Ahmad Joumaa

Si Chen

Sandrine Vincendeau

Florence Gayet

Rinaldo Poli
email: rinaldo.poli@lcc-toulouse.fr

Eric Manoury
email: eric.manoury@lcc-toulouse.fr.

Rhodium-catalyzed aqueous biphasic hydrogenation of alkenes with amphiphilic phosphine-containing core-shell polymers

Keywords: Aqueous biphasic catalysis, Alkene hydrogenation, Water-confined nanoreactors, Rhodium, Recycling

The biphasic aqueous hydrogenation reaction of styrene and 1-octene could be efficiently carried out using phosphine-containing core-shell water-soluble polymers used as nanoreactors in presence of [Rh(COD)Cl]2. The catalytic aqueous phase could be successfully recycled up to six times.

Introduction

Hydrogenation is clearly a major reaction in organic synthesis both at the laboratory and industrial scale. It has been reported that at least one catalytic hydrogenation step is included in around 25% of all chemical processes. 1 Hydrogenation is applied at the very large scale such as, for instance, for the desulfurization of fuels, 2,3 but is also largely used for the production of fine chemicals, 4 including chiral ones. 5,6,7,8 As a way to improve the efficiency of chemical processes 9 in economic and environmental terms, biphasic aqueous catalysis is an elegant approach because it simplifies the catalyst separation by simple decantation and its subsequent recycling. This operational mode has found large scale industrial implementation in hydroformylation, [START_REF] Cornils | Multiphase Homogeneous Catalysis[END_REF][START_REF]Aqueous-Phase Organometallic Catalysis[END_REF][START_REF] Cornils | [END_REF] but has also been used for hydrogenation reactions. 13 Within this approach, the catalyst is usually confined in the aqueous phase [START_REF] Piscopo | For a catalytic system where substrates and products are water-soluble when catalysts are confined in organic phase[END_REF] while substrates and products are in the water-immiscible organic phase. The (pre)catalysts may be water-soluble complexes 15,16,17,18,19,20,21,22,23 or metal nanoparticles dispersed in water. 24,25,26,27,28,29,30,31,32,33 We have recently reported a new approach in aqueous biphasic catalysis, which consists of using stable latexes of unimolecular polymeric nanoreactors, characterized by a well-controlled core-shell structure with a narrow size distribution around 100 nm and a spherical morphology [START_REF] Poli | ACS Symposium Series[END_REF][START_REF] Manoury | Catalysis within Nanoconfined Spaces[END_REF] They were synthesized by controlled RAFT polymerization in water, yielding stable dispersions with a 25-30% polymer content in weight in a one-pot procedure. The polymeric nano-objects remain confined in the aqueous phase by virtue of their hydrophilic shell but contain a phosphine-functionalized core where the (pre)catalyst is covalently bound.

The hydrophobic cores are swollen very rapidly by various organic compounds. [START_REF] Zhang | [END_REF] We have shown that the [Rh(acac)(CO)2] precatalyst can be coordinated inside the polymer core [START_REF] Zhang | [END_REF]37,38 and the resulting metal-loaded nanoreactors were successfully used in the aqueous biphasic hydroformylation of 1-octene with excellent activity, low metal leaching, facile catalyst recovery and efficient recycling. [START_REF] Zhang | [END_REF]39,40,41 We have also shown that other metal complexes, notably [RhCl(COD)]2, can be incorporated inside the polymer core, but the resulting objects were only studied in terms of coordination chemistry and interparticle metal migration dynamics and mechanism. 37,38 In the present contribution, we report the first application of these amphiphilic core-shell polymers to the biphasic aqueous hydrogenation of alkenes.

Experimental

General Considerations.

Chloro(1,5-cyclooctadiene)rhodium(I) dimer, (98%, Strem), 1-octene (99+%, Acros), 1-nonanol (> 99%, Alfa TCI), n-decane (99%, Aldrich), n-dodecane (99%, Acros), toluene (99%, Acros) and anisole (99.0%, Fluka) were used as received. Styrene (99%, Acros) was purified by passing it through a column of active basic aluminum oxide to remove the stabilizer.

Dihydrogen was obtained from Linde Gas. The triphenylphosphine (TPP) containing core cross-linked micelles, TPP@CCM1 [START_REF] Poli | ACS Symposium Series[END_REF] and TPP@CCM2 40 , were synthesized as described in our previous contributions and stored under an inert atmosphere.

Catalytic tests.

General procedure for the hydrogenation of styrene or 1-octene under biphasic conditions.

In a Schlenk tube under argon was added 1mL of latex (for TTP@CCM1: 0.12 mmol of TPP; (for TTP@CCM2: 0.063 mmol of TPP) and 2 mL of H2O. Toluene (0.2 mL) was then added to swell the particles and the reaction mixture was stirred for 1 min. Then, a solution of [Rh(COD)Cl]2 (for TPP@CCM1: 15.4 mg (0.03 mmol; P:Rh = 2); for TPP@CCM2: 3.77 mg (0.0078 mmol; P:Rh = 4) in toluene (0.3 mL) was added and the reaction mixture was stirred for 10 min at RT. The aqueous phase became yellow while the toluene phase became colorless.

Separately, a stock organic phase was prepared with the substrate (for styrene: 3.17 g, 30 mmol; for 1-octene: 3.37 g, 30 mmol) and decane (1.26 g, 9 mmol) as internal standard in 40 mL of the desired solvent to obtain a substrate concentration of 0.75 mmol/ml. For the reaction without solvent, a mixture containing 0.823 g of styrene (7.9 mmol) and 0.766 g of decane (5.38 mmol) was prepared.

In a vial containing a magnetic stirrer under argon was added 0.5 mL of the aqueous phase containing the core cross-linked micelles and the rhodium precursor (see above) and 1 mL of the organic stock solution (see above). The vial was placed in a steel reactor, which was then charged with 20 bar of dihydrogen. The reaction mixture was then stirred at RT for 18 h with magnetic stirring set at a speed of 1200 rpm. After stopping the stirring and an additional 4 h decantation under dihydrogen pressure, the reactor was vented and then flushed with argon.

The organic layer was then withdrawn by syringe and the aqueous phase was extracted by two portions of 0.5mL of diethylether, all these operations being conducted under argon. The combined organic solutions were then analyzed by GC.

For the recycling experiments, a new portion of the substrate stock solution was added to the vial under argon and the reaction was carried out as described above.

Kinetic studies.

The investigations under biphasic conditions were carried out as described in the previous section, using several identical vials. The reaction in each vial was stopped at a different reaction time before the GC analysis.

For the homogenous catalytic reactions, triphenylphosphine (39.3 mg, 0.15 mmol), styrene (1.56 g, 15 mmol) [Rh(COD)Cl]2 (18.5 mg, 0.0375 mmol), and dodecane as GC standard (0.66 mg, 3.75 mmol) were dissolved in deoxygenized anisole (17.6 g) and used as stock solution. Several different vials with 2 ml each of the stock solution were used as described above.

High resolution ICP-MS.

The rhodium catalyst leaching to the organic phase was measured using high resolution ICP-MS on a XR Thermo Scientific Element. For the sample preparation, the recovered organic phase was diluted into water using a 10 5 volumetric dilution factor. In practice, a 1 L volumetric flask was filled at ~2/3 with milli-Q water, then 10 µL of the organic product phase were introduced using a Gilson P20 precision pipette. Borders were rinsed and the flask was introduced into an ultrasound bath for 1 hour. The solution was left overnight and the dilution was then completed with milli-Q water to the 1 L mark. Standards were prepared using solutions of Rh(acac)(CO)TPP in nonanol at various weight fractions of Rh in the (0.001-0.5)•10 -9 range (1-500 ppt).

