertiary amine of DEAEA, 40 some fluctuations were observed. Indeed, this proximity can lead to small changes in the protonation state of the amine when the temperature is changed, that interfere with the temperature response of the polymer. At pH 7 with 0.15 M NaCl (B), the lower pH favors DEAEA protonation, with the resulting repulsion being attenuated by the electrostatic screening due to the salt. At pH 9 (C), there is deprotonation of the tertiary amine of DEAEA, leading to a smaller hydrodynamic size of the expanded polymer chains at low temperature, compared to the other conditions. For (C), up to ca. 37 ºC, there is also a steady decrease of the chain hydrodynamic diameter, followed by a slight increase that might indicate the onset of chain aggregation following the collapse into a globule conformation.

Further studies of the copolymer response to temperature, pH and ionic strength are underway.

Even though the volume phase transition temperature is not sharp, the difference in the hydrodynamic radii of the polymer chains between 37 ºC and lower temperatures (below 20 ºC), indicates a corresponding increase in their hydrophilic character that justifies a decrease in cell adhesion when thermoresponsive hydrogels of these copolymers are used as substrate to culture stem cells. This change enabled a very efficient release of the cells using only a mild temperature stimulus. (DEAEA + ) feature RAFT copolymerization kinetics with both apparent reactivity ratios of about 1. Consequently, whatever the initial AEtMACl/DEAEA + molar ratio, the copolymer chains have the same composition as the feed, regardless of the conversion. This allows us to prepare P(AEtMACl-co-DEAEA + ) copolymers exhibiting not only controlled molecular weights but also highly homogeneous composition and microstructure.

The LCST-type temperature response of the copolymer corresponds to a broad volume phase transition below 30ºC, over which the chains collapse to ca. 50% of their low temperature hydrodynamic diameter. This response is related to the excellent performance in releasing stem cells cultured on hydrogels based on a 75/25 AEtMACl/DEAEA molar ratio.

A hydrogel of this particular copolymer has been previously selected among over 600 different monomer combinations for its ability to release the cultured cells by using only a mild temperature stimulus. The copolymer also responds to pH through protonation/deprotonation of the DEAEA units. The combination of temperature/pH stimuli, in conjugation with the control over molecular weight and composition, opens very exciting opportunities for application in many fields, from controlled delivery to tissue engineering, cell culture, separation, etc.

ORCID

Marie-Thérèse Charreyre: 0000-0002-1303-0888

  0 and M AEtMACl (193.67 g mol -1 ) are respectively the initial molar concentration and the molecular weight of AEtMACl; [DEAEA] 0 and M DEAEA (285.24 g mol - 1 ) are respectively the initial molar concentration and the molecular weight of DEAEA; p AEtMACl and p DEAEA are respectively the individual conversion of AEtMACl and DEAEA determined by 1 H NMR; [CTA] 0 and M CTA (279.38 g mol -1 ) are respectively the initial molar concentration and the molecular weight of the CTA.

Figure 1 .

 1 Figure 1. Monomer conversion versus time plots for AEtMACl RAFT polymerization in ethanol at different temperatures, 70°C (Exp. 5, ♦), 80°C (Exp. 6, ) and 85°C (Exp. 7, ).

Figure 2 .

 2 Figure 2. Monomer conversion versus time plots for DEAEA RAFT polymerization in

Figure 3 .

 3 Figure 3. Influence of TFA on the appearance of parasite resonances ( , due to hydrolysis of DEAEA into acrylic acid and 2-diethylamino ethanol) in the vinyl region of the 1 H NMR

Figure 4 .

 4 Figure 4. Global monomer conversion versus time plots for AEtMACl/DEAEA + 75/25

  (75/25, [M] 0 /[CTA] 0 = 388, [CTA] 0 /[AIBN] 0 = 5, 85°C). Curiously, whatever the initial molar ratio, the global conversion versus time curves were very similar (Figure5), which is quite unusual.

Figure 5 .

 5 Figure 5. Global monomer conversion versus time plots for AEtMACl/DEAEA +

  12 with TFA compared to Exp. 9 without TFA), overlapping with the vinyl proton resonances of AEtMACl (Figures S11-S14 in ESI). Nevertheless, it was possible to integrate the vinyl proton doublet at 6.35-6.55 ppm using a deconvolution method (Figure S15 in ESI), applicable to all the experiments except Exp. 20 (90/10 molar ratio) where the vinyl proton resonances fully overlapped.

Figure 6 .

 6 Figure 6. (left) Individual conversions of AEtMACl ( ), DEAEA + ( ) and global conversion

  resonance relative intensities according to the initial comonomer molar ratio (90/10, 75/25, 60/40 and 25/75), especially visible for the protons c and g (-C(O)-O-CH 2 moiety) and for the protons b and f (-CH 2 -N + moiety) (Figure S17 in ESI).

Figure 7 .

