Francisco Humberto

Eryvaldo Sócrates

Tabosa Do Egito

Andreza Rochelle Do

Vale Morais

Everton Do

Nascimento Alencar

Alexandre Maciuk

Christine Vauthier
email: christine.vauthier@u-psud.fr

Francisco Humberto Xavier-Junior

Andreza Rochelle

Experimental design approach applied to the development of chitosan coated poly(isobutylcyanoacrylate) nanocapsules encapsulating copaiba oil

Keywords: nanocapsules, copaiba oil, chitosan, poly(isobutylcyanoacrylate), experimental design approach

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Abbreviations

CO

the amount of copaiba oil found in the dispersed media of the nanocapsules CO total amount of copaiba oil used in the preparation Adj R 2 adjusted determination coefficient

F model F-value of the model F Tab tabulated F-value F Tab residues
tabulated Fvalue of the residues F residues Fvalue of the residues R 2 coefficient of determination x 1 pH of the polymerization medium x 2 temperature of polymerization x 3 concentration of chitosan in the polymerization medium Y [START_REF] Ponchel | Specific and non-specific bioadhesive particulate systems for oral delivery to the gastrointestinal tract[END_REF] predicted droplet size (nm)

INTRODUCTION

Nanoparticles composed of mucoadhesive polymers are promising systems for oral drug delivery applications [START_REF] Ponchel | Specific and non-specific bioadhesive particulate systems for oral delivery to the gastrointestinal tract[END_REF][START_REF] Petit | The counterbalanced effect of size and surface properties of chitosan-coated poly(isobutylcyanoacrylate) nanoparticles on mucoadhesion due to pluronic F68 addition[END_REF]. Generally, nanoparticles are defined as solid colloidal particles that include both nanospheres and nanocapsules [START_REF] Guterres | Polymeric nanoparticles, nanospheres and nanocapsules, for cutaneous applications[END_REF]. The latter are vesicular systems in which the drug is confined in a liquid/solid cavity surrounded by a polymer envelope.

Proposed to improve drug delivery, nanoparticle systems can modulate drug biodistribution and release in a controlled manner, increase intracellular uptake and improve the stability of active substances [START_REF] Reis | Nanoencapsulation I. Methods for preparation of drug-loaded polymeric nanoparticles[END_REF][START_REF] Leite | Cardiotoxicity reduction induced by halofantrine entrapped in nanocapsule devices[END_REF][START_REF] Anton | Design and production of nanoparticles formulated from nano-emulsion templates -A review[END_REF]. Many types of nanoparticles made of biodegradable polymers, including poly (isobutylcyanoacrylate), were considered to improve drug delivery by oral route. To this purpose, their surface property may be tuned to increase mucoadhesion [START_REF] Bravo-Osuna | Mucoadhesion mechanism of chitosan and thiolated chitosan-poly(isobutyl cyanoacrylate) core-shell nanoparticles[END_REF][START_REF] Bravo-Osuna | Specific permeability modulation of intestinal paracellular pathway by chitosan-poly(isobutylcyanoacrylate) core-shell nanoparticles[END_REF].

Chitosan has been a widely used polysaccharide to formulate mucoadhesive systems [START_REF] Thanou | Oral drug absorption enhancement by chitosan and its derivatives[END_REF][START_REF] Chen | Synthesis of pH-responsive chitosan nanocapsules for the controlled delivery of doxorubicin[END_REF].

In addition, this polysaccharide is biocompatible for oral administration. Its inherent mucoadhesive properties comes from the amino groups included in the chemical structure that can interact with sialic acid groups of mucins composing the mucus via electrostatic interactions [START_REF] Illum | Chitosan as a novel nasal delivery system for vaccines[END_REF]. Besides, the positive charges of chitosan are also believed to play an essential role increasing the permeability of the intestinal epithelium thanks to its capacity to disturb the calcium concentration balance near the tight junction [START_REF] Bernkop-Schnurch | Development of a novel method for the preparation of submicron particles based on thiolated chitosan[END_REF]. Although widely used to improve mucoadhesion of nanospheres, this polysaccharide was not yet used to improve mucoadhesion of polymeric nanocapsules, which are interesting as drug delivery systems for lipophilic drugs.

The copaiba oil-resin (Copaifera langsdorffii) is an oily plant extract used in folk medicine in its in-natura form [START_REF] Sousa | Validation of a gas chromatographic method to quantify sesquiterpenes in copaiba oils[END_REF]. Phytochemical studies on oil-resin reveal that it contains a complex mixture of diterpenes and sesquiterpenes hydrocarbons [START_REF] Alencar | Chemical characterization and antimicrobial activity evaluation of natural oil nanostructured emulsions[END_REF], giving this oil many interesting therapeutic activities. For instance, these include anti-inflammatory, antitumor, anti-tetanus, antimicrobial, antileishmania activities, among others [START_REF] Santos | Antimicrobial activity of Brazilian copaiba oils obtained from different species of the Copaifera genus[END_REF][START_REF] Gomes | Antinociceptive activity of amazonian copaiba oils[END_REF][START_REF] Leandro | Chemistry and biological activities of terpenoids from copaiba (Copaifera spp.) oleoresins[END_REF]. Although used for years in folk medicine, it is believed that pharmacological activities of this oil may be increased by developing appropriate formulations.

Therefore, the aim of the present work was to develop an original formulation of nanocapsules coated with chitosan as mucoadhesive compound and copaiba. The polymer composing the nanocapsule envelope was a critical choice. Poly (isobutylcyanoacrylate) was selected because of its capacity to formulate nanocapsule that resist well to the gastric medium and promote release in the intestinal medium [START_REF] Aboubakar | Insulin-loaded nanocapsules for oral administration: In vitro and in vivo investigation[END_REF]. Although the development of mucoadhesive copaiba oil-containing nanocapsules of poly (isobutylcyanoacrylate) have not been described before. This process was based on the use of an experimental design approach that was never applied so far while developing new formulations of oil-containing nanocapsules prepared by interfacial polymerization of isobutylcyanoacrylate. It is noteworthy that experimental design approach was not so much applied for the development of nanocapsules while such an approach could be helpful to optimize formulations, limiting the number of experiments to perform hence the amount of reagent and time [START_REF] Patel | Development of oral sustained release rifampicin loaded chitosan nanoparticles by design of experiment[END_REF].

MATERIALS AND METHODS

1. Materials

Copaiba resin oil was purchased from Flores & Ervas (Piracicaba, SP, Brazil). Isobutylcyanoacrylate was provided by ORAPI engineered solutions worldwide (Vaulx-en-Velin, France). Water soluble chitosan Mw 20,000 g/mol was purchased from Amicogen (Jinju, South Gyeongsang, South Korea). Ethanol, acetone, 2-propanol, sodium hydroxide, ethyl acetate, nitric acid were provided by Fisher Scientific (Pittsburgh, PA, EUA). Diazomethane and β-caryophyllene were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Ultrapure water was obtained from a Millipore purification system (Milli-Q plus, Millipore, St Quentin en Yvelines, France). All chemicals were reagent grade and used as received.

Copaiba essential oil extraction

Copaiba essential oil was obtained from the hydrodistillation of 400 mL of copaiba resin oil using a Clevenger-type apparatus for 3 h. The extracted essential oil was dried with sodium sulphate, filtered, stored in the refrigerator and protected from light until use.

Preparation of the nanocapsules

Copaiba oil-loaded chitosan-decorated poly (isobutylcyanoacrylate) nanocapsules were elaborated by the method of interfacial polymerization [START_REF] Couvreur | Polycyanoacrylate nanocapsules as potential lysosomotropic carriers: preparation, morphological and sorptive properties[END_REF][START_REF] Fallouh | Development of a New Process for the Manufacture of Polyisobutylcyanoacrylate Nanocapsules[END_REF] that was adapted because of the use of chitosan. A preliminary study investigated the influence of the molecular weight of chitosan (20, 70 and 250 kDa), the type of copaiba oil (resin and essential oil) and the nature of the solvent phase (ethanol, 2-propanol and acetone) in order to identify the best substances to produce nanocapsule. Thus, 0.25 mL of copaiba oil and 0.032mL of isobutylcyanoacrylate were solubilized in 6.25 mL of solvent to produce the organic phase. This phase was slowly injected dropwise in 12.5 mL of the polymerization medium prepared with 0.6% of chitosan at pH 6 and homogenized for 10 min at 250 rpm at 25 °C (Fisher-Bioblock Scientific AM 3001K, Illkirch, France). The obtained colloidal dispersion was concentrated by rotary evaporator for 20 min at 35°C / 43mBar (BÜCHI Rotavapor R-125, Heating Bath B-491, Vacuum pump V-700, recirculating Chiller F-108, Flawil, Switzerland) to eliminate the solvent. Then, the dispersion was filtered through a 5 µm minisart NML membrane (Sartorius GmbH, Goettingen, Germany). The obtained nanocapsule dispersions were purified by dialysis (Spectra/Por Biotech membranes, cellulose ester, 100,000 g/mol molecular weight, Rancho Dominquez, CA, USA) against ultrapure water three times for 60 min and once overnight to remove non-associated chitosan. After dialysis, the nanocapsules were stored at +4°C for 24 hours before characterization.

Experimental design and nanocapsules preparation optimization process

In the present study, a 2 3 full-factorial experimental design with center points leading to 11 experimental randomized runs was used to optimize formulation and process parameters for the preparation of copaiba oil-loaded chitosan decorated-poly (isobutylcyanoacrylate) nanocapsules. Nanocapsules were prepared as described in the section 2.3 using copaiba resin oil, chitosan 20 kDa and ethanol as solvent. For the optimization of the nanocapsules, three independent variables including, the pH of the polymerization media (x 1 ) (3, 6 and 9), the temperature of production (x 2 ) (5, 25, 45 °C) and the concentration of chitosan 20 kDa (x 3 ) (0.3, 0.6, 0.9 %) were selected. For each variable, a low, medium or high level value was attributed (see Table 1). The size and zeta potential of nanocapsules were chosen as the dependent output response variables. Optimization was aimed to obtain small and positively charged nanocapsules. The effects of the studied variables were graphically and statistically interpreted using the Statistic software (Version 7.0, StatSoft Inc., USA) to validate the statistical design. Response surface plots were generated to visualize the simultaneous effect of each variable on each response parameter.

Table 1: Variables and levels chosen to define the experimental region and their corresponding coded values for nanocapsule production

Characterization of the nanocapsules.

Size measurement: Hydrodynamic mean diameter and size distribution of the nanocapsule dispersions were determined at 25°C by quasi-elastic light scattering using a Zetasizer Nano ZS90 (Malvern Instruments Ltd, Orsay, France). The scattered angle was fixed at 90°. The samples were diluted 1:100 before analysis with Milli ® Q water. Each measurement was done in triplicate meaning that the average was calculated from 9 values.

Determination of the zeta potential:

Zeta potential of the nanocapsules was deduced from the determination of the electrophoretic mobility by Laser Doppler Electrophoresis (Zetasizer Nano ZS90 (Malvern Instruments Ltd, Orsay, France). Nanocapsule dispersions were diluted (1:100) with NaCl at 1 mmol/L. Values are presented as mean of measurements performed on three replicate samples.