Results and discussion

Preliminary catalytic tests

Two different polymer samples, TPP@CCM1 and TPP@CCM2, prepared as described in a previous report, [START_REF] Zhang | [END_REF]37 have been used in this study. They contain unimolecular nanoreactors in which the spherical polymer particles are characterized by three distinct zones (see Fig. 1): a hydrophilic shell with an average of 30 monomers (50:50 mixture of methacrylic acid, MAA, and poly(ethylene oxide) methyl ether methacrylate, PEOMA) per chain, linear hydrophobic chains in the intermediate zone with an average of 300 monomer units per chain consisting of a mixture of styrene and diphenylphosphinostyrene (DPPS), and an inner cross-linked core with additional styrene and the cross-linking monomer, diethylene glycol dimethacrylate (DEGDMA) in a 90:10 ratio for a total of 100 monomers per chain. The two samples differ in the DPPS content in the intermediate hydrophobic layer: 10% for CCM1 and 5% for CCM2.

The TPP@CCM1 sample was first used in the hydrogenation of styrene with toluene as organic solvent, after charging the polymer particles with the [Rh(COD)Cl]2 precatalyst as described in the Experimental part. 38 This first test was carried out under the conditions depicted in Scheme 1 with a substrate/P/Rh ratio of 200/2/1. After 18 h at RT under H2 pressure, although the transformation of styrene to ethylbenzene was complete, the decantation process proved very problematic: only a small portion of the organic phase was observed at the top of the reaction vial, while the aqueous phase and most of the organic materials had formed a stable dense phase with the aspect of a gel. The failure of the mixture to properly decant obviously precludes an efficient product recovery and the recycling of the aqueous catalytic phase.

Figure 1. Structure of the reference core cross-linked micelles (TPP@CCM1 (n = 0.10) and TPP@CCM2 (n = 0.05)) from Zhang et al. [START_REF] Zhang | [END_REF] and Chen et al. 37 Scheme 1. Hydrogenation of styrene under biphasic conditions. Solvent = toluene or 1-nonanol.

Styrene/P/Rh=200/2/1.

Hydrogenation reactions of styrene in 1-nonanol

We imagined that the behavior observed in the above preliminary test may result from interparticle coupling with formation of a macrogel. This hypothesis comes from our previous = catalytic site finding of rapid and reversible (in the absence of chemical reaction) interpenetration of the particles, involving core-core contact. 37 This was proven by the irreversible particle aggregation in the presence of reactions leading to products in which the metal centers binds more than one TPP ligand. In the present case, after precatalyst activation involving COD removal as hydrogenated products, the unsaturated metal center with be stabilized by TPP, H2 and substrate coordination, with TPP being the preferred ligand. Since the P/Rh ratio is 2, species such as [RhCl(TPP)2]2, [RhH2Cl(TPP)2]2, or mononuclear olefin adducts should form. In any case, however, the presence of easily available coordination sites and the occurrence of interparticle core-core contact is likely to lead to particle coupling because of Rh binding to TPP ligands that belong to different particle cores resulting in macrogelation. On the basis of this hypothesis, we considered that a slightly coordinating solvent, even though less strongly binding than TPP, could help avoid the nanoparticle coupling by virtue of its large concentration (mass effect) but without completely blocking the catalytic sites. Indeed, using 1-nonanol as solvent, the hydrogenation took place at reasonable speed and a perfect phase separation was witnessed at the end of the reaction. The conversion of styrene was complete under the conditions indicated in Scheme 1, with ethylbenzene as the only product. The aqueous phase could be even recycled once without loss of activity. The reason for choosing a long linear chain alcohol is to maintain a biphasic system, since 1-nonanol has negligible water solubility. Monophasic reaction mixtures would be obtained by using smaller alcohols like methanol or ethanol.

The kinetics of the styrene hydrogenation under these conditions was studied and compared with the homogenous version in anisole with free triphenylphosphine as supporting ligand and carried out with identical temperature, dihydrogen pressure, and substrate/P/Rh ratio (see figure 2). Under the biphasic conditions, 18 h are needed to reach full conversion, which is a rather interesting performance even though the rate is clearly slower than for the corresponding homogenous version, where full conversion is achieved in 10 h. The monophasic version exhibits a classical kinetic profile, with an induction time related to the precatalyst activation phase, which requires cyclooctadiene hydrogenation. 42,43 This induction phase is partially visible also in the biphasic reaction. The subsequent conversion rate after the catalyst activation phase is probably limited by mass transport in and out of the nanoreactor cores.

Hydrogenation reactions of 1-octene in 1-nonanol

The same conditions that allowed us to achieve the biphasic hydrogenation of styrene were next extended to 1-octene (scheme 2). This substrate could be efficiently hydrogenated with the Rh catalyst supported in the TPP@CCM1 nanoreactors and the catalytic aqueous phase could be recycled up to 6 times (see table 1). After 6 recycles, the catalytic activity remained high (full conversion), but the octane selectivity decreased to 90%, suggesting that the catalytically active species may have experience some modification. The rhodium leaching was relatively low, around 0.5 ppm. Note that this leaching level is significantly lower than that observed with the same nanoparticles in the hydroformylation of 1-octene. [START_REF] Zhang | [END_REF]40 This is probably a consequence of the milder reaction temperature. Indeed, we have shown that the Rh leaching in the hydroformylation reaction, which is conducted at 90°C, is directly correlated with the Upper Critical Solution Temperature (UCST) behavior of the POEMA monomers in the hydrophilic shell, which induce a slight compatibilization of the polymer shell with the organic phase at high temperature. 40 Scheme 2. Hydrogenation of 1-octene under biphasic conditions. Styrene/P/Rh=200/2/1.

Hydrogenation reactions of styrene under other conditions

We have imagined that the macrogelation phenomenon observed in the preliminary catalytic test described in section 3.1 could also be limited or suppressed by increasing the P/Rh ratio and by using a less-densely functionalized by triphenylphosphine (TPP) core cross-linked micelles, namely TPP@CCM2 (see figure 1). This way, each Rh atom would be able to find nearby, within the same nanoreactor core, a sufficient number of ligands to stabilize itself.

Using TPP@CCM2 and a P/Rh ratio of 4, the catalytic reaction was carried out with three different organic solvents, as well as without solvent (see table 2). Under all these conditions (table 2, entries 1, 3, 4 and 7), the conversions were high and a clear phase separation was observed. It is worth pointing out that, for the hydrogenation without solvent, the conversion remains high in spite of the greatly increased substrate/Rh ratio (4000), with a quantitative conversion for the first two runs (entries 7 and 8). However, the ethylbenzene selectivity was relatively low under these conditions, as well as in 1-nonanol. A limited number of recycling experiments were also carried out under these conditions. Recycling was especially efficient in terms of catalytic activities and ethylbenzene selectivities for the catalysis with toluene as solvent up to the third run (table 2, entries 4, 5 and 6). Both in 1-nonanol and under solvent-free conditions, the reaction rates were even slightly higher in the second run than in the first run (table 2, entries 1/2 and 7/2), probably because of an induction in precatalyst activation 42,43 during the first runs.

Conclusions

We have shown that the triphenylphosphine-functionalized core-shell polymers TPP@CCM1 and TPP@CCM2 can be successfully used in the biphasic aqueous hydrogenation of simple alkenes (styrene and 1-octene). We have shown that we can avoid the polymer macrogelation during the reaction by either using 1-nonanol as solvent or by increasing the P/Rh ratio to 4. The catalytic aqueous phase can be recycled efficiently at least 6 times, but the activities and the selectivities are slightly eroded in some cases. We are now investigating ways to further stabilize the catalytic species and to optimize the nanoreactor structure in order to improve the catalytic efficiency and recyclability.