 7 Figure 7. 1 H NMR spectrum in D 2 O of P(AEtMACl-co-DEAEA + ) copolymer (Exp. 17, 75/25 AEtMACl/DEAEA + initial molar ratio) showing the different zones (A, B, C, D) chosen for

  poor resolution between A, B and C areas in the corresponding1 H NMR spectrum (FigureS17D in ESI). Moreover, these average composition values determined on the purified final copolymer samples are in good agreement with the ones calculated from the individual monomer conversions, which validate the methods used to follow the kinetics. In addition, since these values are very close to the initial feed compositions (for a global conversion around 75%), they confirm the absence of compositional drift during AEtMACl/DEAEA + RAFT copolymerization for a large range of initial molar ratios.Determination of the purified (co)polymer molecular weights by SLSAnalysis of the homopolymer and copolymer samples by Size Exclusion Chromatography (SEC) evidenced an unimodal molecular weight distribution (Fig S18 in ESI for P(AEtMACl-co-DEAEA + ) copolymer, Exp. 16). However, determination of the molecular weight values was not possible since the polymers showed some affinity with the stationary phase of the different tested columns (see SEC section in ESI). More specifically, a trail was observed on the SEC trace on the low molecular weight side of the polymer peak. As an alternative, the molecular weights were determined by Static Light Scattering (SLS), using a Debye plot (Eq.

1

 1

Figure 8 .

 8 Figure 8. Dynamic Light Scattering measurements of hydrodynamic radius, R H , versus temperature for P(AEtMACl-co-DEAEA + ) copolymer (Exp. 17, 75/25 initial molar ratio) in

1

  Scheme 1. Monomers (AEtMACl and DEAEA) and RAFT CTA (CPADB) used in this study.

	EXPERIMENTAL SECTION
	Materials
	2-(diethylamino)ethyl acrylate (DEAEA, 95%, Sigma-Aldrich, inhibitor: monomethyl ether
	hydroquinone)	was	purified	by	vacuum	distillation.

[2-(acryloyloxy)

ethyl] trimethylammonium chloride solution (AEtMACl, 80 wt% in water, Sigma-Aldrich, inhibitor: monomethyl ether hydroquinone) was purified by passing through an inhibitor removal column (Prepacked column 306312, Sigma-Aldrich). 2,2'-azobisisobutyronitrile (AIBN, >98%, Fluka) was re-crystallized from ethanol. Ethanol (EtOH, absolute 99.9%, Fisher), used as polymerization solvent, was purified by distillation. 4-cyano-4-(phenylcarbonothioylthio)

Table 1 .

 1 Experimental conditions for RAFT (co)polymerization of AEtMACl and/or DEAEA in ethanol, in the presence of AIBN and CPADB (total monomer concentration [M] 0 = 2 M).

		Temperature	AEtMACl/DEAEA	Equiv.of
	Exp	(ºC)	molar ratio	TFA a	[M] 0 /[CTA] 0 [CTA] 0 /[AIBN] 0
	1		0/100	0	-	-b
	2		0/100	0	-	-b
	3		100/0	0	-	-b
	4 c		100/0	0	-	-d
	5		100/0	0	217	5
	6		100/0	0	217	5
	7		100/0	0	388	5
	8 e		100/0	0	388	5
	9		0/100	0	217	5
	10		0/100	1	217	5
	11		0/100	1.25	217	5
	12		0/100	1.25	388	5
	13		0/100	1.25	388	5
	14		75/25	0	217	5
	15		75/25	1.2	217	5
	16		75/25	1.25	388	5
	17 e		75/25	1.25	388	5
	18		60/40	1.25	388	5
	19		25/75	1.25	388	5
	20 e		90/10	1.25	388	5
	21 e		25/75	1.25	388	5

Table 3 .

 3 Average molar composition of the various P(AEtMACl-co-DEAEA + ) copolymers (expressed as AEtMACl/DEAEA + molar ratio).The results from Eqs. 8 and 9 are very similar except for Exp. 19, probably because of the

			Average copolymer composition
	P(AEtMACl-co-DEAEA + )	Initial feed	purified copolymers	calculated from
	copolymers	composition	( 1 H NMR)	kinetics (individual
			(Eq. 8)	(Eq. 9)	conversions)
	Exp. 20 t10	90/10	90/10	89/11	nd
	Exp. 17 t7	75/25	75/25	75/25	75/25
	Exp. 18 t10	60/40	63/37	57/43	60/40
	Exp. 19 t10	25/75	35/65	27/73	25/75
	nd= not determined

Table 4 .

 4 Molecular weights of PAEtMACl and PDEAEA + homopolymers and P(AEtMACl-For Exp. 20, global conversion was calculated using 1 H NMR Method 4 (Eq. S4) and M n was calculated from Eq. 1 assuming that both individual conversions are equal to global conversion.

		Global	Calculated
	Polymer Sample	conversion	M n	a	M w SLS	dn/dc
					(g mol -1 )	(mL g -1 )
		1 H NMR a	(g mol -1 )
	PAEtMACl (Exp. 8 t7 )	56%	42 × 10 3	(43 ± 18) × 10 3 0.19 ± 0.03
	PDEAEA + (Exp. 12 t10 )	60%	40 × 10 3	(41 ± 6) × 10 3	0.15 ± 0.01
	COPO 90/10 (Exp. 20 t8 )	76% b	56 × 10 3 b	(57 ± 10) × 10 3 0.22 ± 0.01
	COPO 75/25 (Exp. 17 t7 )	72%	65 × 10 3	(55 ± 8) × 10 3	0.21 ± 0.01
	COPO 60/40 (Exp. 18 t10 )	74%	57 × 10 3	(49 ± 8) × 10 3	0.20 ± 0.01
	COPO 25/75 (Exp. 21 t11 )	63%	49 × 10 3	(44 ± 4) × 10 3	0.17 ± 0.01

co-DEAEA + ) copolymers determined by Static Light Scattering (SLS), with the used refractive index increment (dn/dc) values. a Global conversion and M n calculated from the corresponding individual monomer conversions, according to Eqs. 7 and 1, respectively. b