Morphology of nanocapsules:

Transmission electron microscopy observation of copaiba oilloaded chitosan-decorated poly (isobutylcyanoacrylate) nanocapsules was performed using a JEOL 1400 electron microscope (JEOL Ltd, Tokyo, Japan) equipped with a Gatan CCD digital camera (Orius SC1000) high-resolution. Samples were observed at 60kV after staining with phosphotungstic acid 2% (pH 7.4) for 30 seconds.

Independent Variable Level

i x i -1 0 +1

Analysis of the encapsulated copaiba oil

Copaiba oil composition was analyzed by PR2100 gas chromatography -Flame Ionization Detector (Alpha MOS, Toulouse, France). A fused silica capillary column (25 m × 0.32 mm i.d., 0.5 µm) coated with cross-linked 5% phenyl polysilphenylene-siloxane (SGE Analytical Science Pty Ltd, Victoria, Australia) was used. The method was previously validated for the analysis of the composition of copaiba oil (Xavier-Junior et al., submitted for publication). Samples were diluted with ethyl acetate and 2.5 µL was injected in the chromatograph. The operating conditions to the samples were: oven temperature program from 90 °C (2 °C min -1 ) to 150 °C, after isothermally heating 20 °C.min -1 to 300 °C, kept for 5 min at the final temperature. Split injection was 1:80, Nitrogen was the carrier gas at a pressure of 166 kPa, flow rate 1 mL.min -1 , temperature of injector and detector fixed at 250 °C and 300 °C, respectively. Composition of the major compounds present in the copaiba oil encapsulated in the nanocapsules was analyzed and compared with that of the oil taken prior to encapsulation.

Determination of encapsulation efficiency, encapsulation rate and concentration in the nanocapsule dispersion

For the determination of the encapsulation efficiency, the encapsulation rate and the concentration of copaiba oil in the nanocapsules, samples were prepared as explained bellow prior to their analysis by gas chromatography.

Copaiba oil-loaded chitosan-decorated poly (isobutylcyanoacrylate) nanocapsules were recovered by an ultrafiltration method. 0.5 mL of the nanocapsule dispersion was centrifuged in a Microcon centrifugal filter unit (Ultracel YM-100, regenerated cellulose, cut-off of 100 kDa, Merck Millipore, Billerica, MA, USA) at a speed of 10,000 rpm for 20 min (Eppendorf centrifuge 5418, Rotor FA-45-18-11, Hamburg, Germany) to remove the dispersion phase.

Copaiba oil-loaded chitosan-decorated poly (isobutylcyanoacrylate) nanocapsules were separated in the different fractions. The parent nanocapsule dispersion, nanocapsules retained on the membrane and dispersion phase (i.e. the ultrafiltrate) were analyzed. These fractions were then resuspended in 1 mL of ethyl acetate, sonicated for 1 hour and filtered through a 0.22 µm Millipore filter. The amount of β-Caryophyllene was determined by gas chromatography as described in section 2.6 to evaluate the amount of copaiba oil in each fraction. The encapsulation efficiency was calculated as follows (Equation 1):

EE (%)= (CO -CO ) CO x 100 (1)
Where CO total was the total amount of copaiba oil used in the preparation and CO dispersed-phase was the amount of copaiba oil found in the dispersed media of the nanocapsules at the end of the preparation.

The nanocapsule concentration in the dispersion was evaluated by gravimetry. 1g of the purified nanocapsule dispersion was freeze-dried and the dry residue was weighted to deduce the percentage of nanocapsules contained in 1g of the dispersion. The encapsulation rate was determined by the ratio between weights of the β-caryophyllene present in copaiba oil loaded in the nanocapsules and the total weight of the nanocapsule analyzed by gas chromatography.

Statistical analysis

The results of these experiments were compared using analysis of variance (ANOVA), which was able to determine if the variables and the interactions between variables were significant. Regression model, t-tests and F-test with a 95% confidence level (p<0.05) were performed. The softwares Graph Pad Prism (Version 5.0, La Jolla, CA, USA) and Statistic software (Version 7.0, StatSoft Inc., USA) were used to perform the statistical analysis of the data.

RESULTS

Copaiba oil nanocapsules were obtained by designing new conditions of interfacial polymerization of poly (isobutylcyanoacrylate), performed with a bioactive natural oil, chitosan and in the absence of surface-active compounds. As a first step of the work that aimed to identify the best conditions to be used to produce copaiba oil containing nanocapsules by interfacial polymerization, several experiments were performed to select suitable materials. To this aim, preparations of nanocapsules were performed with chitosan (0.6 %) of different molecular weight (20, 70 and 250 kDa), with copaiba resin and essential oils and with different solvents including ethanol, 2-propanol and acetone at pH=6 and temperature of 25°C. The solvents were chosen based on their miscibility with water, a condition to achieve the synthesis of nanocapsules by interfacial polymerization of alkylcyanoacrylates. Particle size and zeta potential of the obtained nanocapsules are summarized in Figure 1. The mean diameters of the nanocapsules ranged from 440 ± 8 nm to 932 ± 28 nm when they were prepared with copaiba resin oil and copaiba essential oil, respectively. Nanocapsules obtained with chitosan 20 and 70 kDa were spherical as observed by transmission electron microscopy. The morphology of nanocapsules suggests that they were formed by an oily core surrounded by a polymer envelope. The size of copaiba resin oil-loaded nanocapsules varied with the different solvents. The average particle size were 440 ± 8, 669 ± 8 and 536 ± 7 nm, respectively (p<0.05) for nanocapsules prepared with ethanol, 2-propanol and acetone (Figure 1A).

All nanocapsules were characterized by positive values of zeta potential indicating the presence of chitosan on the nanocapsule surface. The charges of the nanocapsule envelope were statistically different whether they were prepared with copaiba resin oil or copaiba essential oil. Nanocapsules prepared with copaiba essential oil displayed the lowest zeta potential (less than +12 mV).

Based on results obtained from this first series of preparations, optimization of conditions for the preparation of chitosan-coated nanocapsules containing copaiba oil was pursue using chitosan 20kDa, ethanol and copaiba resin oil. The experimental design approach used in this work represented an easy methodology to provide important data about the natural oilcontaining nanocapsules limiting the number of experiments to be performed hence the amount of reagent and time. Thus, the optimization was carried out following a 2 3 fullfactorial experimental design approach with center points. Three independent variables were considered including pH of the polymerization media (x 1 ), the temperature of production (x 2 ) and the concentration of chitosan 20 kDa (x 3 ) (Table 1). The optimal parameters chosen were those which produced small size nanocapsules with positive surface charges.

The mean hydrodynamic diameter of the produced nanocapsules was strongly influenced by the selected variables, emphasizing their relevancy as critical parameters which influence on the preparation process. The size ranged from 200 to 1200 nm. The Pareto chart (Figure 2) gives the relative importance of the individual and interaction effects [START_REF] Zu | Process optimization for the preparation of oligomycin-loaded folate-conjugated chitosan nanoparticles as a tumor-targeted drug delivery system using a two-level factorial design method[END_REF]. Initially, Pareto chart was created to analyses the individual standardized effects of studied variables, and posteriorly their interactions effects on the size and zeta potential, as well as it permits the identification of the statistically significant variables for the process, with a significance level (ρ-value of 0.05). The length of each bar in the chart indicated the standardized effect of the corresponding variable on the response. Negative values indicated unfavorable or antagonistic effect for the development of nanocapsules with the desired characteristics, i.e. small size and positive zeta potential, while positive coefficients showed a favorable or synergistic effect. As shown by the Pareto's chart and considering each variable separately, a decrease of the polymerization medium pH had a statistically positive effect in the decrease of the nanocapsule size. Furthermore, it was observed that the nanocapsule size decreased with an increase of the temperature while the concentration of chitosan had no significant effect on this parameter. Interactions between the different variables can be highlighted by investigating the combined influence of several variables. The Pareto's chart highlighted a clear significant interaction between the pH of the polymerization medium and the other studied variables, i.e. pH  temperature and pH  concentration. In the acid solution at high concentration of chitosan, the increase of the temperature from 5 to 45 °C induced an increase of the nanocapsule size from 250 to 1070 nm. In contrast, in basic polymerization medium, the temperature variation did not show significant size changes (approximately 930 nm). Opposite effect on the nanocapsules size was observed between the concentration of chitosan and the pH of the medium.

The analysis of variance was used to test the significance of independent variables and their interactions. The model's goodness of fit was checked by the coefficient of determination (R 2 ). The R 2 values provide a measure of how much variability in the observed response values can be explained by the experimental variables and their interactions (fluctuation) [START_REF] Cochran | Experimental designs[END_REF]. The matrix of the experiments developed were considered as statistically significant with linear relationship of R²= 0.968 which indicates that the model can explain 96.8 % of the variability in the response variable. In addition, the value of the adjusted determination coefficient (Adj R 2 = 0.942) was also very important to confirm a high significance of the model [START_REF] Montgomery | Design and Analysis of Experiments[END_REF], which value was very close to the experimental R 2 value. Therefore, these matrix of the experiments ensured a satisfactory adjustment of the polynomial model to the experimental data [START_REF] Liu | Optimal production of sulphuric acid by Thiobacillus thiooxidans using response surface methodology[END_REF]. By applying multiple regression analysis on the design matrix and analyzing the responses given in the experiments, the first-order polynomial equation given in Equation 2 in the coded form was established to size droplets:

Y 1 = 834 +214x 1 +58x 2 -104x 1 x 2 -81x 1 x 3 +174x 2 x 3 -169x 1 x 2 x 3 (2)
Where Y 1 was the predicted droplet size (nm), x 1 , x 2 and x 3 were the coded terms for three independent test variables: the pH of the polymerization medium, the temperature at which the interfacial polymerization was carried out and the concentration of chitosan added in the polymerization medium, respectively.

According to the regression model's ANOVA, it was possible to observe that the linear model was significant (p<0.05). This was evidenced from the Fisher's F -test which provided an F-value of the model (F model = 23.5) much greater than the tabulated Fvalue (F Tab = 2.4).

Concerning the statistical analysis used to investigate the significance of independent variables and their interactions, results from the Fisher's F-test suggested that the computed Fisher's variance ratio at 5 % level was large enough to justify a very high degree of adequacy of the linear model and also to indicate that treatment combinations were highly significant [START_REF] Sen | Response surface modeling and optimization to elucidate and analyze the effects of inoculum age and size on surfactin production[END_REF].

The normal (percentage) probability plot of the residuals was an important diagnostic tool to detect and explain the systematic departures from the assumptions that errors were normally distributed and were independent of each other and that the error variances are homogeneous. In this study, a plot of normal probability of the residuals indicated almost no serious violation of the assumptions underlying the analyses (F residues = 3.5). This value was found to be lower than the tabulated Fvalue (F Tab residues = 4.26) at the 5% level, indicating that the experiment exhibited predictive results (F residues / F Tab residues < 1). This satisfactory result confirmed the normality distribution assumptions previously made and the independence of the residuals.

Aiming the straightforward examination of the experimental variables on the responses, the three-dimensional response surfaces were drawn. Figure 3 (A) shows a three-dimensional diagram of calculated size response surface relating both pH and temperature to copaiba oilcontaining nanocapsule size.