Figure 2 .

 2 Figure 2. Styrene conversion during the Rh-catalyzed hydrogenation under homogeneous (free triphenylphosphine in anisole) and biphasic (TPP@CCM1 and 1-nonanol as substrate solvent) conditions. P(H2) = 20 bar; styrene/P/Rh = 200/2/1; room temperature.

Table 1 .

 1 Results of recycle runs for the 1-octene hydrogenation under biphasic conditions with TPP@CCM1. a Reaction conditions as shown in Scheme 2. b As a mixture of cis and trans isomers.

	Recycle	1-octene	Octane	2-octenes b	[Rh]org/ppm
	number	conversion/%	yield/%	Yield/%
	-	100	99	0	0.55
	1	100	96	0	0.63
	2	100	98	0	0.45
	3	100	93	2	0.45
	4	100	92	6	0.46
	5	100	91	6	0.30
	6	100	90	9	0.59

a

Table 2 .

 2 Hydrogenation of styrene under biphasic conditions with TPP@CCM2 and a P/Rh ratio of 4. a

	Entry	solvent	Styrene conversion	Yield of
				ethylbenzene
	1	1-nonanol (first run)	95.2%	85.3%
	2	1-nonanol (second run)	100%	83.6%
	3	1-nonanol/toluene	99.6%	98.3%
		(1/1, v/v)
	4	toluene (first run)	100%	99.8%
	5	toluene (second run)	100%	98.9%
	6	toluene (third run)	100%	99.5%
	7	no solvent (first run)	99.4%	92.4%
	8	no solvent (second run)	100%	84.2%
	9	no solvent (third run)	79.4%	79.0%

a Conditions as in Scheme 1, except styrene/P/Rh = 400/4/1 for entries 1-6 and 4000/4/1 for entries 7-9.

Acknowledgment

We are grateful to the "Agence Nationale de la Recherche" (ANR) for support of this work through grant "BIPHASNANOCAT" (ANR-11-BS07--025--01). AJ thanks the City Council of Houmine El Fawka (Lebanon) for a Ph.D. fellowship. Additional support from the "Centre National de la Recherche Scientifique" (CNRS) and from the "Institut Universitaire de France" (IUF) is also gratefully acknowledged.


Dr Sanjib Banerjee

Dr Vincent Ladmiral

Antoine Debuigne

Dr Christophe Detrembleur

S M Wahidur Rahaman

Rinaldo Poli

Dr Bruno Ameduri
email: bruno.ameduri@enscm.fr

S M Wahidur Rahaman

Dr A Debuigne

Dr S M Wahidur Rahalan

Prof R Poli

Organometallic-Mediated Alternating Radical Copolymerization of tert -Butyl-2-Trifluoromethacrylate with Vinyl Acetate and Synthesis of Block Copolymers Thereof

Keywords: alternating copolymerization, block copolymer, organometallic mediated radical polymerization, fluoromonomer, one pot synthesis

published or not. The documents may come

Prof. R. Poli Institut Universitaire de France, 1, rue Descartes, 75231 Paris Cedex 05, France ---------Organometallic-mediated radical polymerization (OMRP) has given access to well-defined poly(vinyl acetate-alt-tert-butyl-2-trifluoromethacrylate)-b-poly(vinyl acetate) and poly(VAcalt-MAF-TBE) copolymers composed of two electronically distinct monomers: vinyl acetate (VAc, donor, D) and tert-butyl-2-trifluoromethacrylate (MAF-TBE, acceptor, A), with low dispersity (≤1. 24) and molar masses up to 57,000 g/mol. These copolymers have a precise 1:1 alternating structure over a wide range of comonomer feed compositions. The reactivity ratios are determined as rVAc = 0.01 ± 0.01 and rMAF-TBE = 0 at 40 °C. Remarkably, from a feed containing >50% molar VAc content, poly(VAc-alt-MAF-TBE)-b-PVAc block copolymers are produced via a one pot synthesis. Such diblock copolymers exhibit two glass transition temperatures attributed to the alternating and homopolymer sequences. The OMRP of this a Supporting Information is available online from the Wiley Online Library or from the author. fluorine-containing alternating monomer system may provide access to a wide range of new polymer materials.

FIGURE FOR ToC_ABSTRACT

Introduction

Fluorinated homopolymers are unique materials with outstanding properties for high value added applications. The incorporation of sequences of these polymers into a di-or multiblock [1] structure is expected to lead to materials of even greater value. To this end, the use of reversible deactivation radical polymerization (RDRP) synthetic techniques should be ideal in most cases. Indeed, Daikin was the first company to develop such materials over 35 years ago, [2] leading to the industrial scale production of segmented thermoplastic elastomers. [3] Since these pioneering works, interest in fluorinated block copolymers [4] has surged in the last decade. This led to the synthesis of a variety of fluorinated block copolymers prepared using telomerization, [5][6][7][8] conventional radical polymerization from functionalized initiators, [9][10][11][12] click chemistry [13] and using different RDRP techniques [4,14] : iodine transfer polymerization (ITP), [15][16][17] photoinitiated ITP, [18] reversible addition fragmentation chain transfer (RAFT) polymerization, [9,[19][20][21][22] and atom transfer radical polymerization (ATRP.) [5,7,8] However, for all their merits, telomerization only affords oligomers, and functional initiators lead to broad dispersity.

Alternating copolymers have been reported to constitute a valuable tool to build copolymers with some degree of sequence control. [23] To date, only a few comonomer pairs have been reported to undergo alternating radical copolymerization such as styrene/maleic anhydride, [24] styrene/maleimide derivatives, [25] styrene/methacrylates (in the presence of Lewis acids), [26] N-substituted maleimides/1-methylenebenzo-cycloalkanes, [27] chlorotrifluoroethylene/vinyl ether [28] and vinyl ethers/methacrylates. [29] For other monomer pairs, highly ordered monomer sequence control is still challenging. [30] Regarding fluorinated polymers, recently, Studer and coworkers [31] and the Scherf's team [32] attempted the synthesis of predominantly alternating copolymers composed of hexafluoroisopropyl acrylate with 7-octenyl vinyl ether and Ndialkylated benzothiadiazolodithienopyrrole with a dibrominated terephthalophenone derivative, respectively. Recently, Jordan's group reported (phosphinoarenesulfonate)Pd fluoride complex catalyst reactivation following -F elimination in ethylene/vinyl fluoride copolymerization. [33] 2-(Trifluoromethyl)acrylic acid (MAF) and alkyl 2-trifluoromethacrylate (MAF-esters) are very interesting monomers. Although homopolymers can be produced anionically, [34] these monomers do not homopolymerize under radical conditions, [35][36][37] but can be copolymerized together with electron-donating monomers such as vinyl ethers. [35] MAF-and MAF-esterscontaining fluorinated copolymers led to suitable materials for molecularly imprinted polymers, microlithography, polymer electrolyte membranes for fuel cells and for Li-ion batteries. [35] Recently, a nitroxide-mediated alternating copolymerization of VAc and MAF-TBE has been achieved. [38] However, the polymerization control was not entirely satisfactory and attempts to synthesize block copolymers failed even with styrene, probably because the C-ON bond at the chain-ends cannot be cleaved at a reasonably low temperature. [39] .