A. M. Santiago, T. Ribeiro, A. S. Rodrigues, B. Ribeiro, R. F. M. Frade, C. Baleizão, J. P.

Arnaud Favier: 0000-0002-7482-7874 Carlos Baleizão: 0000-0002-7810-1747 José Paulo S. Farinha: 0000-0002-6394-5031 Sofia Rodrigues: 0000-0003-2328-1549 ACKNOWLEDGEMENTS We thank Agnès Crépet (Laboratory IMP) and Dr. Sergio Alves (CQE) for SEC measurements. This work was partially supported by CNRS (France), Fundação para a Ciência e a Tecnologia (FCT-Portugal) and COMPETE (FEDER), projects UID/NAN/50024/2013, PTDC/CTM-POL/3698/2014 and UltraFluoPol (FCT-Portugal/CNRS-France, PICS nº 6411). S. Rodrigues also thanks FCT for PhD grant (SFRH/BD/89615/2012).

Conflicts of interest

There are no conflicts to declare.


You-Quan Zou

Dr Niklas Von Wolff

Dr Michael Rauch

Dr Moran Feller

Quan-Quan Zhou

Aviel Anaby

Dr Yael Diskin-Posner

Dr Linda J W Shimon

Dr Liat Avram

Yehoshoa Ben-David

You-Quan Zou

] Quan-Quan

Zhou

David Milstein
email: david.milstein@weizmann.ac.il

Dr Y.-Q Zou

Dr Q.-Q Zhou

Dr Y A Anaby

Prof. Dr D Ben-David

Milstein

Homogeneous Reforming of Aqueous Ethylene Glycol to Glycolic Acid and Pure Hydrogen Catalyzed by Pincer-Ruthenium Complexes Capable of Metal-Ligand Cooperation

Keywords: homogeneous catalysis, clean and sustainable, ethylene glycol, glycolic acid, hydrogen

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

The development of green and atom efficient protocols in organic synthesis is an important goal. [1] Indeed, over the last decade, progress has been made in the design and discovery of highly atom efficient transformations in organic redox chemistry. In this respect, transition metal complexes that are able to activate strong bonds via metal-ligand cooperation (MLC) are particularly fruitful in catalysis, [2] enabling the dehydrogenation of readily available starting materials, such as alcohols and amines under mild conditions and very high atom efficiency in the synthesis of useful amides and esters via acceptorless dehydrogenative coupling (ADC) reactions. Additionally, these complexes have been utilized to hydrogenate a variety of oxidized substrates, such as amides, esters, carbonates, carbamates and ureas to fine and useful chemicals. [START_REF]Selected reviews: a) D. Milstein[END_REF] Given the great potential of ADC catalysts in greener synthetic routes, it is desirable to extend the possible field of application to key molecular targets. In addition, gaining a more thorough understanding of their reactivity and bond activation patterns may help the design of new reaction schemes in the future to access desirable molecules in an efficient manner.

Being used today in a variety of fields, [START_REF] Kubitschke | Carboxylic Acids, Aliphatic in Ullmann's Encyclopedia of Industrial Chemistry[END_REF] ranging from skin care, adhesives and polymers to the textile industry, glycolic acid is an essential chemical commodity with a forecasted US market share of over 400 million USD by 2024. [START_REF]Glycolic Acid Market-Forecasts from 2018 to[END_REF] Nevertheless, the current protocols for its synthesis rely on high pressure formaldehyde carbonylation using CO at high temperatures, [START_REF] Barnicki | [END_REF] or hydrolysis of chloroacetic acid or methyl 2-hydroxyacetate; [7,8] processes that are associated with the generation of stoichiometric amounts of waste, multiple synthetic steps, and the use of toxic reagents. The environmentally benign, direct generation of glycolic acid from readily accessible starting materials under mild and atom efficient conditions is highly desirable.

Given the outstanding performance of pincer-type complexes in the ADC reactions of alcohols, [START_REF]Selected reviews: a) D. Milstein[END_REF] we are interested in understanding if and how pincer complexes might enable the generation of glycolic acid from readily available alcohols. In this respect, ethylene glycol [9] as a feedstock is of particular interest, as it is potentially biomass-derived and already used on an industrial scale. [10] Pioneering work by the Dumesic group elegantly demonstrated that aqueous ethylene glycol can be reformed to hydrogen, carbon dioxide and short alkanes at elevated temperatures using Pt/Al2O3 [11] and Raney-NiSn [12] catalysts at 225 °C or 265 °C. Despite the great interest in the use of atom efficient reforming of ethylene glycol, only a single heterogenous system developed by Bitter and co-workers has been described to catalyze the transformation of ethylene glycol to glycolic acid and hydrogen. At 150 or 180 °C using carbon nanofiber (CNF)-supported copper and nickel nanoparticles, glycolic acid could be obtained from ethylene glycol, [13] whereas a Ni/CNF catalyst led to further decomposition to formic acid, hydrogen and carbon dioxide (Scheme 1a).