Figure 3: 3D Response surface for size droplets (A) and zeta potential (B) variable dependency with temperature and pH independent variables to production of nanocapsules.

This diagram indicated that the size of the nanocapsules can be reduced decreasing the pH and the temperature applied during preparation. The response considering the size of the nanocapsules varied linearly with each of the variables demonstrating that there were considerable interactions between each of the independent variables and the size of the nanocapsules obtained from the different tested conditions.

Regarding the zeta potential, the copaiba oil nanocapsules showed values ranging from + 15 to + 55 mV. The Pareto's chart showed a higher increase of the zeta potential of the nanocapsules increasing the temperature and decreasing the pH of the polymerization medium comparatively to the smaller influence revealed by varying the concentration in chitosan (Figure 2). The first-order polynomial equation was established to zeta potential analyzing the responses given in the experiments (Equation 3):

Y 2 = 31.8 -3.8x 1 +6.4x 2 -3.4x 1 x 2 +4.2x 1 x 3 -5.9x 2 x 3 +1.9x 1 x 2 x 3 (3)
Where Y 2 was the predicted zeta potential (mV), x 1 , x 2 and x 3 were the coded terms for three independent test variables: the pH of the polymerization medium, the temperature at which the interfacial polymerization was carried out and the concentration of chitosan added in the polymerization medium, respectively.

These experiments presented the linearity regression of R²=0.94, therefore this model explains 94% of the variability in the response. The value of the adjusted determination coefficient (Adj R 2 = 0.92) was accepted as high significance of the model. The F test applied in the mathematical model shows the significant (Fmodel > F tab ) and predictive results (F residues / F Tab residues < 1) of these experiments. In addition, the full first-order response surface was plotted for analysis of the optimal zeta potential on the nanocapsules (Figure 3B). Nanocapsules with high positive values of the zeta potential can be obtained at low pH of the polymerization medium and performing the polymerization at high temperature. However, increasing the temperature of the polymerization increased the size of the nanocapsules that was not a desired result. To define optimal conditions, it was taken into account that the size decreased more than the zeta potential increased in the same range of variation of the temperature at which the polymerization was performed.

The result of the optimization study following a 2 3 full-factorial experimental design approach allowed to identify optimal conditions for nanocapsule preparation that have both a small particle size and positive zeta potential. The optimal characteristics of the nanocapsule preparation were pH 3, temperature 4 °C and a concentration of chitosan of 0.9 % (w/v). The samples prepared under these conditions showed a size of 473 ± 1 nm, the size distribution was unimodal with a polydispersion index of 0.20 ± 0.02 and a zeta potential of +34.8 ± 0.2 mV. Observation of the sample by transmission electron microscopy showed that the optimal nanocapsules prepared by interfacial polymerization were spherical and well individualized (Figure 4). They showed typical images of nanocapsules composed of an oily core surrounded by a polymer envelope.

The composition of copaiba oil recovered from the nanocapsules was determined by gas chromatography and compared with the oil prior to its nanoencapsulation (Figure 5). All components found in the original oil were also found in the nanocapsules. Differences in concentrations of components present in copaiba oil versus the copaiba oil extracted from the nanocapsules were in the range of precision (bellow 2%) of the gas chromatography method used to perform the analysis (Figure 5C). Results indicated that the encapsulated oil was identical in term of its composition than the initial oil. The nanoencapsulation process fully preserved the complexity of composition of the oil. Therefore, the encapsulation efficiency of copaiba oil was high at 75.8 ± 3 %. It could be determined that the nanocapsule dispersion contained 2.5 mg of β-caryophyllene encapsulated in the nanocapsules per mL of the dispersion while the β-caryophyllene payload was 55.5 µg/mg of nanocapsules.

DISCUSSION

This work aimed to identify optimal conditions for the encapsulation of copaiba oil in chitosan-coated nanocapsules by the method of interfacial polymerization of isobutylcyanoacrylate. Although the method of the obtaining of the nanocapsules is well known, in the present work it was applied to the encapsulation of copaiba oil, a natural complex oil mixture, that was never described to our knowledge. The second originality of the work was to design those innovatively obtained nanocapsules with positive charge on their surface. It was assumed that stable dispersion of nanocapsules could be prepared in the absence of surfactants thanks to the incorporation of chitosan in the nanocapsule envelope conferring the surface with the desired positive charges. Because chitosan include chemical groups that are able to initiate the polymerization of isobutylcyanoacrylate monomers [START_REF] Zandanel | Characterization of fluorescent poly(isobutylcyanoacrylate) nanoparticles obtained by copolymerization of a fluorescent probe during Redox Radical Emulsion Polymerization (RREP)[END_REF], it was postulated that chitosan could be incorporated in the nanocapsule envelope thanks to a reaction with isobutylcyanoacrylate that lead to the synthesis of an amphiphilic copolymer including poly(isobutylcyanoacrylate) chains grafted on chitosan. Thus, the grafted poly(isobutylcyanoacrylate) chains could serve as anchor to attach chitosan at the nanocapsule surface.

Preparations of nanocapsules were investigated changing the type of solvents used to produce the oil-in-water emulsion based on a solvent diffusion method. The nature of the solvent is an important factor as it conditions the nucleation of emulsion droplets of the oil that formed by a mechanism of homogenous nucleation (Ouzo effect) [START_REF] Ganachaud | Nanoparticles and nanocapsules created using the Ouzo effect: Spontaneous emulsification as an alternative to ultrasonic and high-shear devices[END_REF]. The feasibility of the encapsulation of copaiba oil was assessed considering the resin and the essential oil fractions. Small and highly positively charged nanocapsules containing copaiba resin oil could be obtained working with ethanol as the dispersing solvent. The results are consistent with those of the literature where ethanol is widely used as a suitable solvent to produce nanocapsules by interfacial polymerization of alkylcyanoacrylate [START_REF] Gallardo | Study of the mechanisms of formation of nanoparticles and nanocapsules of polyisobutyl-2cyanoacrylate[END_REF]. On the other hand, nanocapsules produced with the essential oil were of much larger size and showed a lower zeta potential. Copaiba resin and essential oils show different composition which volatile components were extracted from the resin to prepare the essential oil fraction. This difference in composition may explain the difference in the size of droplets that were formed by the Ouzo effect during the preparation of the emulsion in the beginning of the process and hence, affecting the nanocapsules size. Regarding the effect of the molecular weight of chitosan, at constant temperature and concentration, viscosity of polymer solutions increases with the molecular weight of the polymer [START_REF] Desbrieres | Viscosity of semiflexible chitosan solutions: influence of concentration, temperature, and role of intermolecular interactions[END_REF] and the smallest nanocapsules were obtained with the shorter chitosan. They were obtained with the aqueous phase of the expected lower viscosity favoring the dispersion of the organic phase during formation of the emulsion by the solvent diffusion technique. Data obtained from these preliminary experiments provided with enough information to make the selection of the components producing positively charged copaiba oil-loaded nanocapsules.

For the optimization step, temperature, pH of the polymerization medium and concentration of chitosan were the variables analyzed with a full-factorial 2 3 experimental design approach. In all tested conditions, the nanocapsules showed a positive zeta potential. Nanocapsules from the literature that were prepared in the absence of chitosan were characterized with a negative zeta potential [START_REF] Aboubakar | Insulin-loaded nanocapsules for oral administration: In vitro and in vivo investigation[END_REF][START_REF] Cournarie | Evidence for restrictive parameters in formulation of insulin-loaded nanocapsules[END_REF]. The positive value of the zeta potential acknowledged the presence of chitosan on the nanoparticle surface consistently with the assumption drawn for this work. Formation of nanocapsules with a small hydrodynamic diameter was promoted in conditions of preparation favoring a slow polymerization of isobutylcyanoacrylate. These included acid pH and lower temperature. In contrast, submitted to conditions in which the polymerization happened more rapidly because of the basic pH, high temperature, and low degree of protonation of chitosan, the nanocapsules size was markedly increased. These results were consistent with those of the literature suggesting that slowing down the polymerization carried out in presence of chitosan is needed to obtain well define nanoparticles [START_REF] Mccarron | Response surface methodology as a predictive tool for determining the effects of preparation conditions on the physicochemical properties of poly(isobutylcyanoacrylate) nanoparticles[END_REF][START_REF] Ravikumara | Chitosan nanoparticles for tamoxifen delivery and cytotoxicity to MCF-7 and Vero cells[END_REF][START_REF] Yang | Formation of positively charged poly(butyl cyanoacrylate) nanoparticles stabilized with chitosan[END_REF][START_REF] Aboubakar | Study of the mechanism of insulin encapsulation in poly(isobutylcyanoacrylate) nanocapsules obtained by interfacial polymerization[END_REF].

The high encapsulation efficiency of copaiba resin oil indicated that the oil immediately diffused to the internal phase of nanocapsules, where it was entrapped by the newly polymer formed nanocapsules envelope. The maintenance of oil composition during the nanoencapsulation process was an important parameter to evaluate because the oil contains biologically active components that are believed to promote a synergistic effect when they are associated with classical anticancer drugs with a high potential for the development of new treatments against cancer. The synergistic effect may arise from the accumulation of βcaryophyllene in membranes of cancer cells that can promote membrane permeability to bioactive compounds as suggested in a recent report [START_REF] Legault | Potentiating effect of beta-caryophyllene on anticancer activity of alpha-humulene, isocaryophyllene and paclitaxel[END_REF]. A simulation of the dosage of nanocapsules required to fulfill a therapeutic dose of β-caryophyllene in human adults (0.16-3.3 mg/kg for a 60kg human) was calculated from the amount of this compound found in the nanocapsules. The requested dose range of β-caryophyllene can be achieved by administering 0.18 to 3.6 g of nanocapsules. This seems reasonable to achieve in practice emphasizing the interest of the proposed nanocapsules that are easy to produce. Since the developed nanocapsules were coated with chitosan, they are expected to display mucoadhesive properties that are of interest to improve drug delivery by mucosal routes of administration, including the oral route.

CONCLUSION

Poly (isobutylcyanoacrylate) nanocapsules incorporating copaiba oil could be prepared by interfacial polymerization in a surfactant free polymerization medium thanks to the use of chitosan. As assumed, chitosan was associated with the nanocapsules and provided positives charges to the nanocapsules surface. An experimental design approach was used to optimize the formulation aiming to prepare nanocapsules with small diameter and highly positive zeta potential. The three variables of the process, i.e. pH, concentration of chitosan in the polymerization medium and the temperature of polymerization, were identified influencing both the size and zeta potential of the prepared nanocapsules. These variables are known to affect the polymerization of isobutylcyanoacrylate. Copaiba oil was efficiently encapsulated while the composition of the encapsulated oil was identical to that of the original oil. These nanocapsules are expected to be mucoadhesive and suitable to serve as carrier system for lipophilic anticancer drugs by the oral route with possible synergistic anticancer activity between the oil and the drug. The overall work presented itself as a rich information source for further research aiming to develop potential mucoadhesive nanoparticles containing natural oils by performing a small amount of experiments, saving time and reagents on development.

Figure 1 :

 1 Figure 1: Particle size (A) and zeta potential (B) of copaiba oil nanocapsules with chitosan coated in the surface at pH=6 and temperature of 25°C. Wherein RO and RE corresponds to nanocapsules produced with copaiba resin and essential oils, respectively; C20, C70 and C250 referred to the three different chitosan (0.6 %) molecular weights of 20, 70 and 250 kDa; and E, iP and A indicated the type of organic solvent used, ethanol, 2-propanol and acetone respectively, used to nanocapsule production.