This contribution aims at developing a well-controlled radical copolymerization of VAc and MAF-TBE by organometallic mediated radical polymerization (OMRP) using cobalt(II) acetylacetonate, [Co(acac)2] as a controlling agent. [40,41] It is focused on this particular RDRP technique due to its ability to efficiently control the polymerization of several non-conjugated monomers, [42,43] in particular VAc. [42,43] Notably, RDRP of VAc was only achieved using a few techniques: reversible addition fragmentation chain transfer (RAFT) polymerization, [44] iodine transfer polymerization (ITP), [45] and organometallic-mediated radical polymerization (OMRP). [42] Following a straightforward "one-pot" approach, a series of well-defined fluorinated copolymers was prepared including 1:1 alternating VAc-MAF-TBE copolymers as well as block copolymers where this poly(VAc-alt-MAF-TBE) alternating sequence is followed by a PVAc homopolymer block (Scheme 1). Although the "one-pot" strategy to prepare copolymer-b-polymer architectures was reported previously, [46][47][48] its implementation in combination with OMRP to synthesize a novel block copolymer (containing a fluorinated alternating block) is unprecedented.

Results and Discussion

The homopolymerization of VAc initiated by 2,2-azobis (4-methoxy-2,4dimethylvaleronitrile) (V-70) in the presence of [Co(acac)2] at 40 °C produced PVAc with controlled molar mass (Mn = 28,200 g/mol) and low dispersity (Đ = 1.16), as previously described [49] (entry 1 in Table S1 and Figure S1). In contrast, attempts to obtain a MAF-TBE homopolymer under the same conditions failed (entry 2, Table S1), as expected, since MAF-TBE does not homopolymerize under radical conditions. [35][36][37] The OMRP of VAc with MAF-TBE was initiated by V-70/Co(acac)2 in bulk at 40 °C (Scheme 1) using various MAF-TBE feed contents (fMAF-TBE) ranging from 0.1 to 0.9) (entries 3-8, Table S1). The reaction progress (conversions of VAc and MAF-TBE) was monitored by 1 H (equation S1) and 19 F (equation S2) NMR spectroscopies. Figures S2-S3 show representative stack plots of two such 1 H NMR kinetic experiments using different VAc/MAF-TBE comonomer feed ratios, fMAF-TBE = 0.5 and 0.1, respectively. Figure S4 exhibits a corresponding time evolution of the 19 F NMR spectra for fMAF-TBE = 0.5. In all cases, equimolar conversions of VAc and MAF-TBE were recorded, until the depletion of MAF-TBE (Figure 1 andFigures S5-S10), suggesting highly alternating copolymerizations (entries 3-8, Table S1). The 1 H NMR spectra (Figure S2) corresponding to the copolymerization with fMAF-TBE = 0.5, display a complex signal around 5.20 ppm, assigned to the -CH group of VAc in the VAc-MAF-TBE alternating dyads. This complex shape probably results from the adjacent stereocenters in VAc and MAF-TBE units. In contrast, copolymerizations using higher VAc feed contents (fMAF-TBE = 0.1, 0.2 and 0.3) and continued beyond the complete MAF-TBE consumption, led to poly(VAc-alt-MAF-TBE)-b-PVAc block copolymers, as evidenced by the appearance of an additional NMR signal centered at 4.80 ppm characteristic of the -CH group of VAc in the PVAc block [50] (Figure S3).

The semilogarithmic kinetic plots of these copolymerizations (carried out at fMAF-TBE = 0.1, 0.2 and 0.3, Figures S11-S13) displayed three distinct regimes: a short (30-45 min) induction period (such induction period was already observed for the CMRP of VAc), a relatively fast copolymerization reaction (until complete MAF-TBE consumption), and a slower homopolymerization of VAc, as confirmed by entry 1, Table S1 (fVAc = 1.0). Both segments of the plots are linear indicating that both the copolymerization and the homopolymerization followed first order kinetics. As expected, for copolymerizations using 1:1 VAc:MAF-TBE and an excess of MAF-TBE (fMAF-TBE = 0.7 and 0.9), the semilogarithmic plots (Figures S14-S16) exhibited only one regime corresponding to the alternating copolymerization. When there is no more VAc in the comonomer feed, the polymerization stops. The apparent propagation rate constant of the copolymerization (kp(app)) was determined from the slope of the first order kinetic plots (ln([M]0/[M]) vs. t, Figure S17a). [51] The kp(app) vs. fMAF-TBE plot (Figure S17b) reveals a bell-shaped curve with a maximum at fMAF-TBE = 0.5 (kp(app) = 3.9 × 10 -4 s -1 ) that is in further agreement with an alternating copolymerization. The SEC traces (Figure 2a and Figures S18a-S22a) remained monomodal throughout the polymerization and the Mn vs. conversion plots (Figure 2b andFigures S18b-S22b) show a regular increase of Mn with monomer conversion, maintaining narrow Đ. These features are consistent with RDRP.

To gain further insight on the controlling mechanism, we have compared the bond dissociation free energy (BDFE) for the putative (acac)2Co-VAc-coP and (acac)2Co-MAF-TBE-coP dormant species (coP = copolymer) by DFT calculations, using model systems where the coP chain beyond the Co-bonded monomer was replaced by an H atom (details are in SI, Pages S7-S8 and Table S2). These new calculations integrated thermal and dispersion corrections, relative to those previously published for (acac)2Co-VAc-H. [52] The resulting BDFE for the Co-VAc bond is 31.4 kJ/mol, whereas that of the Co-MAF-TBE is only 8.4 kJ/mol, suggesting a much higher lability of the latter.

In order to study the nature of the chain ends, the controlled copolymerization of VAc/MAF-TBE (80/20) was initiated from a PVAc<4-Co(acac)2 initiator and monitored by spin trapping electron paramagnetic resonance (EPR) experiments (Figures 3 andS23-S24).

Along the polymerization, the dormant species were isolated from aliquots of the reaction mixture and reacted with the tert-butyl nitroso Confirmation of the alternating structure was obtained by 1 H, 19 F, 13 C and 2D NMR characterizations. The detailed NMR assignments are available in the supporting information (Figures S25-S32). MALDI-TOF spectrometry of both poly(VAc-alt-MAF-TBE) alternating copolymer and poly(VAc-alt-MAF-TBE)-b-PVAc block copolymer was also attempted to confirm and determine the monomer sequences but these analyses gave inconclusive results.

Actually, NMR spectroscopy evidences and the determination of the reactivity ratios (see below) constitute sufficiently solid proofs to claim alternating microstructures of these polymers.

The OMRP kinetics enabled us to determine the reactivity ratios of these monomers (details are in SI, Page S7). The copolymer-monomer composition curve (Figure 4) reveals that the copolymer composition is always 1:1, independent of the feed composition. These data points, fitted with the Mayo-Lewis copolymerization equation [53] (S3) provide the following reactivity ratios: rVAc = 0.01 ± 0.01 and rMAF-TBE = 0 at 40 °C. These values are close to zero, which illustrates exclusive cross-propagation leading to alternating copolymerization, independent of the comonomer feed.

The thermal properties of these copolymers were studied by thermogravimetric analysis and differential scanning calorimetry (DSC). The 21% weight loss of the poly(VAc-alt-MAF-TBE) copolymer just above 150 °C (vs. a reduced weight loss for the block copolymer and no loss for PVAc, Figure S33) matches the value calculated for the loss of the carbo-tert-butoxy groups of the MAF-TBE monomers. It is likely due to the tert-butyl ester group decomposition into a carboxylic acid function with release of isobutene, followed by the decarboxylation of the carboxylic acid group (Scheme S1). [54] The polymers exhibited glass transition temperatures (Tg's) depending on both the monomer compositions and sequences (Figure 5). PVAc (Tg = 40 °C) and poly(VAc-alt-MAF-TBE) alternating copolymer (Tg = 72 °C) displayed a single Tg. The poly(VAc-alt-MAF-TBE)-b-PVAc copolymer exhibited two Tg's at 43 °C and 67 °C, which correspond to those of the PVAc homopolymer and of the poly(VAc-alt-MAF-TBE) alternating copolymer, respectively.