Homogeneous reforming of ethylene glycol to generate hydrogen was reported by the groups of Cole-Hamilton [14] and Beller, [15a] although the generation of glycolic acid was not confirmed. The group of Grützmacher reported the oxidative dehydrogenation of alcohols and polyalcohols, including ethylene glycol, to carboxylic acids using a homogeneous rhodium catalyst under mild conditions, albeit in the presence of sacrificial hydrogen acceptors such as ketones or methyl methacrylate. [16] Reforming of ethylene glycol to glycolic acid homogeneously catalyzed by an iridium complex (4 mol%) was reported recently, although the catalytic mechanism was not studied. [17] Very recently, homogeneous iridium-catalyzed dehydrogenative crosscoupling of ethylene glycol and methanol to lactic acid was also disclosed by Tu, Xu and co-workers. [18] The scarcity of reports on catalytic reforming of aqueous ethylene glycol to glycolic acid, despite its significant promise, demonstrates the need for new catalytic systems as well as a deeper understanding of the reaction mechanism. Herein, we report a fully characterized homogeneously catalyzed reforming of aqueous ethylene glycol to glycolic acid and exclusively hydrogen gas at low catalyst loading (0.1 mol% on large scale). Mechanistic studies, supported by synthesis of potential intermediates, X-ray crystallography, nuclear magnetic resonance (NMR) experiments and density functional theory (DFT) calculations are presented to highlight the key steps of this transformation.

Results and Discussion

During recent years, our group has reported several acceptorless dehydrogenative coupling reactions of alcohols with alcohols, [19] amines [20] and water [21] to the corresponding esters, amides and acids. Very recently, we described a liquid organic hydrogen carrier system based on ethylene glycol, which enabled the efficient and reversible loading and discharge of hydrogen using a single ruthenium pincer complex (Scheme 1b). [22] Based on this report, we envisioned that ethylene glycol would undergo dehydrogenation to glycolaldehyde (GAL) in the presence of a ruthenium pincer complex via MLC, accompanied by hydrogen release (Scheme 1c). Subsequently, the interception of glycolaldehyde by water and base could liberate a second molecule of hydrogen and enable access to glycolic acid (GAC) after acid workup.

To examine the feasibility of the above-proposed reforming process, we evaluated the acridine-type PNP complexes, Ru-1 and Ru-2, that gave excellent results in the dehydrogenative coupling of ethylene glycol to oligoesters (Table 1). [22] The reforming of aqueous ethylene glycol was attempted using 0.5 mol% catalyst loading and 5 equivalents of potassium hydroxide at 115 °C (oil bath temperature) in a 1:1 mixture of water (1.0 mL) and THF (1.0 mL). [23] Somewhat surprisingly, these catalysts showed only sluggish reactivity (Table 1, entries 1 and 4) giving a maximum of 14 mL of hydrogen. Increasing the temperature to 150 °C resulted in increased volumes of H2 up to 71 mL (entries 3 and6). On the other hand, the PNN and PNP ruthenium complexes Ru-3 to Ru-7, developed by our group, gave more promising results (entries 7-11). Complex Ru-4 performed best, delivering 195 mL of hydrogen gas (81% yield, theoretical volume of hydrogen: 240 mL) after 48 hours at 115 °C (entry 8). Gas chromatographic (GC) analysis of the gas phase indicated that the evolved hydrogen was pure (Figure S28). NMR analysis of the crude reaction mixture indicated that only potassium 2hydroxyacetate was formed (see Figures S12-13 for details). After workup with 6 M HCl, glycolic acid was formed in 78% yield (entry 8, Figures S20-21).

Table 1. Optimization of the reaction conditions using PNN and PNP pincer complexes Ru-1 to Ru-7

Entry [a] Ru T (ºC) V (H2, mL) [b] Yield (1, %) [c] 1 Ru-1 115 14 [a] Reaction conditions: ethylene glycol (5.0 mmol), Ru cat. (0.5 mol%), KOH (25 mmol), H2O (1.0 mL) and THF (1.0 mL) at 115 o C (bath temperature) for 48 hours. [b] Hydrogen gas was collected every 24 hours. [c] Yields are based on the volume of collected hydrogen gas. [d] Yield of glycolic acid is shown in parentheses, as determined by 1 H NMR of the crude reaction mixture after acid workup using acetonitrile as an internal standard. N.D.: not determined.