Figure 2 :

 2 Figure 2: The Pareto Chart of standardized effects to size and zeta potential dependent variables (p<0.05).

Figure 4 :

 4 Figure 4: Transmission electron microscopy analysis of the optimal formulation of copaiba oilloaded chitosan-poly (isobutylcyanoacrylate) nanocapsules stained with phosphotungstic acid (2%) at 60 kV (Imagif). Scale bar of 100nm.

Figure 5 :

 5 Figure 5: Major compounds encapsulated in copaiba resin oil-nanocapsules coated with chitosan. Figures A and B represent the copaiba resin oil chromatograms before and after recovery from the nanocapsule. Figure C gives the difference in the area of the peaks between the copaiba resin oil in native form and the oil encapsulated in the nanocapsules. The gray color represents the precision of the method to determination of copaiba oil compounds

ACKNOWLEDGEMENTS

Authors acknowledge financial support from CAPES COFECUB 721/11 and would like to thank Imagif Cell Biology Unit of the Gif campus (www.imagif.cnrs.fr) which is supported by the Conseil Général de l'Essonne for providing facilities for transmission electron microscopy analysis.


Pavel A Dub

Miguel Baya

Jennifer Houghton

Natalia V Belkova

Jean-Claude Daran

Rinaldo Poli
email: poli@lcc-toulouse.fr

Lina M Epstein

Elena S Shubina

Jean- Claude Daran

Solvent control in the protonation of [Cp*Mo(dppe)H3] by CF3COOH

Keywords: Molybdenum, hydride ligands, proton transfer, dihydrogen bonding, trifluoroacetate ligand Molybdenum / hydride ligands / proton transfer / dihydrogen bonding / trifluoroacetate ligand

The outcome of the reaction between Cp*Mo(dppe)H3 [dppe = 1,2bis(diphenylphosphino)ethane] and trifluoroacetic acid (TFA) is highly dependent on the solvent and the TFA/Mo ratio. The new dihydride compound Cp*Mo(dppe)H2(O2CCF3) is obtained selectively when the reaction is carried out in aromatic hydrocarbons (benzene, toluene) using ≤1 equiv of TFA, and is also the end product in THF or MeCN independent of the TFA/Mo ratio. In benzene/toluene, use of an excess amount of the acid has a profound effect, resulting in the formation of the tetrahydrido complex [Cp*Mo(dppe)H4] + , which does not further evolve to the dihydrido product. Monitoring the reaction by NMR and IR under different conditions (solvent, temperature, TFA/Mo ratio) reveals the rapid establishment of an equilibrium between the dihydrogen bonded adduct, Cp*Mo(dppe)H3•••HOOCCF3, the ion paired proton transfer product, Cp*Mo(dppe)H4 + ••• -OOCCF3, and the separated ions, followed by a slower irreversible transformation to the final dihydride product with H2 evolution. The activation parameters of the H2 evolution and M-OR product formation have been determined. Excess TFA in low polarity solvents stabilizes the separated charged species by forming the homoconjugate anion [CF3COO(HOOCCF3)n] -. The effect of the solvent on the course of the reaction is interpreted in terms of the differing polarity, H-bonding ability, and coordinating power of the different solvent molecules.

Introduction

It is now well established that a common proton transfer pathway from a proton donor HA to a transition metal hydride complex involves the initial attack of HA on the hydride ligand with formation of a dihydrogen bonded adduct, MH•••HA, followed by proton transfer and production of a non-classical dihydride (dihydrogen complex) product, M( 2 -H2) + . The establishment of a hydrogen bonded ion pair between the non-classical cation and its counteranion decreases the stability of the M( 2 -H2) + complex and favors its transformation into a classical polyhydride or organyloxo (if X = OR) species (Scheme 1). [1] MH + HX MH

•••HX M( 2 -H) + •••X - M( 2 -H) + //X - MX M(H) 2 + //X - -H 2 I II III IV V VI VII M(H) 2 + •••X - Scheme 1
The formation of M( 2 -H2) + •••X -ion pairs (III, X -= CF3COO -, ArO -, R F O -), stabilized by hydrogen bonds between the dihydrogen ligand and counteranions, was detected for CpRuH(CO)(PCy3), [2,3] PP3MH2 [PP3 = P(CH2CH2PPh2)3] [4] and Cp*MH(dppe) (M = Fe, [5] Ru [6] ) complexes by IR and UV-visible spectroscopy. The ion pair stability increases with the proton accepting ability of the anion. [3] Proton transfer may also be assisted by a second molecule of the proton donor HX, depending on the acid strength. In this case, the conjugate base X -in species III-VI may be present in the form of the homoconjugate anion, XHX -. We have recently shown that the irreversible isomerization of Cp*M( 2 -H2)(dppe) + complexes (M = Fe, [5] Ru [6] ) into classical trans-dihydride species occurs upon dissociation of the M( 2 -H2) + •••XHX -ion pair (i.e. III to IV). This is indicated by difference in temperature at which the M( 2 -H2) + → M(H)2 + isomerization process can occur depending on the counteranion: higher temperatures are necessary to induce isomerization in the presence of more basic anions (greater ion pair stability). Additionally, the isomerization rate constant does not depend on the anion nature. [7,8] In some cases, the non-classical to classical rearrangement (III to V, or IV to VI) is a reversible process. [9] In many cases, however, basic anions (X -= RO -, RCOO -) tend to displace H2 from the transition metal coordination sphere, yielding organyloxo products (i.e. VII). This is the case for the protonation of (triphos)ReH(CO)2, [10] Re(CO)H2(NO)(PR3)2, [11] and the above mentioned CpRuH(CO)(PCy3), [2] for which corresponding M( 2 -H2) + X -complexes could be isolated as BF4 -salts which evolved into MX species in the presence of fluorinated alcohols or carboxylic acids. This reaction proceeds intra-molecularly without the hydrogen bonded ion pair dissociation. [2] Most of the M-OR complexes were only characterized in solution, two examples of isolated compounds being ReH(OC6H4NO2)(CO)(NO)(PMe3)2 [11] and (triphos)Re(CO)2-(OCOCH2Cl). [10] In addition to the counteranion nature, temperature and solvent polarity could be used to control the stability of the M( 2 -H2) + species and the proton transfer equilibrium position. For example, the substantial increase of the dielectric constant of the Freon mixture (CDCl2F/CDF3 2:1) at low temperatures was reported to assist the proton transfer to Cp*RuH3(PCy3) yielding [Cp*Ru(H)2( 2 -H2)(PCy3)] + OR -. [12] However, the influence of the medium on the H2 release process was not studied in detail.

In contrast to all the above described systems, the protonation of the trihydridomolybdenum complex Cp*Mo(dppe)H3 gives, via the intermediate formation of a dihydrogen bonded complex, the cationic classical tetrahydride complex [Cp*Mo(dppe)H4] + (V/VI), without the detection of a nonclassical isomer, [Cp*Mo(dppe)( 2 -H2)H2] + (III/IV).

Theoretical calculations, however, located the latter complex on the proton transfer potential energy surface, with a very low energy barrier for the intramolecular rearrangement. [13] Facile access to a nonclassical isomer is evidenced by the evolution of H2 from [Cp*Mo(dppe)H4] + at ambient temperature. We now report new results on the protonation of Cp*Mo(dppe)H3 using trifluoroacetic acid (TFA) in various solvents of different coordinating (in particular H-bonding) ability and polarity: benzene, toluene, THF, CH2Cl2 and MeCN. These reactions lead to the formation of a new hydride product with a coordinated trifluoroacetate anion. We will show how subtle changes of the protonation medium may drastically effect the reactivity of Cp*Mo(dppe)H3 and the stability of its protonation product.

Results

Since the results of previous investigations into the protonation of Cp*Mo(dppe)H3 [13][14][15] serve as basis for the new findings described in this paper, we summarize here the salient points that emerge from those studies.

H2(O2CCF3).

The room temperature reaction between Cp*MoH3(dppe) and strictly 1 equivalent of TFA in toluene led selectively to a new dihydride complex, Cp*Mo(dppe)H2(O2CCF3), as seen in Equation 1. The parallel formation of free H2 was confirmed by 1 H NMR monitoring (characteristic resonance at  4.58). The new hydride compound was isolated and fully characterized. It is quite stable in aromatic solvents, decomposing very slowly with generation of free dppe (identified by its 31 P resonance at  -12.9) and other unidentified products that do not contain hydride ligands. Ca. 13 % of the product decomposed after 22 hours at room temperature.

Cp*Mo(dppe)3H3 + TFA Cp*Mo(dppe)H2(O2CCF3) + H2

Equation 1

The compound is characterized by a hydride resonance centered at  -5.08, with a relative intensity corresponding to two protons, only slightly displaced with respect to the hydride resonance of Cp*Mo(dppe)H3 at  -5.16. The 31 P{ 1 H} spectrum shows a resonance at  76.9, which becomes a triplet when selectively decoupled from the dppe proton resonance, confirming the presence of two hydride ligands. The shape of the hydride resonance suggests the occurrence of a dynamic process. A variable temperature NMR investigation gave further information about this process and also provided longitudinal relaxation times for the hydride resonances. The and S ‡ = 10 ± 2 cal K -1 mol -1 were calculated using an Eyring analysis (see Supporting Information). The line shape analysis of the 1 H resonance was complicated by the lack of knowledge of JHP data due to broadness in the low temperature spectra, but a simulation using the rate constant obtained from the 31 P spectrum and a reasonable guess for the JHP values gave a reasonable fit above coalescence, suggesting that the same mechanism is responsible for both H and P exchange processes. A possibility for this mechanism is illustrated in Scheme 3. The T1 value for the hydride resonance remains rather high throughout the temperature range (see Supporting Information), characteristic of a classical dihydride, with a minimum of 510 ms before decoalescence. For comparison, the T1min of complex [Cp*Mo(dppe)H4] + is 174 ms for the BF4 -salt and 191 ms when obtained by proton transfer from TFE (these measurements were carried out at 400 MHz). [13] -9 -8 -7 -6 -5 -4

 (ppm) 193 K 213 K 233 K 253 K 273 K 293 K (a) -9 -8 -7 -6 -5 -4  (ppm) 193 K 213 K 233 K 253 K 273 K 293 K (

Scheme 3

The chemical composition of the product was confirmed by a single crystal X-ray analysis.

The coordination geometry, shown in Figure 2 [16] 2.102(3) and 2.092(3) Å found in CpMo III ( 4 -C4H6)(O2CCF3)2, [17] and 2.113 (4) and 2.102 (4) Å found in Cp2Mo IV (O2CPh)2. [18] Another interesting feature is the similar C8-O1 ( [16] 1.276(5) and 1.255(5) vs. 1.200(5) and 1.192(6) Å ( = 0.076 and 0.063 Å) for CpMo III ( 4 -C4H6)(O2CCF3)2, [17] and 1.281(8) and 1.300(8) vs. 1.208(8) and 1.210(8) Å ( = 0.073 and 0.090 Å) for Cp2Mo IV (O2CPh)2. [18] The above combined evidence indicates a significant ionic contribution to the bond between the Mo center and the monodentate anion, which then continues to promote some electronic delocalization between the two C-O bonds.