Conclusions

A simple approach for the preparation of well-defined 1:1 alternating VAc (electrondonating): MAF-TBE (electron-accepting) copolymers and a novel diblock copolymer (composed of one alternating block and one PVAc homopolymer sequence) by OMRP has been described. This is the first report on the synthesis of a fluorinated copolymer using OMRP. The relative length of the two blocks can be varied by simply tuning the comonomer feed ratio. The very low reactivity ratios account for the nearly perfect alternating copolymer structure over the whole tested range of comonomer feeds. Due to the remarkable properties of the fluorinated groups (low refractive index/dielectric constant, and interesting surface properties), the resulting copolymers are of interests for potential applications in high value added materials (e.g., coatings for optics, lithography, to prepare graft copolymer surfactants and fluorinated hydrophilic copolymer for membranes for water purification. This one-pot synthetic strategy will further contribute to the development of high-performance sequencespecific copolymers, a step toward advanced materials. S1). S1). S1).

Scheme &

-15 -Scheme 1. Synthesis of either poly(VAc-alt-MAF-TBE) alternating copolymers or poly(VAcalt-MAF-TBE)-b-PVAc block copolymers by "one-pot" OMRP. S1). S1).

  ( t BuNO) spin trap. ESR-a and ESR-c spectra corresponding to PVAc<4-( t Bu)NO• and to the final poly(VAc-alt-MAF-TBE)-b-PVAc-NO • , respectively, appear interpretable as the overlap of two different triplets of doublets (dt), with similar hyperfine splittings for the doublets but slightly different ones for the triplets. Each of these dt envelopes results from the coupling of the radical with N (1:1:1 triplet) and H of CHOAc (doublet) and probably derives from a different stereochemistry (m and r) of the last dyad (components A and B). Simulation of ESR-a (Figure S23) using this model and isotropic parameters gave a satisfactory fit, with component A (64.5%) having aN = 36.1 MHz and aH = 4.38 MHz, and component B (45.5%) having aN = 41.2 MHz and aH = 3.81 MHz. In contrast, the intermediate ESR-b spectrum corresponding to the trapping of the intermediate poly(VAcalt-MAFTBE)-Co(acac)2 species that is present during the alternating copolymerization regime appears as a mixture of two signals: a major 1:1:1 triplet assigned to PMAF-TBE( t Bu)NO•, in which only the coupling of the unpaired electron with the N nucleus is visible, and a smaller feature corresponding to PVAc-NO • . Although no quantitative information can be deduced, this observation suggests that both types of chain ends (P-MAF-TBE • and P-VAc • ) might be reversibly trapped by Co(acac)2 in the alternating copolymerization regime.

Scheme 1 .

 1 Scheme 1. Synthesis of either poly(VAc-alt-MAF-TBE) alternating copolymers or poly(VAcalt-MAF-TBE)-b-PVAc block copolymers by "one-pot" OMRP.

Figure 1 :

 1 Figure 1: No. of mmol monomer conversion vs. time plot of VAc and MAF-TBE during the OMRP of VAc and MAF-TBE (fMAF-TBE = 0.1) mediated by V-70/Co(acac)2 at 40 °C in bulk (entry 3, TableS1).

Figure 2 :

 2 Figure 2: a) Evolutions of the SEC traces vs. time and b) plot of Mn and Ɖ vs. the total monomer conversion for the OMRP of VAc and MAF-TBE (fMAF-TBE = 0.5) mediated by V-70/Co(acac)2 at 40 °C in bulk (entry 6, TableS1).

Figure 3 :

 3 Figure 3: EPR spectra of the spin trapped PVAc-Co(acac)2, poly(VAc-alt-MAFTBE)-Co(acac)2 and poly(VAc-alt-MAFTBE)-b-PVAc-Co(acac)2 (see Experimental Section in the SI for the detailed experimental conditions).

Figure 4 :

 4 Figure 4: Evolution of copolymer-monomer composition curve for the OMRP of VAc with MAF-TBE mediated by V-70/Co(acac)2 in bulk at 40 °C.

Figure 5 :

 5 Figure 5: DSC thermograms of pure PVAc, poly(VAc-alt-MAF-TBE) and poly(VAc-alt-MAF-TBE)-b-PVAc copolymers (entries 1, 6 and 3, respectively, TableS1).

Figure 1 .

 1 Figure 1. No. of mmol monomer conversion vs. time plot of VAc and MAF-TBE during the OMRP of VAc and MAF-TBE (fMAF-TBE = 0.1) mediated by V-70/Co(acac)2 at 40 °C in bulk (entry 3, TableS1).

Figure 2 .

 2 Figure 2. a) Evolutions of the SEC traces vs. time and b) plot of Mn and Ɖ vs. the total monomer conversion for the OMRP of VAc and MAF-TBE (fMAF-TBE = 0.5) mediated by V-70/Co(acac)2 at 40 °C in bulk (entry 6, TableS1).

Acknowledgements: The authors thank Tosoh F-Tech Company (Shunan, Japan) and Akzo Nobel (Chalons sur Marne, France) for providing MAF-TBE and BTBCP, respectively. Financial support from the French National Agency (ANR grant FLUPOL) is greatly appreciated. C.D. and A.D. are Research Director and Research Associate by F.R.S.-FNRS, respectively, and thank FNRS for financial support. We also thank Lionel Rechignat for the simulation of the EPR spectrum. This work was granted access to the HPC resources of IDRIS under the allocation 2016-086343 made by GENCI (Grand Equipement National de Calcul Intensif) and to the resources of the CICT (Centre Interuniversitaire de Calcul de Toulouse, project CALMIP). Received: Month XX, XXXX; Revised: Month XX, XXXX; Published online:

Supporting Information

Supporting Information is available from the Wiley Online Library or from the authors.

The table of contents entry

Organometallic-mediated radical copolymerization has been successfully employed for 1:1 alternating controlled copolymerization (independent of the monomer feed) of VAc (electron-donating) and MAF-TBE (electron-accepting). By simply tuning the comonomer feed ratio, well-defined poly(VAc-alt-MAF-TBE) alternating copolymers or novel poly(VAcalt-MAF-TBE)-b-PVAc diblock copolymers (comprising of one alternating block and one homopolymer block) can be prepared. Sanjib Banerjee, Vincent Ladmiral, Antoine Debuigne, Christophe Detrembleur, S. M. Wahidur Rahaman, Rinaldo Poli, and Bruno Ameduri*

Organometallic-Mediated Alternating Radical Copolymerization of tert-Butyl-2trifluoromethacrylate with Vinyl Acetate and Synthesis of Block Copolymers Thereof

ToC figure


Dr Pascal Guillo

Dr Jean-Claude Daran

Dr Eric Manoury

Pr Rinaldo Poli

Synthesis and Characterization of First Row Metal Complexes Derived from a Pyridinophane Ligand Functionalized by Fluoroalcohol

A new tetradentate N-donor ligand (L) based on 2,11diaza[3.3](2,6)pyridinophane with N-bonded (CF3)2(OH)CCH2 groups is presented, along with the coordination compounds [(L)FeCl2][FeCl4], [(L)MnCl2], [(L)Cu(OAc)2] and [(L)Zn(OTf)(H2O)](OTf) (OAc = acetate; OTf = trifluomethylsulfonate). All metal complexes possess a distorted octahedral geometry around the metal center.