To our delight, upon screening PNNH-based complexes (Table 2) we found that using Ru-8 the yield of potassium 2hydroxyacetate was increased to 89% and 214 mL of pure hydrogen (Figure S29) were collected after 48 hours (Table 2,entry 2). At the same time, glycolic acid was formed in 88% yield after acid workup of the crude reaction mixture (Figures S22-23) indicating an excellent correlation between the yield of glycolic acid and the amount of hydrogen evolved. Additionally, Ru-9 and Ru-10 also catalyzed the reaction, albeit in moderate yields of hydrogen and potassium 2-hydroxyacetate (entries 3 and 4). Notably, doubling the water loading liberated 223 mL of pure hydrogen (Figure S30) and further increased the yield of glycolic acid to 91% (entry 5, Figures S24-25). The glycolic acid was isolated in 84% yield after removing the water, followed by extraction with THF. Lowering the reaction temperature to 100 °C led to decreased reaction efficiency (entry 6). Using NaOH instead of KOH provided comparable results, yielding 94% of hydrogen and 93% of glycolic acid after workup (entry 7, Figures S26-27). Lower loadings of NaOH resulted in formation of less hydrogen gas (193 mL, entry 8). Using water as the only solvent (single phase) resulted in a sluggish reaction (entry 9), but it proceeded well at higher temperatures, generating 48% and 70% yields of potassium 2-hydroxyacetate at 135 °C and 150 °C, respectively (entries 10 and 11). Entry [a] Ru MOH V (H2, mL) [b] Yield (1, %) [c] 1 Ru-4 KOH 195 81(78) [d] 2

Ru-8 KOH 214 89(88) [d] 3

Ru-9 KOH 138 58 4 Ru-10 KOH 166 69

5 [e] Ru-8 KOH 223 93(91) [d] 6 [f] Ru-8 KOH 79 33

7 [e] Ru-8 NaOH 226 94(93) [d] 8 [e,g] Ru-8 NaOH 193 80

9 [h] Ru-8 KOH 29 12

10 [h,i] Ru-8 KOH 116 48

11 [h,j] Ru-8 KOH 167 70

[a] Reaction conditions: ethylene glycol (5.0 mmol), Ru cat. (0.5 mol%), MOH (25 mmol, M = K, Na), H2O (1.0 mL) and THF (1.0 mL) at 115 o C (bath temperature) for 48 hours. [b] Hydrogen was collected every 24 hours. [c] Yields are based on the volume of collected hydrogen gas. [d] Yield of glycolic acid is shown in parentheses, as determined by 1 H NMR of the crude reaction mixture after acid workup using acetonitrile as an internal standard.

[e] H2O (2.0 mL) was used. [f] Reaction was performed at 100 ˚C. [g] NaOH (15 mmol) was used.

[h] H2O (3.0 mL) was used as the reactant and solvent without THF. [i] Reaction was performed at 135 °C. [j] Reaction was performed at 150 °C. Grey rows are indicative of optimal conditions.

After establishing the optimized reaction conditions, we investigated the scalability of our methodology. As shown in Figure 1, using only 0.1 mol% of Ru-8, 10 mmol of ethylene glycol were efficiently dehydrogenated to form hydrogen gas and sodium 2-hydroxyacetate in a mixture of THF (2.0 mL) and water (4.0 mL). During the first 48 hours, the reaction was relatively fast, releasing 282 mL of hydrogen. Subsequently, the reaction slowed down, likely a result of the decreased concentration of ethylene glycol. After 120 hours, 349 mL pure hydrogen gas (yield of hydrogen at this stage: 73%) was collected (red curve, see Figure S4 for details). The reforming reaction also proceeded quite well on a 30 mmol scale, delivering 921 mL of pure hydrogen gas (yield of hydrogen at this stage: 64%) after 120 hours (blue curve, 3 mL THF and 6 mL water were used, see Figure S5 for details). A catalyst recycling experiment indicated that the catalytic species was still active after 72 hours, albeit at a reduced rate (Figure S6, see SI for details). Even though complexes Ru-1 and Ru-2 were shown to be efficient in the dehydrogenative coupling of ethylene glycol to oligoesters, [22] the PNNH complex Ru-8 showed superior performance in the reforming reaction. In order to understand the efficiency of the PNNH-family of complexes, we sought to investigate its possible modes of bond activation. An entry point to a possible catalytic cycle is the deprotonation of Ru-8 by base. Previous experimental and theoretical reports have shown that in the presence of excess base (and absence of a protic medium), a double deprotonated anionic species was formed, in which the CH2-group of P-arm was still present. [24] On the other hand, using only 1.1 equivalents of base 1 H, 31 P{ 1 H}, 13 C{ 1 H} DEPTQ and 1 H{ 13 C} HSQC NMR clearly showed that among the three acidic sites (P-arm, N-arm and NH-group) only P-arm deprotonation took place (Figures S33-37). This finding is further corroborated by DFT calculations, showing that the P-arm deprotonated species 3a is 3.6 and 6.8 kcal.mol -1 more stable than the NHdeprotonated species 3b and the N-arm deprotonated 3c, respectively (Scheme 2b, see the SI for details). Furthermore, in a protic medium (such as the crude reaction mixture), no double deprotonated species is observed even in the presence of excess base. Treatment of the resulting P-arm deprotonated mixture with 1 atmosphere of H2 at room temperature in THF converged to the aromatized trans-dihydride complex 4 (Scheme 2a). Complex 4 was only stable under an atmosphere of hydrogen, and was fully characterized under H2 (Figures S38-42, see SI for details). Since attempts to crystallize the product of the reaction between dearomatized intermediate 3 and ethylene glycol were unsuccessful, we used the PNN complex Ru-4 as surrogate (which exhibits good catalytic performance, see Table 1, entry 8). Deprotonating Ru-4 (using 1.2 equivalent of t BuOK) followed by reaction with ethylene glycol led to formation of the aromatized complex 6 via the dearomatized species 5, [25] as witnessed by NMR spectroscopy (Scheme 3). In the 1 H NMR spectrum (Figure S43), the hydride is shifted downfield to -16.14 ppm (doublet, 2 JPH = 25 Hz), consistent with the vacant position trans to the hydride of 5, now being occupied by the alkoxo group. A broad singlet appeared at δ = 103.4 ppm in the 31 P{ 1 H} NMR spectrum (Figure S46), which is also consistent with a saturated aromatic complex. The IR spectrum showed a strong absorption band at 1907 cm -1 (Figure S47). Slow evaporation of the mixture led to formation of crystals suitable for X-ray diffraction. As shown in Figure 2, a neutral distorted octahedral ruthenium complex was formed, with the alkoxide of ethylene glycol coordinated to the metal center. In the unit cell of the crystal, H-bonding with another molecule of ethylene glycol to the alkoxide ligand is observed (see SI for details). 7); Ru1-N1, 2.123( 5); Ru1-N2, 2.081( 5); Ru1-P1, 2.2738( 17); Ru1-H1A, 1.68( 8); Ru1-O2, 2.243(4); H4A-O2, 1.867; H5-O2, 1.787; C20-Ru1-N2, 178.3(2); P1-Ru1-N1, 157.83( 14); N2-Ru1-O2, 80.60 (17).