Other relevant structural parameters are listed in Table 1. Compound Cp*Mo(dppe)H2(OCOCF3) exhibits two intense overlapping  as OCO bands in the IR spectrum, the frequencies and relative intensities of which are solvent dependent (see Table 2). As the solvent polarity increases, Alow/Ahigh decreases and  = (high -low) increases.

The same behavior was observed previously for CpM(CO)2(OCOR) complexes (M = Fe, Ru) [20] and was explained by the presence of a Fermi resonance between the fundamental  as OCO stretching vibration and an overtone or combination band of very close frequency. The larger intensity ratio observed in CH2Cl2 could be explained by the different nature of the solutesolvent interaction, since this solvent could act as a proton donor and form hydrogen bonds with carboxyl group coordinated to the metal.

(b) Protonation in benzene/toluene: evolution with time.

The 1 H NMR monitoring of the Cp*Mo(dppe)H3-TFA reaction in C6D6 at different TFA/Mo ratios at room temperature was most enlightening. Figure 3a shows the spectrum of the starting material before TFA addition. [21] -6 -5 -4 -3

/ppm (a) (b) (c)
x 30

x 30 When using a 10-fold excess of TFA, the tetrahydrido complex [Cp*Mo(dppe)H4] + was the only observed product, Figure 3b. This is characterized by a hydride triplet resonance at -3.57 ppm (JHP = 37.9 Hz) in the 1 H NMR spectrum and by a 31 P resonance at 72.3 ppm in C6D6, quite close to the values previously reported for the BF4 -salt in CDFCl2 at -60°C. [14] In agreement with the literature, decomposition takes place at room temperature (90% after ca. 25 min), without yielding any hydride containing product. This decomposition was not investigated in any further detail.

~~~~(

For the 1:1 reaction, a broad resonance was initially present in the hydride region at an intermediate position between those of the trihydride and tetrahydride complexes, in addition to the final product resonance (Figure 3c). The position and shape of the broad resonance depends on the TFA/Mo ratio and on the temperature (vide infra). The 31 P{ 1 H} spectrum at room temperature exhibits two small and broadened resonances at the characteristic positions of the trihydride complex ( 91.4) and the tetrahydride protonation product ( 72.3), [22] in addition to the sharp resonance of the Cp*Mo(dppe)H2(O2CCF3) product at  76.9 and a minor contaminant resonance. [21] This suggests that the broad 1 H NMR resonance results from a rapid degenerate exchange between the starting complex Cp*Mo(dppe)H3, which is not completely consumed, and its protonation product, Cp*Mo(dppe)H4 + . This exchange will be addressed in further detail in the next section. Given the larger chemical shift difference between the 31 P resonances, the shape of 31 P NMR spectrum is much closer to the slow exchange limit than that of the 1 H NMR spectrum. After a few hours at room temperature (or upon brief warming to ca. 40°C), the broad band disappears and the resonance corresponding to Cp*Mo(dppe)H2(OCOCF3) increases in intensity (Figure 3d). These observations reveal the presence of an equilibrium between the tetrahydride complex and the starting trihydride complex, followed by a slow irreversible conversion to the final product by H2 loss.

A variable temperature study of the reaction mixture in C6D5CD3, carried out immediately after mixing the reagents in a 1:1 ratio, confirmed the above assignments. The hydride resonance of the dihydride product evolved as discussed above (Figure 1a), whereas the broad resonance at -3.63 ppm decoalesced at T < 270 K to yield two resonances at the expected positions of Cp*Mo(dppe)H3 and Cp*Mo(dppe)H4 + . The incomplete protonation in the presence of a stoichiometric amount of TFA shows that the protonation process is at equilibrium using this particular proton donor. Both complexes exhibit sharp resonances when alone and also when mixed together with Cp*Mo(dppe)H2(OCOCF3). Interestingly, the hydride resonance of Cp*Mo(dppe)H4 + broadens again at temperatures < 240 K, but does not decoalesce. On the other hand, the BF4 -salt of this complex was previously reported to retain sharp lines in the 1 H and 31 P NMR spectra down to -90°C, indicating rapid equilibration between the inequivalent H sites. [14] This suggests that the hydride scrambling process is slowed down by hydrogen bonding between Cp*Mo(dppe)H4 + and CF3COO -.

The reaction with 3 equiv of TFA resulted in the observation of a sharp resonance at the chemical shift of the tetrahydride complex (indicating essentially complete disappearance of the starting trihydride reagent) and no significant amount of the dihydride product. The subsequent addition of two equivalents of Et2NH to this solution led to the formation of complex Cp*Mo(dppe)H2(OCOCF3) cleanly and quantitatively. When using 1.5 equivalents of TFA, rather sharp resonances for the tetrahydrido complex and the final dihydrido product were initially observed by 1 H NMR monitoring, but the latter was once again the only observable species remaining at end of the reaction (ca. 30 min at 290 K). When using only 0.5 equiv of TFA, on the other hand, a broad resonance was again initially present, but its chemical shift was closer to that of the starting hydride complex, see We have also investigated the reaction between Cp*Mo(dppe)H3 and CF3COOD (1 equivalent) by 1 H NMR in C6D5CD3. The reaction progresses in an essentially identical manner to that when CF3COOH is used: immediate observation of the virtual triplet at  -5.25 due to the final product and a broad resonance centred at ca.  -3.6. The latter disappears upon brief warming to ca. 40°C. However, the NMR shows the formation of H2 and HD in ca. 3:1 ratio. This is not surprising, since all hydride positions in the tetrahydride complex are readily scrambled, therefore the elimination of H2 from the [Cp*Mo(dppe)H3D] + cation will be statistically favored. The molybdenum product resulting from this reaction should therefore contain both H and D. The hydride resonance does not show evidence for the presence of isotopomers, probably because the isotope shift is too small. The formation of deuterated product(s), Cp*Mo(dppe)HD(O2CCF3) and/or Cp*Mo(dppe)D2(O2CCF3), however, was confirmed by a 2 H NMR study, which revealed a binomial triplet resonance at  -5.9 with JPD = 7.7 Hz.

(c) Protonation in other solvents: NMR study.

The solvent has a significant effect on the course of the protonation reaction. The behavior in THF-d8 is similar but not identical to that in aromatic hydrocarbons (see Figure 5): use of a (sub)stoichiometric amount of the acid initially yields a broad hydride resonance (Figure 5b). Time evolution at these ratios leads to sharpening of the signals as the irreversible H2 elimination and Cp*Mo(dppe)H2(O2CCF3) formation takes place, a similar process to that shown in Figure 4 for the benzene solution. When the sample corresponding to Figure 5b was treated with additional TFA (total: 1.5 equiv) quantitative formation of the dihydride product occurred (Figure 5c). Use of a large excess (10 equiv) of CF3COOH affords a sharp triplet corresponding to the tetrahydrido cation and a strong resonance for the Cp*Mo(dppe)H2(O2CCF3) product (Figure 5d). As in C6D6 or C6D5CD3, a broad resonance is not obtained at this ratio. However, with time, the tetrahydride resonance disappears and the dihydride resonance increases in THF-d8 (Figure 5e), whereas in the analogous benzene solution decomposition occurs without formation of the dihydride product, as stated above.

-6 -5.5 -5 -4.5 -4 -3.5 -3 The doublet resonance at  -3.73 belongs to the Cp*Mo( 1 -dppe)H5 contaminant. [14] THF was found to be a suitable solvent in which to probe the nature of the degenerate although the broadening effect was limited to a few Hz in most cases. [2,23] ( The doublet resonance at  -3.73 belongs to the Cp*Mo( 1 -dppe)H5 contaminant. [14] The new triplet resonance at  -5.65 in Figure 6 Cp*Mo(dppe)H3 by HBF4 in MeCN, [14] displays a proton resonance at  -5.50 (JP1 = 51 Hz, JP2 = 52 Hz) and a phosphorus resonance at 78.1. NMR monitoring shows that this resonance grows while the resonance of the tetrahydride product simultaneously decreases. Interesting features to note are: (i) this cationic THF adduct is not observed in the protonation experiment with CF3COOH; (ii) the rate of disappearance of the tetrahydride complex is much slower in the presence of the BF4 -anion than in the presence of the CF3COO -anion (cf. Figure 6 with Figure 5); (iii) conversion to the THF solvent adduct appears to be much slower than the analogous reaction in MeCN, which was reported to lead to vigorous gas evolution and quantitative production of the MeCN complex within 60 min. [14] When using CH2Cl2 as solvent, the initially formed tetrahydride complex displayed a sharper resonance, even when as little as 0.5 equiv of the acid was used. The formation of minor amounts of Cp*Mo(dppe)H2(O2CCF3) was also detected (ca. 10% for the 1 equiv experiment).

/ppm (a) (b) (c) (d) (e)
In this solvent, however, the interpretation of the data is difficult because of the instability of the starting material, the cationic tetrahydride intermediate, and the product (24% decomposition within 1 hour at RT).

Finally, the results of the protonation with TFA (0.5 and 1 equiv) in MeCN-d3 at room temperature are shown in Figure 7.

In both cases, the immediate formation of Cp*Mo(dppe)H2(O2CCF3) and [Cp*Mo(dppe)H4] + was observed. When using 0.5 equivalents, the resonances corresponding to the tetrahydride complex and trihydride residual starting compound were separate and relatively sharp, see Figure 7a, a similar situation to that seen in CD2Cl2 using TFA as proton donor or in THF-d8 using HBF4. This indicates that the degenerate exchange is slow under these conditions contrary to the situations in THF-d8 and C6D6 using TFA as proton donor. In turn, this suggests that weak ion pairing occurs, if any, between the tetrahydrido cation and the trifluoroacetate anion in MeCN-d3. The conversion of the tetrahydride complex to Cp*Mo(dppe)H2(O2CCF3) is relatively fast and quantitative, see Figure 7b-d. As already noted, when the reaction is carried out in THF the final product is the trifluoroacetate compound rather than the solvent adduct [Cp*Mo(dppe)H2(MeCN)] + (previously shown to result from the interaction with HBF4), implying that the ligand exchange shown in Equation 2 is completely displaced toward the right. A first comparison of the behavior of [Cp*Mo(dppe)H4] + in non coordinating solvents confirms the complex instability, as previously shown for the BF4 -salt, leading to hydride-free decomposition products. [14] However, in the presence of either coordinating solvents or the CF3COO -anion, efficient trapping of the 16-electron product of H2 dissociation, [Cp*Mo(dppe)H2] + , is achieved. The coordinating CF3COO -anion is an even better trap than THF and MeCN. The nature of the solvent has a dramatic effect on two related features: (i) the ability of the CF3COO -anion to attack the cationic complex and yield the neutral dihydridetrifluroacetate product; (ii) the rate of degenerate exchange between Cp*Mo(dppe)H3 and [Cp*Mo(dppe)H4] + . The way in which these two processes are affected by the solvent properties will be addressed in more detail in a later section.