Introduction

Access to new molecular complexes inspired by the active sites of metalloenzymes that involve metal-oxo intermediates during their catalytic action is of great interest for the inorganic community. [1] Indeed, these complexes allow better understanding of processes and mechanisms taking place in these metalloenzymes [2] and some of them have also proven very efficient catalysts in numerous catalytic reactions. [1b, 3] Most of these molecular models are inspired by the first coordination sphere of the active sites of metalloenzymes. Only few take into account the influence of the second coordination sphere. [4] This aspect is however crucial. Indeed, the local environment engendered by the second coordination sphere is responsible for establishing non-covalent interactions, mainly by the presence of a hydrogen-bond network. [5] In the realm of metalloenzyme reactions involving highly unstable species such as metal-oxo intermediates, H-bond networks have been proven essential to control dioxygen binding and activation and the stabilization of intermediates. [6] In order to better understand the role of the second coordination sphere, synthetic systems with a second coordination sphere able to create an H-bond network have been synthesized. Initial works have focused on the functionalization of porphyrin-based systems, which indeed allowed the stabilization of highly reactive intermediates. [7] However, the synthesis and the selective functionalization of this ligand family is often multi-step and low yielding. A breakthrough has been achieved by the Borovik's group [8] with the development of systems based on a tripodal urea-based ligand. These systems provide an appropriate H-bond network able to stabilize highly reactive species and thus to characterize them. [9] Thanks to this approach, Fe IV -oxo [10] and Mn V -oxo [11] complexes have notably been isolated and even characterized by X-ray crystallography for the Fe IV -oxo compound. [10a] More recently, Fout's group also used C3-symmetric ligands, based on a pyrrolide platform and modified by an imine fragment acting as an H-bond acceptor. [12] With this approach, Mn III -oxo [12d] and Fe III -oxo [12b, 12e] complexes have notably been synthesized. Interestingly, the Fe III -oxo complex is able to catalytically reduce nitrate and perchlorate anions. [12e] Goldberg's group has focused on the development of amide group-functionalized polypyridyl ligands, [13] leading to an accelerated oxygen-atom transfer in comparison to the non-modified systems.

Given that the utilization of H-bonds in the design and preparation of biomimetic metal complexes is still in the early stages of development, we have focused on a new strategy that consists of introducing fluoroalcohol groups. This is motivated by the fact that fluoroalcohols are strong H-bond donors. [START_REF] Berkessel | Modern Oxidation Methods[END_REF] Notably, fluorinated alcohol solvents are able to activate hydrogen peroxide toward electrophilic oxidation by hydrogen bonding. [START_REF] Berkessel | Modern Oxidation Methods[END_REF][15] In the context of reactions involving oxygen atom transfer, this feature is particularly interesting.

In the present contribution, we report the synthesis and characterization of a new tetradentate N-donor ligand L possessing two fluoroalcohol group-functionalized nitrogen atoms and a few first row metal complexes (metal = manganese, iron, copper and zinc) obtained from this ligand.

Results and Discussion

The 2,11-diaza[3.3](2,6)pyridinophane molecule was targeted as an interesting precursor for our studies. Indeed, this tetradentate ligand possesses two secondary amine groups that can be functionalized. Moreover, the amine functionalization by alkyl groups has already been reported for this system and the syntheses of octahedral metal complexes possessing two cis labile sites have also been reported. [16] The ligand L was obtained as a white powder in 79 % yield by reaction between 2,11diaza[3.3](2,6)pyridinophane and 2 equiv of 3,3,3-trifluoro-2-(trifluoromethyl)-1,2-propenoxide via a regioselective epoxide ring opening in absolute ethanol (scheme 1) at the expected 1position. [17] The X-ray structure of L (Figure 1) shows significant differences relative to that containing tert-butyl groups instead of the fluoroalcohol groups. [18] Indeed, the 12 atoms of the macrocyclic ring constitute a syn chair-chair conformation for the ligand with tert-butyl groups whereas L adopts a syn boat-boat conformation, which is already properly disposed to complex The reaction of L with various first row metal salts led to the formation of the complexes listed in Scheme 2. Reaction with 2.5 equiv of FeCl3,6H2O in CH3CN at 25 °C resulted in the formation of [(L)FeCl2](FeCl4) (1) as a yellow solid in 90 % yield. The presence of FeCl4 -as counteranion instead of Cl -was not anticipated, but this behavior has already been reported for similar systems in the literature. [19] Starting from only one equiv of FeCl3,6H2O led to the formation of the same metal-complex 1 in 40 % yield. Reaction with 1 equiv of MnCl2,4H2O in CH3CN at 25 °C resulted in the quantitative formation of (L)MnCl2 (2) as a pale yellow powder. (L)Cu(OAc)2 (3) was obtained in 77 % yield as a blue powder by reaction between L and 1 equiv of Cu(OAc)2,H2O in a CH3CN:CH3OH (1:1) mixture at 25 °C. The reaction between Zn(OTf)2 and L in CH3CN resulted into the quantitative formation of a product that was identified as a monoaquo monotriflate complex [(L)Zn(OH2)(OTf)](OTf) (4) in the solid state, according to the X-ray analysis. The 1 H NMR spectrum of 4 in acetone-d6 reveals a symmetrical species in solution that exhibits only one doublet and one triplet respectively for the pyridine ring m-H and p-H nuclei. Due to the presence of water in the solvent, this likely results from an equilibrium and rapid exchange between the three possible complexes [(L)Zn(OH2)n](OTf)2 (n = 0, 1, 2) in solution.

A broad signal at 4.39 ppm, attributed to the H2O protons and integrating for 4H per complex molecule, confirms the presence of rapid exchange reactions involving water. Crystals suitable for X-ray diffraction analysis were obtained, in all cases, by slow diffusion of diethyl ether into concentrated solutions in CH3CN. The ORTEP plots for compounds 1, 3 and 4 are given in Figure 2 and selected parameters are shown in Table 1. For complex 2 (Supporting Information, figure S1), the analysis was of sufficient quality to establish the atom connectivity, but the obtained structural parameters should be interpreted with caution because of the large R values. All complexes display the same syn boat-boat conformation as described for free L. However, while the two pyridyl rings are roughly parallel for L (vide supra), this is no longer the case for the metal complexes, especially for 1 and 3 (dihedral angles of 51,5(1)°, 65.70(7)° and 34.81(11)° for 1, 3 and 4, respectively). All metal complexes possess a distorted octahedral geometry around the metal center with an Nam-M-Nam angle between 147° and 150° and an Npy-M-Npy angle between 77° and 82°. The M-Nam distances are longer than the M-Npy distances in all complexes (2.263 to 2.4230 Å vs. 2.0009 to 2.124 Å). Similar trends have been previously observed for similar complexes obtained from pyridinophane based ligands. [16a, 16c] Concerning the Cu II -metal complex 3, elongated M-Nam bond lengths are observed due to a Jahn-Teller effect as usually observed for d 9 ions. The UV-Visible spectra of L and 1-4 were recorded in CH3CN solution. The free ligand L exhibits an intense transition at 259 nm attributed to a →* transition with an extinction coefficient of 4 600 M -1 cm -1 . The metal-complexes 1-4 exhibit similar transitions with extinction coefficients in the 4600 -21 200 M -1 cm - 1 range. In addition, the spectrum of 1 also displays two bands at 315 nm ( = 12 900 M -1 cm -1 ) and 361 nm ( = 11 600 M -1 cm -1 ) originating from the FeCl4 -counteranion, as previously reported. [20] Concerning (L)Cu(OAc)2 (3), a weak d-d transition observed at 710 nm ( = 700 M -1 cm -1 , see insert of Figure 3) is responsible for the blue color of the complex.

Conclusions

In conclusion, in this study we have disclosed the synthesis of a new ligand L possessing a nitrogen based environment for the first coordination sphere and a second coordination sphere constituted of fluoroalcohol groups. L is able to coordinate first row metal centers, as shown by the synthesis of four metalcomplexes 1-4 (metal = iron, manganese, copper and zinc), all of them characterized by X-ray crystallography. The ability of the fluoroalcohol groups to act as H-bond donor for the activation of substrates such as H2O2 and the stabilization of reactive intermediates such as oxo complexes are now being evaluated and the results will be reported in due course.