Reaction of the dearomatized complex 5 with ten equivalents of ethylene glycol in a 1:1 mixture of THF (0.25 mL) and water (0.25 mL) with no added base was also performed at 115 ˚C in an open system, resulting in formation of the glycolic acid adduct 7 as the major product along with minor byproducts (Scheme 4, Figures S53-54, see SI for details). Complex 7 was also obtained by the addition of two equivalents of glycolic acid to complex 5 [25] in THF at room temperature, and its structure was further confirmed by X-ray crystallography (Figure 3). The results of the base-free experiment indicate that the first cycle of the reforming reaction to generate the glycolic acid adduct does not require base to promote the C-O bond formation. [23] Moreover, upon treatment of sodium 2-hydroxyacetate as a substrate under the optimal conditions no hydrogen evolution occurred, clearly showing that further dehydrogenation of sodium 2-hydroxyacetate did not take place (see SI 6.6 for details). 9); Ru(1)-H, 1.56 (5); C( 22)-Ru(1)-N(1), 178.50 (14); N(2)-Ru(1)-P(1), 159.96 (9); N(1)-Ru(1)-O(1), 77.43 (11).

Following the reaction under standard conditions by 31 P{ 1 H} NMR showed the initial formation of a new species, assigned to be the hydrido-hydroxo complex 8 shown in Scheme 5A (see SI, Figure S55-57, previously also reported for an analogous PNNbased ruthenium complex). [26] Importantly, this shows that even in the presence of excess base, no double deprotonation takes place, excluding a previously proposed anionic reaction surface. [27] This is also in agreement with the base-free reaction outlined earlier (Scheme 4) and might be expected given the protic reaction conditions. In an earlier study, we conjectured that a PNNH-pincer complex might enable challenging reaction schemes by switching between appropriate MLC modes (dearomatization and amido-formation). [24] Employing DFT calculations, we tried to shed light into plausible reaction pathways for this transformation, in particular on three key events: 1) liberation of H2, 2) formation of the C-O bond, as well as 3) C-H bond scission. DFT calculations were performed at the ωB97M-V/def2-TZVPP/RIJCOSX/SMD//M06-L/def2TZVP/GD3/W0 level of theory (see SI for details). In order to correctly address possible concertedness of transition states, all structures were optimized in solution (THF). [28,29] The trans dihydride 4 is connecting both the H2 liberation step, as well as the C-H bond activation step and was thus chosen as a starting point of the mechanistic investigation. Transition states not involving side-arm protons (see SI 8.5) are linked to unreasonably high barriers and were thus excluded. As observed for many other pincer complexes, a proton shuttle is needed for the liberation of H2 from dihydride 4 (Step 1, TS1A, Scheme 5B). Although highly asynchronous, extended IRC-calculations confirm the concertedness of this step (see SI 8.7). Indeed, no stable intermediate is formed after initial protonation of the hydride by the proton shuttle, with subsequent side-arm deprotonation by the conjugated base being linked to the highest energy of this step. [28b] A more basic proton-shuttle (ethylene glycol) thus gives slightly lower barriers (Scheme 5B, TS1A vs TS1C). As for related PNPcomplexes, the dearomatized species will quickly react with the present protic species, to give either the alkoxo or the hydroxo compounds. The reversibility of the H-H and O-H activation events is demonstrated experimentally by running the reaction in D2O instead of H2O. D2 liberation was observed by GC-MS, and 2 H-NMR after 12h indicates formation of deuterated deuteriodeuterioxo complex 8 (see SI, Figure S58). After a first dehydrogenation event (see SI), [START_REF]Step 2 resembles Step 4, but is significantly lower in energy[END_REF] the hydroxo complex 8 is nucleophlic enough to attack the aldehyde intermediate (Step 3, TS3A). This transition state is thus somewhat reminiscent of C-N bond cleavage events in Fe-and Mo-catalyzed hydrogenation of amides. [31] The subsequent final C-H bond dissociation (Step 4, Scheme 5B) from the geminal diol is found to be step-wise, i.e. the C-H bond abstraction involves formally the alkoxide and a cationic Ru-species (TS4B) to regenerate the dihydride 4. It is important to note that no base is needed for the transient generation of the alkoxide in the TOF-determining (TDTS) TS4B. This is in agreement with our experimental results (Scheme 4), where the addition of water and ethylene glycol (but no base) to complex 5 generates the carboxylate 7. As was already postulated in the case of Ru-4, [32] excess base is still necessary in the catalytic system with a twofold role. On the one hand, the overall reaction is endergonic (+5.5 kcal/mol) and formation of the carboxylate salt would serve as a driving force for the reaction. On the other hand, the formation of carboxylate 10 from 4 and glycolic acid is highly exergonic (-18.7 kcal/mol) and efficient quenching of glycolic acid by excess base might help to prevent the formation of this off-cycle product, while at the same time increasing the concentration of the hydrido hydroxo complex 8. Several groups highlighted the importance of explicit solvent models for accurate DFT assessment of alcohol dehydrogenation reactions. [28a,33] Following the approach of Dub and Gordon, we computed selected key intermediates and transition states in the presence of an explicit water cluster (Scheme 6A). [28b] Expectedly, the hydrido hydroxo compound 8 is highly stabilized by the Hbonding network (-9.9 kcal/mol with respect to the dihydride 4, stabilization of 16.1 kcal/mol). The H-bonding has also a stabilizing effect on the TDTS TS4BEXP (17.1 kcal/mol, stabilization of 11.8 kcal/mol) and the H2 liberating TS1DEXP (11.7 kcal/mol, stabilization of 12.4 kcal/mol). As observed in DFT-MD calculations, the H-bond network will decrease the hydricity of the alkoxide species, explaining the lesser degree of stabilization in TS4BEXP. Including the explicit water cluster yields an overall energetic span dE of 27.0 kcal/mol, in good agreement with the observed experimental reactivity. Furthermore, the explicit model predicts the hydrido hydroxo compex to be the resting state, which is confirmed experimentally (see SI) and predicted for Ru-4 theoretically. [32] The inclusion of explicit solvent molecules is thus critical to correctly identify rate-limiting states on the potential energy surface.