(d) IR studies.

As equilibrium positions and reaction rates are expected to strongly depend on the solvent ability to influence hydrogen bonding and ion pairing equilibria, we considered that additional useful information could be gathered from infrared investigations, especially at low temperature where ion pairing and hydrogen bonding are favored. The dielectric permittivity, , increases at ambient temperature in the order toluene (2.4) < THF (7.8) < CH2Cl2 (8.3) << MeCN (35.9). [START_REF] Reichardt | Solvents and Solvent Effects in Organic Chemistry[END_REF] It increases substantially upon cooling for some solvents, e.g. a values of 15.5 for CH2Cl2 [START_REF] Morgan | [END_REF]26] and 11.9 for THF [27,28] are recorded at 200 K, but not for others, e.g. 2.47 at 240 K and 2.71 at 180 K for toluene. [26] On the other hand, these solvents have different coordinating and acid/base properties. THF is quite an efficient hydrogen bond acceptor (Ej = 1.04), better than MeCN (Ej = 0.75), [29] although the latter is generally a better ligand for transition metals.

Benzene and toluene are much weaker bases (Ej = 0.4), [29] whereas CH2Cl2 tends to behave more like a weak proton donor than a proton acceptor. [START_REF] Reichardt | Solvents and Solvent Effects in Organic Chemistry[END_REF] We start by presenting the spectral features of the TFA proton donor in the CO region in various solvents, since the TFA/solvent interaction certainly plays an important role in the proton transfer equilibria involving the hydride complex.

The relevant spectra are shown in Figure 8. Two bands at similar frequencies are observed in CH2Cl2 and toluene (1804 and 1785 cm -1 for the former, 1803 cm -1 and 1788 cm -1 for the latter).

The higher frequency band is due to the acid monomer, whereas the lower frequency one is typically assigned to the corresponding dimer. [30] Note that the equilibrium is shifted towards the dimer in toluene, as is evident from the different relative intensities. In THF the wide, asymmetric CO band of the acid monomer is greatly red-shifted to 1780 cm -1 due to the formation of stronger hydrogen bonds to solvent molecules. The shoulder at 1762 cm -1 (which becomes a distinct band at 200 K), is assigned to the CF3COOH dimer. The low-frequency asymmetry of the acid bands in all solvents suggests the presence of higher order TFA associates. Thus THF can form hydrogen bonds with the CF3COOH, lowering the activity of the acid, which is one of the reasons for the evolution of Cp*Mo(dppe)H4 + CF3COO -to the final dihydride product in this solvent, as will be shown below. d1. In dichloromethane. Although the starting material and products are unstable in dichloromethane at room temperature, they do not decompose at low temperatures and this solvent has many advantages for infrared studies (it is not a strong proton acceptor, therefore does not compete with the basic hydride complexes for hydrogen bonding, and has good solvent properties for ionic compounds). An IR monitoring at 200 K in the acid CO and conjugated anion  as OCO vibration region yielded the results shown in Figure 9. At this temperature the dihydride complex Cp*Mo(dppe)H2(O2CCF3), which has a  as CO at 1692 cm -1 (vide infra), does not form. A tentative assignment of all observed bands is possible on the basis of previous IR studies into the interaction between CF3COOH and various bases, [30][31][32] in combination with the results shown in the previous section. At 200K the acid self-association equilibrium is shifted to the dimer, exhibiting a strong band at 1780 cm -1 with a shoulder at 1800 cm -1 , see Figure 9a. The spectrum recorded for the solution with a MoH3/TFA ratio of 1:0.5, Figure 9c, shows a prominent band at 1686 cm -1 , due to the  as OCO vibration of the trifluoroacetate anion. Its relatively sharp nature and almost negligible high frequency shift relative to the position of the free CF3COO -anion [31,32] indicates the formation of either a weakly H-bonded contact ion pair or a solvent separated ion pair, consistent with the relatively high polarity of CH2Cl2 at low temperatures and the poor proton donor ability of the [Cp*Mo(dppe)H4] + cation. In reference to Scheme 1, this means that species V is extensively dissociated to VI or that the interaction is loose under these conditions of solvent and temperature. There is no significant intensity in the region characteristic for the free homoconjugate anion, [CF3COO(CF3COOH)n] -. According to the literature, this species should display a low intensity broad band with a maximum at 1630-1620 cm -1 . [33] Given the presence of an excess of the trihydride complex, most of the acid is presumably consumed and little is left to yield the homoconjugate anion. The relative intensity of the 1686 cm -1 band indicates that the proton transfer equilibrium is highly shifted to the tetrahydride product, and the presence of the unreacted trihydride complex is indicated by the shoulder at 1816 cm -1 (cf. the starting spectrum of free Cp*Mo(dppe)H3 in Figure 9b). There are two additional weak bands in this spectrum. The first one at 1782 cm -1 could be assigned to free CH3COOH, but is also consistent with an H-bonded adduct where the acid acts as a proton donor (a CF3COO-H•••B interaction does not significantly perturb the CO normal mode). The second band at 1736 cm -1 indicates the presence of an H-bonded adduct where the acid acts as a proton acceptor (the CF3C(OH)O•••HA interaction significantly red-shifts the CO normal mode). [34,35] Thus, these bands constitute evidence for the presence of a 2:1 H-bonded adduct, Cp*Mo(dppe)H3

Scheme 4

An increase in the CF3COOH/MoH3 ratio is expected to shift the hydrogen bonding equilibrium position towards the 2:1 adduct. At the same time, equilibria involving the anionic species (either hydrogen bonded or free) are expected to shift towards the homoconjugate anion (equilibria drawn vertically in Scheme 4).

On the other hand, an increase in the CF3COOH/MoH3 ratio will not affect the equilibria shown horizontally in Scheme 4. Indeed, upon addition of a second 0.5 equiv of TFA (final MoH3/TFA ratio of 1:1), Figure 9d, the IR spectrum reveals a significant increase in intensity for the bands assigned to the neutral 2:1 adduct and a dramatic decrease for that assigned to the free anion. The growth of a broad shoulder at lower frequencies signals the formation of anionic aggregates, as expected.

d2. In benzene/toluene-d8. The low temperature IR spectral picture in toluene-d8 in the presence of a stoichiometric amount of TFA is similar to that shown above in dichloromethane, see Figure 10. The band at 1742 cm -1 observed for the 1:1 mixture (Figure 10c) is assigned to the CO of the hydrogen bonded complex (cf. 1736 cm -1 in CH2Cl2), whereas the bands at 1690 and 1674 cm -1 are assigned to the CF3COO -anion ( as OCO, cf. 1686 cm -1 in CH2Cl2). The complex pattern attained for this absorption is possibly related to the presence of different hydrogen bonded species (H-bonding should be favored more in low polarity toluene). The anion band did not show a significant increase in intensity at greater MoH3/TFA ratios (1:0.5 or 1:0.2), indicating that the proton transfer equilibrium is shifted further toward the neutral hydrogen bonded species in this solvent in comparison to CH2Cl2. This agrees with the results of the NMR investigation. Upon warming above 270 K, the bands corresponding to the Cp*Mo(dppe)H2(O2CCF3) product at 1702 and 1690 cm -1 quickly grow, while the shoulder at 1674 cm -1 disappears. The final spectrum is shown in Figure 10d. Monitoring the growth of this band for the 1:1 reaction in C6H6 at 298 K (Figure S3 in the Supporting Information) yielded a first order rate constant of (1.8 ± 0.1)•10 -3 s -1 . In the presence of a 3-5fold excess of acid, the broad, low intensity  as OCO band of the homoconjugated anion was observed at ca 1640 cm -1 . No formation of Cp*MoH2(CF3COO)(dppe) was observed under these conditions, in agreement with the NMR data. d3. In THF. At low temperature in this solvent and in the presence of 1 equiv of TFA, the dominant species are the neutral hydrogen bonded complexes, as indicated by the major bands at 1784 and 1744 cm -1 , see Figure 11c. No significant amounts of the CF3COO -ion can be seen. This implies that proton transfer occurs to a smaller degree than in toluene. No homoconjugated anion band was observed upon raising the TFA excess to 5 equiv, in contrast to the results in aromatic hydrocarbon solution. Upon warming, the spectral changes were similar to those observed in toluene (see previous section), yielding Cp*Mo(dppe)H2(O2CCF3), Figure 11d. We have also investigated the reverse proton transfer reaction by mixing together [Cp*Mo(dppe)H4] + BF4 -(generated in situ at 200 K from Cp*Mo(dppe)H3 and HBF4) and CF3COONa at 200 K in THF. This experiment confirms the reversible nature of the proton transfer process and the position of equilibrium. The essentially complete disappearance of the trifluoroacetate anion  as OCO bands at 1702 and 1690 cm -1 and the growth of the CO band at 1752 cm -1 corresponding to the H-bonded adduct [36] were observed at 200K in the presence of Since the NMR study shows that this conversion also occurs in the presence of excess acid, a kinetics investigation was carried out at 298K using different TFA/Mo ratios. The reaction was found to be zero order with respect to the acid (see Table 3). Interestingly, the rate constant in THF is identical to that measured in benzene (vide supra) within experimental error. The study was also extended to different temperatures (using a TFA/Mo ratio of 1:1), the subsequent Eyring analysis (see Supporting Information) giving the following activation parameters: H ‡ = 31.8 ± 0.5 kcal mol -1 and S ‡ = 36 ± 2 e.u. The large positive activation entropy suggests that the transition state is dissociative in nature. At low temperatures, the rearrangement becomes too slow; for instance, reaction rate constants of 5.2•10 -8 s -1 and 5.8•10 -15 s -1 are calculated at 250K and 200K, respectively.

Discussion

The collective observations that will be detailed below are summarized in Scheme 5.

Scheme 5