Supporting Information Summary

Synthetic and spectroscopic details corresponding to L, 1, 2, 3 and 4 are available in the Supporting Information. Crystal data and refinement parameters are shown in Table S1. CCDC 1527381 (for L), 1527382 (for 1), 1527383 (for 3) and 1527384 (for 4) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre.

  Scheme 1. Ligand L synthesis.

Figure 1 .

 1 Figure 1. Molecular structure of L displaying thermal ellipsoids at the 50 % probability level.

Scheme 2 .

 2 Scheme 2. [(L)FeCl2](FeCl4) (1), (L)MnCl2 (2), (L)Cu(OAc)2 (3) and [(L)Zn(OH2)(OTf)](OTf) (4) synthesized.

Figure 2 .

 2 Figure 2. Molecular structures of 1, 3 and 4 displaying thermal ellipsoids at the 50 % probability level. H-atoms have been omitted for clarity for 4.

Figure 3 .

 3 Figure 3. UV-Visible spectra of CH3CN solutions of Lblue), 1 (black), 2 (red), 3 (green) and 4 (orange). Insert: Zoom on the UV-Visible spectrum of 3.

Table 1 .

 1 Selected bond distances (Å) and angles (deg.) for complexes

1, 3 and 4.

		1	3	4
	M-N1 (N11)	2.263(3)	2.4230(19)	2.315(2)
	M-N2 (N12)	2.124(3)	2.0266(19)	2.080(2)
	M-N3 (N13)	2.274(2)	2.420(2)	2.309(2)
	M-N4(N14)	2.104(2)	2.0009(19)	2.098(2)
	N1(N11)-M-N2(N12)	76.81(9)	78.40(7)	79.08(9)
	N3(N13)-M-N4(N14)	76.79(9)	78.75(7)	78.32(8)
	N2(N12)-M-N4(N14)	77.30(9)	82.00(8)	82.13(10)
	N1(N11)-M-N3(N13)	146.94(9)	149.54(6)	149.51(8)

Acknowledgements

P.G thanks l'Université Fédérale Toulouse Midi-Pyrénées (UFTMiP) for a starting grant IDEX "Nouveaux Entrants" 2014. Additional support from the CNRS and the IUF is gratefully acknowledged.

Keywords: fluoroalcohol • metal complexes • molecular models • pyridinophane

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FULL PAPER

Fluoraoalcohol groups have been introduced on a tetradentate N-donor ligand.

Four metal complexes containing this ligand are described.

Metal complexes


Frédéric Delsuc
email: delsuc@isem.univ-montp2.fr

Mariella Superina

Guillermo Ferraris

Marie-Ka Tilak

Emmanuel J P Douzery

Hybridisation between South-American Ratites

In a private wildlife research facility and rhea farm of Argentina, artificially incubated eggs of putative hybrid origin between the Greater Rhea (Rhea americana) and the Lesser Rhea (Rhea pennata) hatched and gave birth to healthy chicks. Molecular genotyping by the analysis of mitochondrial Cytochrome b (Cyt b) and nuclear Chromo-Helicase DNA binding (CHD1) gene sequences confirmed the hybrid origin of these chicks which were molecularly sexed as females. The possibility of hybridisation argues for careful management of captive populations of these species, especially if individuals are to be released in the wild for conservation purposes.

Introduction

Ratites (order Struthioniformes) are large flightless birds represented by kiwis, emus, cassowaries, ostriches and rheas [START_REF] Folch | Order Struthioniformes[END_REF]). Among this order, rheas are endemic of South America and are represented by two living species: the common or greater rhea (Rhea americana) and Darwin's or lesser rhea (Rhea pennata formerly Pterocnemia pennata). Five and three sub-species are recognized within the geographical range of R. americana and R. pennata, respectively. Among these, the Andean endemic puna rhea (R. p. tarapacensis/garleppi) may merit consideration as a full species which is critically endangered since its population is thought to comprise only several hundred individuals at the most [START_REF] Folch | Order Struthioniformes[END_REF]. Both greater and lesser rheas have seen their populations declining dramatically in the recent past as the result of combined effects of high hunting pressure and habitat loss induced by the conversion of plains into farmland and pastures [START_REF] Bellis | Home range of greater and lesser rhea in Argentina: relevance for conservation[END_REF]). As a consequence, both species are classified as Near Threatened (NT) in the 2006 IUCN Red List (BirdLife International 2004).

In natural populations of both rhea species, the tasks of incubation and chickrearing are left exclusively to the male, after all females from its harem have laid their eggs in a previously prepared nest [START_REF] Folch | Order Struthioniformes[END_REF]. Some orphan eggs laid outside active nests, long thought to be unfertile, have been shown to produce viable chicks after artificial incubation [START_REF] Navarro | Fertility of greater rhea orphan eggs: conservation and management implications[END_REF]. This property has led to the conservation strategy of using captive reared yearlings for reintroduction in declining natural populations [START_REF] Bellis | Experience of release of yearlings of greater rhea reproduced artificially[END_REF]. Indeed, both species of rheas are commonly bred in South American farms and zoos. In captivity, some eggs are routinely collected and artificially incubated to increase successful reproduction. After hatching in the incubator, the chicks are brought together with the offspring produced by natural incubation and are reared naturally by males. In the first days after the laying, eggs from greater rheas are yellow colored whereas lesser rheas lay greenish eggs, and thus can be easily distinguished. Consequently, greater and lesser rheas are often bred in the same enclosures.

A private wildlife research facility (Estación de Incubación y Cría de Aves Silvestres -EICAS) in Mendoza (Argentina) maintains about 30 greater rheas together with a single female lesser rhea. Surprisingly, in October 1999, six green lesser rhea-like eggs were found in the enclosure. These eggs were artificially incubated, and two of them hatched 40 days later giving birth to healthy chicks (Figure 1A). Given the absence of male lesser rheas in the ratite farm, these chicks were suspected to represent viable hybrids of the lesser rhea female and one of the greater rhea males. Hybridisation is actually common in birds as [START_REF] Grant | Hybridization of bird species[END_REF] estimated that 9.2% of bird species hybridise either in nature or in captivity. However, the authors did not mention this possibility between species of the rhea family (Rheidae). The only mention of such a possibility was briefly noted by Folch (1992: plate 2).

To test for hybridisation, we studied the two putative hybrid chicks by DNA sequencing of two widely used molecular markers: the nuclear Chromo-Helicase DNA binding (CHD1) [START_REF] Miyaki | The last Spix's Macaw and an Illiger's Macaw produced a hybrid[END_REF]) and the mitochondrial Cytochrome b (Cyt b) genes [START_REF] Lee | The phylogeny of ratite birds: resolving conflicts between molecular and morphological data sets[END_REF]. Potential conservation implications of hybridisation between these near threatened species are discussed in the context of reintroduction plans from captive animals.