Finally, we were interested in analyzing if the proposed model could account (and explain) the observed difference between PNNH complex Ru-8 and PNN Ru-4 (Scheme 6B). Indeed, the proposed mechanism reproduces qualitatively the slightly higher activity of Ru-8, compared to Ru-4 (TS4B vs. TS4Bbpy). Importantly, a step-wise deprotonation/hydride transfer sequence can be excluded in the case of Ru-4, highlighting the importance of branching points on the potential energy surface. [34] Whereas in the case of the PNNH compound, deprotonation of the sidearm NH group is energetically accessible (Scheme 5B, TS4A, 26.1 kcal/mol), this is not the case for Ru-4 (TS4Abpy = 44.1 kcal/mol, see SI). Instead, the C-H bond activation via TS4Bbpy (Scheme 6B) might be reached after C-O bond formation (see SI 8.4, TS3Abpy = 28.0 kcal/mol) via a O/H slippage, as proposed previously. [35] Although the ligand framework in the bipyridyl dihydride 11 is overall sterically less crowded, compared to 4 (Scheme 6B, %Vfree = 41.6 vs. %Vfree = 36.1) [36] and the Rucenters have similar positive NBO-charges of +0.33, the hydride abstraction in the case of the PNNH-ligand is around 4 kcal/mol lower in energy compared to the bipyridyl-analogue (Scheme 6B, TS4B = 26.9 vs TS4Bbpy = 30.9 kcal/mol). This difference might be explained by the additional stabilization of the formal alkoxide by the N-H moiety in 4. Indeed, the N-H proton is not only more polarized (NBO-charge of +0.41 vs. +0.27 in 11) but also more accessible in the parent dihydride compound 4, as demonstrated by the steric topographic analysis. In the PNNH-ligand, the northwest (NW) quadrant accommodating the N-H-moiety has an accessible free volume of %Vfree = 22.9, whereas the quadrants occupied by the ligand backbone PCH2 protons in both ligands are sterically more hindered (e.g. Scheme 6B, %Vfree (NE) = 10.6 and %Vfree (SE) = 14.6 in 11). Indeed, these electronic and steric analyses together indicate that a variety of factors likely govern the difference in activity of the catalysts employed. It might be argued, that the N-H bonding is made redundant in the presence of protic reagents/solvents. We nevertheless observe that the stabilization persists in the presence of explicit solvent molecules, with TS4Bexp being around 4 kcal/mol lower than the corresponding PNN-analogue (TS4Bexp = 17.1 kcal/mol vs. TS4Bexp,bpy = 21.9 kcal/mol, see SI 8.6) [34] with the ruthenium atom as the center of the sphere (3.5), scaled bond radii (1.17) and 0.1 mesh spacing.