Proton transfer to Cp*Mo(dppe)H3, via the H-bonded adduct A to yield the classical tetrahydride complex, [Cp*Mo(dppe)H4] + , C/D, is the only reaction that may occur at low temperatures. The formation of the 2:1 H-bonded adduct A' has also been evidenced by low temperature IR, but one molecule of TFA is sufficient for proton transfer to occur, as also indicated by previous kinetics studies. [13] The proton transfer probably proceeds via a nonclassical intermediate (species B in Scheme 5), although we have not found any direct experimental evidence to support this proposition. Our recently reported theoretical calculations [13] indicate that the nonclassical complex lies in a very shallow minimum with a low barrier for conversion to the classical tetrahydride product, the isomerization taking place without counteranion dissociation. The competitive dihydrogen evolution yielding Cp*Mo(dppe)H2(O2CCF3) occurs only above 250 K and is possible (in non coordinating solvents) via the reverse reaction between D and CF3COO -. This forms the ion pair C which subsequently isomerizes to the non-classical species B.

The formation of [Cp*Mo(dppe)H4] + is reversible below 250 K in contrast to the irreversible formation of trans-Cp*MH2(dppe) + (M = Fe, Ru). [6,8] One important difference between these systems is the reversibility of the nonclassical/classical isomerization for [Cp*Mo(dppe)H4] + (according to the DFT calculations), whereas this process is irreversible for [Cp*M(dppe)H2] + (M = Fe, Ru). However, proton transfer to the hydride ligand is less thermodynamically favorable for Cp*Mo(dppe)H3 than for Cp*Fe(dppe)H: a significant amount of TFA remains unreacted (in the H-bonded form) for a TFA/Mo ratio of 0.5 in CH2Cl2 at 200K (see Figure 9), whereas it is completely consumed under the same solvent, temperature and stoichiometry conditions in the presence of Cp*Fe(dppe)H. [5] These results contrast with the higher basicity factor of Cp*Mo(dppe)H3 (Ej = 1.42±0.02) [13] relative to Cp*Fe(dppe)H (Ej = 1.36±0.02). [7] The corresponding proton transfer to Cp*Ru(dppe)H (Ej = 1.39), however, is also an equilibrium process. [6] The basicity factor correlates with the strength of the hydrogen bond electronically saturated nature of system B. The rate of formation in MeCN has not been determined with accuracy, but the NMR data in Figure 7 allow us to estimate a rate constant of ca. 2•10 -3 s -1 , i.e. close to that determined in THF and benzene (see Table 3) , if we assume a first order decay process is in operation. The high polarity of MeCN favors proton transfer and ion pair dissociation through the stabilization of the charge-separated species, but the instability of [Cp*Mo(dppe)H4] + leads to its ultimate decomposition by H2 elimination. In this case, the process may be solvent-assisted, leading to the solvent adduct G, as previously established by the protonation with HBF4. [14] This phenomenon also occurs in THF as verified by using HBF4. However, the CF3COO -anion has a stronger coordinating power than both THF and MeCN and the ultimate product is once again E.

Species A and C/D exhibit a dynamic exchange process, as revealed by a broad 1 H NMR resonance at room temperature which decoalesces at 270 K in C6D5CD3 for the mixture containing 1 equiv of TFA. This results from the fast and direct unimolecular proton transfer involving the dihydrogen bonded complex A and the H-bonded ion pair C. It is not possible, unfortunately, to use this information to derive an accurate value for the proton transfer rate constant, because of the complexity of the coupled equilibria and the irreversible transformation to product E. Assuming, however, that only species A and C are present in an approximately equimolar ratio, an activation barrier G ‡ of 11.7 kcal mol -1 may be estimated for the proton transfer in C6D5CD3 at 270 K, from the coalescence temperature and the chemical shift difference. [START_REF] Sandström | Dynamic NMR Spectroscopy[END_REF] This activation barrier is lower than that of the proton transfer by HFIP in toluene (G ‡ 293K = 15.8 kcal mol -1 ), [13] in agreement with the higher proton donating ability of TFA. It is also lower than G ‡ 270K for the proton transfer from HFIP to Cp*Fe(dppe)H in CH2Cl2 (14.7 kcal mol -1 ) or from PFTB to CpRuH(CO(PCy3) in hexane (16.1 kcal mol -1 ), calculated for 270 K using the published activation parameters. [3,5] IR kinetic data (vide supra) give G ‡ 270K = 22.1 kcal mol -1 for the dihydrido-trifluoroacetate formation. The irreversibility of the H2 evolution process renders Cp*Mo(dppe)H2(O2CCF3) the sole end product.

A final point of interest is a comparison between the results shown here and those reported previously for the decomposition of the proton transfer product obtained with TFE, which led to a diamagnetic hydride-free decomposition product, Cp*Mo(dppe)(OCH2CF3)(TFE)x (x = 0 or 1).

The mechanism is likely to be the same for both systems, leading to a first H2 elimination assisted by the coordination of the alkoxide ligand, see Scheme

Conclusion

We have explored the protonation of Cp*Mo(dppe)H3 by trifluoroacetic acid in various solvents which display different proton accepting ability, polarity, and coordinating power toward transition metals: dichloromethane, benzene/toluene, THF and MeCN. The nature of the solvent and the amount of excess acid determine the nature of the product by delicately controlling the position of the proton transfer and ion pairing equilibria. In agreement with previous investigations, the classical tetrahydrido cation is produced following the initial formation of a dihydrogen bonded intermediate without the observation of a nonclassical tautomer (dihydrogen complex); it is stable at low temperatures, and can exist as solvent separated or contact ion pair. However, the use of suitable conditions of solvent, temperature and hydride/acid ratio has led to the selective formation of a new product, the dihydrido complex Cp*Mo(dppe)H2(O2CCF3). The trifluoroacetate anion is a sufficiently strong ligand to coordinate the cationic complex and saturate its coordination sphere after loss of H2, even in the presence of coordinating solvent molecule such as MeCN or THF. However, it remains coordinated in a monodentate fashion and, contrary to the CF3CH2O -anion, is not capable of inducing the elimination of the residual hydride ligands as H2 to form a hydride-free product.

Experimental section

General. All manipulations were performed under an argon atmosphere by standard Schlenk techniques. All solvents were dried over appropriate drying agent (Na/benzophenone for benzene, toluene or THF, Na for pentane, CaH2 for CH2Cl2) and freshly distilled under an argon atmosphere prior to use. C6D6, C6D5CD3, THF-d8, CD3CN, CD2Cl2 by Euriso-Top for NMR experiments and C6D5CD3 by Aldrich for IR experiments were degassed by three freeze-pumpthaw cycles, and then purified by vacuum transfer at room temperature. Cp*Mo(dppe)H3 was synthesized according to literature. [14] Spectroscopic studies. (a) NMR investigations. Samples of the hydride Cp*Mo(dppe)H3 in each solvent were prepared under an argon atmosphere in 5 mm NMR tubes. The 1 H and 31 P{ 1 H} data were collected with Bruker AMX300 and Bruker AV500 spectrometers, operating at 300.1 (500.3) MHz and 121.5 (202.5) MHz, respectively. The temperature was calibrated using a methanol chemical shift thermometer; the accuracy and stability was ±1 K. All samples were allowed to equilibrate at every temperature for at least 3 min. The spectra were calibrated with the residual solvent resonance ( 1 H) and with external 85% H3PO4 ( 31 P). The conventional inversion-recovery method (180-τ-90) was used to determine the variable-temperature MoK radiation (= 0.71073). The structure was solved by direct methods (SIR97) [START_REF] Altomare | [END_REF] and refined by least-squares procedures on F 2 using SHELXL-97. [START_REF] Sheldrick | SHELXL97. Program for Crystal Structure refinement[END_REF] All H atoms attached to carbon were introduced in calculation in idealised positions and treated as riding models. The two hydrides were located in difference Fourier syntheses and they were freely refined with isotropic thermal parameter. The drawing of the molecule was realised with the help of ORTEP32. [START_REF] Farrugia | [END_REF] Crystal data and refinement parameters are shown in Table 2. Crystallographic data (excluding structure factors) have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 632317. Copies of the data can be obtained free of charge on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: (+44)

1223-336-033; e-mail: deposit@ccdc.cam.ac.uk).

acknowledged. NB thanks Russian Science Support Foundation for an individual grant. MB thanks the Spanish Ministerio de Educación y Ciencia for a post-doctoral fellowship.

Supporting Information

  (i) The trihydride complex has a high basicity factor (Ej = 1.42±0.02), placing it in the category of the most hydridic complexes known. (ii) As mentioned in the introduction, the interaction with HBF4 in non coordinating or weakly coordinating solvents (ether, THF) leads directly to the tetrahydride complex [Cp*Mo(dppe)H4] + , without the detection of a nonclassical intermediate, although its presence is suggested by the theoretical calculations. (iii) [Cp*Mo(dppe)H4] + BF4 -is unstable and rapidly loses dihydrogen; in MeCN, this process leads to the formation of the solvent stabilized complex [Cp*Mo(dppe)H2(MeCN)] + . (iv) The interaction with weaker proton donors (the fluorinated alcohols CH2FCH2OH, CF3CH2OH, and (CF3)2CHOH) in low polarity solvents (e.g. THF, CH2Cl2) at low temperatures leads to the observation of intermediate hydrogen bonded adducts. Infrared spectral analysis, in combination with theoretical calculations, indicates that one of the hydride ligands of Cp*Mo(dppe)H3 takes part in hydrogen bonding. (v) Proton transfer from the dihydrogen-bonded adduct yields, without the observable intervention of a second HA molecule in the rate determining step, the tetrahydride product as a 1:1 ion pair stabilized by a hydrogen bond between the cation and the anion, [Cp*Mo(dppe)H4] + •••A -, as established for HA = p-nitrophenol and CF3CH2OH. The proton transfer reaction is equilibrated for these weaker proton donors. (vi) On a longer time scale, the tetrahydride product [Cp*Mo(dppe)H4] + obtained using HA = CF3CH2OH evolves at ambient temperature by release of two H2 molecules to form the hydridefree product Cp*Mo(dppe)(OCH2CF3), probably stabilized by coordination of a second alcohol molecule, Cp*Mo(dppe)(OCH2CF3)(CF3CH2OH

shape of the 1 H

 1 Figure 1(a). Cooling resulted in broadening and eventual decoalescence (225 K), yielding two

Figure 1 .

 1 Figure 1. Variable temperature spectra for compound Cp*Mo(dppe)H2(O2CCF3) in C6D5CD3. (a) 1 H NMR (500 MHz). (b) 31 P{ 1 H} NMR (202 MHz).

Figure 2 .

 2 Figure 2. ORTEP view of compound Cp*Mo(dppe)H2(OCOCF3). For the sake of clarity, the H atoms are not shown, except for the two hydride ligands. The thermal ellipsoids are drawn at the 30% probability level.

Figure 3 .

 3 Figure 3. 1 H NMR (300 MHz) monitoring of the reaction between Cp*Mo(dppe)H3 and TFA in C6D6 (290 K). (a) Starting material before the TFA addition; (b) immediately after the TFA addition (10 equiv) (c) immediately after the TFA addition (1 equiv); (d) after 3.5 h from spectrum (c).

Figure 4 .