Methods

Complete genomic DNA was extracted from blood samples of two putative hybrids (H332 and H055) and a greater rhea R. americana (Ram) from the farm, and from muscle tissue of an unrelated lesser rhea R. pennata (Rpe) using standard procedures [START_REF] Sambrook | Molecular Cloning: a Laboratory Manual[END_REF]. Because standard molecular sexing protocols based on the study of the CHD genes do not work in ratites which have undifferentiated sex chromosomes [START_REF] Griffiths | A simple DNA test to sex most birds[END_REF][START_REF] Ellegren | Evolution of the avian sex chromosomes and their role in sex determination[END_REF], we used the test especially designed by [START_REF] Huynen | A DNA test to sex ratite birds[END_REF]. The sex-specific sequence (kW1) was PCR amplified in the two putative hybrids using primers w1' (forward): 5'-ACCAGCCTTTAAACAAGCTATTAA-3' and k7' (reverse): 5'-TCTCTTTTGTTTTAGACACCCT-3' slightly modified from the ones defined in the original publication to specifically target rhea sequences [START_REF] Huynen | A DNA test to sex ratite birds[END_REF]. A region of the CHD1 nuclear gene, encompassing approximately equal parts of a complete intron and portions of its flanking exons (García-Moreno & Mindell 2000) was also amplified with the universal sexing primers P8 and P2 [START_REF] Griffiths | A simple DNA test to sex most birds[END_REF] for the two putative hybrids, the greater rhea, and the lesser rhea. Finally, a 337 base pair (bp) fragment of the mitochondrial Cyt b gene from the two putative hybrids was directly PCR amplified using the primers L2 (forward): 5'-TACCATGAGGACAAATATC-3' and H10 (reverse): 5'-CTGGGGTGTAGTTCTCTGGGTC-3'. PCR products for CHD1 and Cyt b genes were purified from 1% agarose gels using Amicon Ultrafree-DA columns (Millipore), and directly sequenced with PCR primers on both strands using automatic sequencing (Big Dye Terminator cycle kit) on an ABI 310 (PE Applied Biosystems).

Sequences were manually aligned with the ED editor of the MUST package [START_REF] Philippe | MUST: a computer package of management utilities for sequences and trees[END_REF]). The CHD1 gene was 319 bp long for all four individuals analyzed and no gaps needed to be introduced in the alignment. Sequences have been deposited in the EMBL data bank under Accession Numbers AJ430514 to AJ430519.

Results and Discussion

The two putative hybrids were molecularly sexed as females by the presence of the female specific 150 bp fragment of the kW1 region in the two PCR amplifications (Figure 1B). Alignment of the mitochondrial Cyt b sequences from the two putative hybrids with previously published sequences of R. americana and R. pennata has shown a perfect identity with R. pennata (Figure 2). Results from this maternally inherited gene confirmed that the artificially incubated lesser rhea-like eggs, giving birth to the putative hybrids, were laid by the lesser rhea female of the breeding facility. No lesser rhea males were kept on the farm and accidental mating with a wild living male was highly unlikely, as the facility is located in a semi-urban area of Mendoza free of wild rhea populations. This result thus strengthened the possibility that the chicks arose from hybridisation with a captive greater rhea male. Definitive confirmation of this suspicion came from the comparison of CHD1 gene sequences (Figure 3). Only three variable sites on the 319 sequenced sites of the CHD1 alignment were detected, of which two were diagnostic for the hybrids (positions 104 and 109). These variable sites were all situated within the intronic region of the gene. When looking at these sites in chromatograms of the hybrids, we observed two exactly superimposed fluorochrome peaks corresponding to the nucleotides found in the parental species R. americana and R. pennata (Figure 3). As CHD1 appeared as non sex-linked in ratites with an autosomal mode of inheritance [START_REF] Ellegren | Evolution of the avian sex chromosomes and their role in sex determination[END_REF], these sites indicated that the chicks possessed the two parental alleles: one likely inherited from the lesser rhea female and the other from one of the greater rhea males. The third variable site between the two parental sequences (position 149) was not diagnostic since hybrids unambiguously possessed an Adenine like their lesser rhea mother whereas a Guanine was found in the putative greater rhea father sequence. This observation suggests that the greater rhea male we randomly sampled in the farm is not actually the father of the hybrids. However, our results provide clear molecular evidence that hybridisation in captivity between the two living species of South American ratites led to viable first generation hybrids (F1 hybrids).

As expected, the hybrids showed mixed morphological characters from both maternal and paternal inheritance. Shortly after hatching, the hybrid chicks had greyish brown feathers, resembling offspring of greater rheas. A white line at the tip of the feathers could be observed at one year, which made the chicks look more similar to lesser rheas. Approximately two years after hatching, the hybrids showed a steel grey plumage that reaches the tarsus. The head is rounded as in lesser rheas, whereas the beak is long and flattened as in greater rheas (Figure 1C). The hybrids are slightly larger in size than greater rheas of the same age. Very unfortunately, we have been unable to collect any data on the age at which the described hybrids may reach sexual maturity, since they were sold after two years by the owner of the farm for commercial purposes. Therefore, we cannot make any prediction about their potential fertility.

As demonstrated by the occurrence of occasional hybridisation, Rhea americana and Rhea pennata seem to be phylogenetically closely related species. At the molecular level, we observed only three differences on 319 nucleotides in the CHD1 gene (0.9 % divergence) and 23 differences on 337 nucleotides in the mitochondrial Cyt b gene (6.8 % divergence). Our results confirm that the genus-level taxonomic distinction introduced with the long use of the name Pterocnemia pennata Gray 1870 for the lesser rhea might not be justified. This gives credit to the recent taxonomic revision coming back to Rhea pennata as originally proposed by d'Orbigny in 1834. It also provides a good example of the trend for taxonomical splitting observed in birds where species have tended to be placed into separate genera at a lower level of genetic divergence than is often true for other vertebrates [START_REF] Johns | A comparative summary of genetic distances in the vertebrates from the mitochondrial Cytochrome b gene[END_REF].

The fact that hybridisation can occur in captivity points out that the differentiation between the two species is not complete and raises questions about the possibility of its occurrence in natural populations. The two species have a parapatric distribution range with a sympatric zone North of Patagonia in Argentina [START_REF] Folch | Order Struthioniformes[END_REF] suggesting that hybridisation could potentially occur in this area. However, it is clear that captive living conditions are far from the natural ones. In the wild, it is likely that a number of prezygotic barriers, which are relaxed in captivity, prevent hybridisation between the two species. In particular, the complex courtship behaviour displayed by both species of rheas [START_REF] Folch | Order Struthioniformes[END_REF]) constitutes an ethological barrier to hybridisation. Continuing habitat fragmentation, reduction of population sizes by illegal hunting and isolation of subpopulations of both species could, however, increase the encounters between greater and lesser rheas [START_REF] Bellis | Experience of release of yearlings of greater rhea reproduced artificially[END_REF][START_REF] Bouzat | The population genetic structure of the greater rhea (Rhea americana) in an agricultural landscape[END_REF]. It can not be excluded that wild rheas would hybridise with individuals of the other species if conspecifics are rare or absent during the mating season.

Numerous rhea populations are managed by man in farms and zoos and our results argue for a careful management of captive breeding populations in order to avoid hybridisation between greater and lesser rheas. In private husbandries and zoos, the two species must therefore be strictly separated and meaningful captive breeding programs should be followed to conserve the integrity of both species. This latter point is of primary importance in cases where captive bred animals are thought to be released in the wild for reintroduction or population increase purposes [START_REF] Bellis | Experience of release of yearlings of greater rhea reproduced artificially[END_REF][START_REF] Allendorf | The problems with hybrids: setting conservation guidelines[END_REF].

Figure legends

Figure 1 .

 1 Figure 1. A. Putative hybrid egg hatching after 40 days of artificial incubation. Note the

Figure 2 .

 2 Figure 2. Alignment of the 24 variable sites over 337 base pairs from the mitochondrial

Acknowledgments

Mr. Caesar Claude (Zoologisches Museum Zürich) generously provided us with Pterocnemia pennata tissue samples. Jaime García-Moreno kindly shared CHD alignment files and chromatograms. Lura Williamson provided helpful comments on the manuscript. This work is the contribution ISEM 2006-39 of the Institut des Sciences de l'Evolution de Montpellier (UMR 5554 -CNRS).