Conclusion

In conclusion, we have developed a homogeneous, ruthenium catalyzed, efficient reforming of aqueous ethylene glycol under mild conditions. This reforming process produces glycolic acid (up to 93% yield) and pure hydrogen gas, without CO2 or CO emission. The reaction can also be performed on a large scale of 30 mmol of ethylene glycol. Moreover, this reforming process can also proceed well in water with no additional organic solvent. These characteristics make this protocol environmentally friendly, atomeconomical, renewable and sustainable. A plausible reaction mechanism is proposed based on stoichiometric reactions, NMR studies, X-ray crystallography and DFT calculations. The mechanistic studies allow rationalization of the high reactivity of the PNNH ligand framework. It is envisioned that the understanding of the reaction pathways at play might enable future development of new reaction schemes and catalysts.

Entry for the Table of Contents

RESEARCH ARTICLE

A homogeneous ruthenium-catalyzed reforming of aqueous ethylene glycol to glycolic acid and hydrogen is described. A plausible reaction mechanism, involving metal ligand cooperation is proposed and supported by stoichiometric reactions, NMR studies, X-ray crystallography and computational studies.

Y.-Q. Zou, N. von Wolff, M. Rauch, M. Feller, Q.-Q. Zhou, A. Anaby, Y. Diskin-Posner, L. J. W. Shimon, L. Avram, Y. Ben-David, and D. Milstein

Scheme 1 .

 1 Scheme 1. (a) Representative examples of heterogeneously catalyzed reforming of aqueous ethylene glycol. (b) Ethylene glycol as a reversible hydrogen carrier. (c) Homogeneously catalyzed reforming of aqueous ethylene glycol to glycolic acid and H2.

Figure 1 .

 1 Figure 1. Reforming aqueous ethylene glycol to hydrogen and sodium 2hydroxyacetate using 0.1% mol of Ru-8.

Scheme 2 .

 2 Scheme 2. (a) Deprotonation of Ru-8 followed by reaction with H2. (b) Site of deprotonation of Ru-8 by DFT calculations. Values are Gibbs free energies in kcal.mol -1 with respect to 3a in THF (Values in parentheses are gas phase values, 298K)

Scheme 3 .

 3 Scheme 3. Dearomatization of complex Ru-4 and reaction with ethylene glycol.

Figure 2 .

 2 Figure 2. X-ray crystal structure of complex 6. Atoms are presented as thermal ellipsoids at 50% probability level. Hydrogen atoms are omitted for clarity except the hydride H1A, H5, H3A and H4A. Selected bond lengths (Å) and angles (deg): Ru1-C20, 1.834(7); Ru1-N1, 2.123(5); Ru1-N2, 2.081(5); Ru1-P1, 2.2738(17); Ru1-H1A, 1.68(8); Ru1-O2, 2.243(4); H4A-O2, 1.867; H5-O2, 1.787; C20-Ru1-N2, 178.3(2); P1-Ru1-N1, 157.83(14); N2-Ru1-O2, 80.60(17).

Figure 3 .

 3 Figure 3. X-ray crystal structure of complex 7. Atoms are presented as therm al ellipsoids at 50% probability level. Hydrogen atoms are omitted for clarity except the hydride and H3. Selected bond lengths (Å) and angles (deg): Ru(1)-C(22), 1.845(4); Ru(1)-N(1), 2.086(3); Ru(1)-N(2), 2.118(3); Ru(1)-O(1), 2.207(3); Ru(1)-P(1), 2.2653(9); Ru(1)-H, 1.56(5); C(22)-Ru(1)-N(1), 178.50(14); N(2)-Ru(1)-P(1), 159.96(9); N(1)-Ru(1)-O(1), 77.43(11).

  Scheme 5. (A) Proposed catalytic cycle for the reforming of ethylene glycol with water. Values correspond to Gibbs free energies in kcal.mol -1 with respect to the starting material (0.0). Level of theory: M06-L/def2TZVP/W06/GD3//ωBP97M-V/def2TZVPP/RIJCOSX, structures optimized in THF (SMD). (B) Key transition states of the proposed mechanism. Bond lengths in the computed structures in Å.

Table 2 .

 2 Production of glycolic acid and hydrogen from aqueous ethylene glycol catalyzed by PNNH complexes Ru

-8, 9, 10

* Page No. -Page No. Homogeneous Reforming of Aqueous Ethylene Glycol to Glycolic Acid and Pure Hydrogen Catalyzed by Pincer- Ruthenium Complexes Capable of Metal-Ligand Cooperation

Scheme 4. Dearomatization of complex Ru-4 and reaction with glycolic acid, and reaction with ethylene glycol without base.

Acknowledgements D.M. holds the Israel Matz Professorial Chair of Organic Chemistry. Y.-Q.Z. acknowledges the Sustainability and Energy Research Initiative (SAERI) of Weizmann Institute of Science for a research fellowship. N.v.W. is supported by the Foreign Postdoctoral Fellowship Program of the Israel Academy of Sciences and Humanities. M.R. acknowledges the Zuckerman STEM Leadership Program for a research fellowship. N.v.W and M.R. thank Dr Mark Iron (department of chemical research

calculations. Computations were performed using HPC resources from GENCI-CINES (Grant 2019AP010811227).

Data availabilty:

All computed structures and energies can be accessed on the ioChem-BD.org online repository under the following link: https://doi.org /10.19061/iochem-bd-6-66