 4 This is as expected, because the equilibrium mixture of the rapidly exchanging Cp*Mo(dppe)H3 and [Cp*Mo(dppe)H4] + complexes is now richer in the former. The formation of the final dihydride product was slower (equilibrium was attained in ca. 2 h at 290 K), and ca. 50% of the initial trihydride remained unreacted. As the [Cp*Mo(dppe)H4] + reacted to yield Cp*Mo(dppe)H2(OCOCF3), the resonance corresponding to Cp*Mo(dppe)H3 sharpened. The addition of excess TFA to a solution containing Cp*Mo(dppe)H2(OCOCF3) does not lead to any reaction, in particular no tetrahydride resonance was observed under these conditions.

Figure 4 .

 4 Figure 4. 1 H NMR (300 MHz) monitoring of the reaction between Cp*Mo(dppe)H3 and TFA (1:0.5 ratio) in C6D6 (290 K). (a) Recorded immediately; (b) after 7 min; (c) 28 min; (d) 155 min.

Figure 5 .

 5 Figure 5. 1 H NMR (300 MHz) monitoring of the reaction between Cp*Mo(dppe)H3 and TFA in THF-d8 at 290K: (a) before the TFA addition; (b) TFA/Mo = 0.5, 8 min; (c) further addition of 1 equiv to sample (b): TFA/Mo = 1.5, 30 min; (d) TFA/Mo = 10, immediately after the addition; (e) after 13 min from sample (d).The doublet resonance at  -3.73 belongs to the Cp*Mo( 1 -dppe)H5 contaminant.[14]

  exchange between CpMo(dppe)H3 and [Cp*Mo(dppe)H4] + , by repeating the protonation in the presence of HBF4 instead of CF3COOH (the BF4 -salt of the tetrahydride product is soluble in THF, but precipitates from toluene or benzene). If the exchange takes place bimolecularly via a symmetrical [Cp*(dppe)H3Mo•••H•••MoH3(dppe)Cp*] + transition state, the lineshape of the signal will not depend on the nature of the acid. On the other hand, a unimolecular pathway involving the conjugate base of the proton donor should yield a slower exchange rate for the BF4 - sample, because this is a much weaker base than CF3COO -. The NMR study of a 2:1 Cp*Mo(dppe)H3/HBF4 mixture provides evidence in favor of the unimolecular pathway. As can be seen in Figure 6, addition of 0.5 equiv of HBF4 results in a decrease in the Cp*Mo(dppe)H3 resonance and the appearance of the [Cp*Mo(dppe)H4] + resonance, plus a weak triplet resonance at  -5.65 (JHP = 53.5 Hz), whilst the doublet resonance of the Cp*Mo( 1 -dppe)H5 contaminant remains unchanged. The trihydride and tetrahydride complexes give separated sharp resonances, contrary to the same situation in the presence of trifluoroacetate anion in the same solvent (Figure 5b). This clearly demonstrates that the anion determines the rate of degenerate exchange. This exchange process therefore involves the hydrogen bonded ion pair [Cp*Mo(dppe)H4] + ••• -OCOCF3 and the dihydrogen bonded trihydride Cp*Mo(dppe)H3•••HOCOCF3, the latter being in further rapid equilibration with free trihydride and TFA. Analogous broadening phenomena for hydride signals as a consequence of dihydrogen bonding and equilibrated proton transfer were noted previously in other cases,

Figure 6 . 1 H

 61 Figure 6. 1 H NMR study of a Cp*Mo(dppe)H3/HBF4 mixture in THF-d8: (a) starting compound; (b) 21 min after the addition of 0.5 equiv of HBF4.The doublet resonance at  -3.73 belongs to the Cp*Mo( 1 -dppe)H5 contaminant.[14]

  is assigned to complex [Cp*Mo(dppe)H2(THF)] + . It is associated with a new 31 P{ 1 H} resonance at  70.7. For comparison, the related [Cp*Mo(dppe)H2(MeCN)] + complex, obtained from the protonation of

Figure 7 . 1 H

 71 Figure 7. 1 H NMR study of the Cp*Mo(dppe)H3-TFA reaction in MeCN-d3 at room temperature: (a) 0.5 equiv of TFA, recorded immediately; (b) 1 equiv of TFA, recorded immediately; (c) after 9 min from spectrum (b); (d) 40 min after spectrum (b).

Figure 8 .

 8 Figure 8. IR spectra of trifluoroacetic acid solutions (0.027 M, pathlength 0.04 mm) in various

Figure 9 .

 9 Figure 9. Low temperature (200 K) IR spectra for the reaction between Cp*Mo(dppe)H3 and

Figure 10 .

 10 Figure 10. IR spectra for the reaction between Cp*Mo(dppe)H3 and TFA in toluene-d8. (a)

either 1 or 2 Figure 11 .

 211 Figure 11. Low temperature (200K) IR spectra for the reaction of Cp*Mo(dppe)H3 with TFA in

3 C[

 3 Cp*Mo(dppe)H 4 ] + CF 3 COO - [CF 3 COO(CF 3 COOH) n ] -

  longitudinal relaxation time T1. Standard Bruker software was used for the calculation of the longitudinal relaxation time.(b) IR Investigations. The IR measurements were performed on the "Infralum 801" FT-IR spectrometer using CaF2 cells of 0.04 cm path length. All IR measurements were carried out by use of a home-modified cryostat (Carl Zeiss Jena) in the 190-290 K temperature range. The cryostat modification allows operating under an inert atmosphere and transferring the reagents (premixed either at low or room temperature) directly into the cell pre-cooled to the required temperature. The accuracy of the temperature adjustment was ±1 K. This setup was used both for the variable-temperature studies and for the kinetics investigations.Synthesis of Cp*Mo(dppe)H2(O2CCF3).A CF3COOH solution (9.5 L, 0.13 mmol) in THF (3 mL) was slowly added to a solution of Cp*Mo(dppe)H3 (80 mg, 0.13 mmol) in 15 mL THF. The reaction mixture was stirred at RT for ca. 1.5 h. The solvent was evaporated and the resulting solid was recrystallized from pentane. Yield: 75 mg (78%).1 H NMR (300.13 MHz, C6D6, 290K):  -5.09 (Mo-H2), 1.79 (s, 15 H, Cp*), 3.2-2.5 (m, 4 H, -CH2-CH2-), 7.10-7.77 (m, 20 H, Ph).31 P { 1 H} NMR (121.5 MHz, C6D6, 290K): 76.9 (s). The 1 H hydride and the31 P resonances decoalesce at low temperatures (see Results and discussion section). IR (KBr), cm -1 : 1682 and 1702 (sh) (νCO), 1818 and 1850 (Mo-H2). Anal. Calc. for C38H41F3P2O2Mo: C, 61.29; H, 5.56. Found: C, 61.26; H, 5.54. Crystals were grown by slow diffusion of a pentane layer into a saturated solution of the mixture Cp*Mo(dppe)H3 and ca. 1 equiv TFA in toluene.Kinetics of the Cp*Mo(dppe)H2(O2CCF3) formation.The reagents were mixed at low temperature (ca. 270 K) and the solution was transferred into the cryostat. The kinetics data were obtained by following the increase of the band at 1692 cm -1 corresponding to the Cp*Mo(dppe)H2(O2CF3) complex at the desired temperature. The first-order rates were obtained by plotting ln(A∞ -At) vs. time.X-ray analysis of compound Cp*Mo(dppe)H2(O2CCF3).Single crystal was mounted under inert perfluoropolyether on the tip of glass fibre and cooled in the cryostream of the Oxford-Diffraction XCALIBUR CCD diffractometer. Data were collected using the monochromatic

Scheme 2 (a) Protonation in benzene/toluene: formation of compound Cp*Mo(dppe)-

  ). The above observations are summarized in

	Scheme 2.
										[Cp*Mo(dppe)(OCH 2 CF 3 )(CF 3 CH 2 OH) x ]
										HA = CF 3 CH 2 OH
										(excess)	-2H 2							+
										slow		ether, THF
												or CH 2 Cl 2
	H	P	Mo P	H H	HA	H	P	Mo P	H H H	MeCN [Cp*Mo(dppe)( 2 -H 2 )H 2 ] + A -?	fast	H	H	P	P Mo	H H	A	-
									A	HA = HBF 4 (1 equiv)	-H 2		quantitative if HA strong (e.g. HBF 4 )
										fast
										[Cp*Mo(dppe)(MeCN)H 2 ] + BF 4	-

  •••HOOCCF3•••HOOCCF3. The simultaneous presence of a 1:1 adduct, Cp*Mo(dppe)H3•••HOOCCF3, cannot be established because it would also afford a band at ca. 1780 cm -1 . A general picture of the proton transfer process can be drawn as shown in Scheme 4.

	Cp*Mo(dppe)H 3 HO 2 CCF 3			Cp*Mo(dppe)H 4 O 2 CCF 3	+ Cp*Mo(dppe)H 4 + CF 3 COO -
	+CF 3 COOH	-CF 3 COOH			+CF 3 COOH	-CF 3 COOH	+CF 3 COOH	-CF 3 COOH
			CF 3 COOH		CF 3 COOH	Cp*Mo(dppe)H 4 +
										+ CF 3 COO(CF 3 COOH) -
	Cp*(dppe)H 2 Mo H	H	O	C	O	Cp*(dppe)H 3 Mo H	O	C	O
					CF 3			CF 3

Table 2 .

 2 Table of longitudinal relaxation times at different temperatures for the hydride resonance of Cp*Mo(dppe)H2(O2CCF3) and figures showing IR spectra of this compound in various solvents, the IR study of its formation kinetics in toluene, and an Eyring plot of the rate constants in THF (4 pages). Characteristics of the  as OCO band of Cp*Mo(dppe)H2(OCOCF3) in the solid state and in solution.

	solvent	 as OCO /cm -1		intensity ratio
				(Alow/Ahigh)
	solid (KBr)	1700 sh, 1685 s 15	1.8
	C6H6	1700 sh, 1690	10	1.2
	C6D5CD3	1700 sh, 1690	10	1.1
	THF	1702, 1690	12	1.0
	CH2Cl2	1706 sh, 1688	18	2.2

Table 3 .

 3 Rate constants for the Cp*Mo(dppe)H2(O2CCF3) formation reaction in THF. C(MoH3) = 0.025 M.

	T, K	TFA/Mo	k , 10 -3 s -1
	280	1	0.064 ± 0.0015
	290	1	0.63± 0.01
	298	0.5	1.7 ± 0.1
	298	1	1.8 ± 0.1
	298	3	1.9 ± 0.1
	298	5	1.80 ± 0.05
	308	1	13.7 ± 0.2

Acknowledgment

We thank the European Commission through the HYDROCHEM program (contract HPRN-CT-2002-00176) for support of this work. Additional bilateral support (PICS, France-Russia) and National support from the CNRS (France), from the RFBR (05-03-22001, 05-03-32415) and the Division of Chemistry and Material Sciences of RAS (Russia), is also gratefully

(formation enthalpy), whereas the protonation equilibrium is determined by the free energy of the proton transfer process. Obviously, the two parameters do not necessarily correlate quantitatively.

Both the A ↔ C/D equilibrium position and rate of the irreversible Cp*Mo(dppe)H2(O2CCF3) formation depend on the temperature, the solvent and the MoH3/TFA ratio. Polar solvents (e.g. dichloromethane at low temperatures, acetonitrile) weaken the H-bond in C and lead to the formation of a solvent separated ion pair (D in Scheme 5), which stabilizes the tetrahydrido complex against the H2 loss. The proton transfer equilibrium is shifted to species D in CH2Cl2 even in the presence of stoichiometric amounts of the acid. Low polarity solvents (e.g. toluene) provide less stabilization for the charged species, thus the proton transfer equilibrium is shifted to the left when near stoichiometric amounts of the acid are used. The tetrahydride product exists as an ion pair stabilized by hydrogen bonding to the CF3COO -anion (species C in Scheme 5), as reflected for instance in a reduced rate of hydride site exchange measured by 1 H NMR below 240 K. The formation of hydrogen bonded ion pairs of 1:1 composition was also confirmed for weaker proton donors. [13] At room temperature and above, the competitive irreversible H2 elimination process eventually leads to the quantitative formation of the final product E. However, excess acid favors ion pair dissociation through formation of the thermodynamically more stable homoconjugate anion, thereby stabilizing the proton transfer product D and precluding the H2 evolution.

In solvents with proton accepting ability (THF, MeCN) the activity of the acid is reduced by H-bonding with solvent molecules, formation of the homoconjugated anion is not observed and the reaction yields E even in the presence of excess acid. The attainment of the same formation rate constant in THF and in benzene and the zero order dependence on TFA agree with the proposed unimolecular generation of E from the same intermediate B.

Furthermore, the large positive activation entropy is consistent with a dissociative mechanism (i.e. H2 must leave before the trifluoroacetate anion can coordinate), as may be expected from the