Lucie-Marie Scailteux
email: luciemarie.scailteux@chu-rennes.fr

Frédéric Balusson

Emmanuel Oger

Sebastien Vincendeau

Romain Mathieu

Sébastien Vincendeau

Androgen deprivation therapy prescription, blood and bone-density testing in a French population-based study exploring adherence to the French prostate cancer guidelines

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Short communication

Androgen deprivation therapy (ADT) is a cornerstone therapy in prostate cancer (PCa) treatment.

This treatment is associated with several adverse effects such as bone-mineral density decrease and related fractures, insulin resistance with an increased risk of diabetes pseudo-metabolic syndrome, and cardiovascular events [START_REF] Rhee | Adverse effects of androgen-deprivation therapy in prostate cancer and their management[END_REF][START_REF] Nguyen | Adverse effects of androgen deprivation therapy and strategies to mitigate them[END_REF] .

Before ADT initiation, the French Committee of Urologic Oncology (CCAFU) [START_REF] Rozet | Recommandations françaises du Comité de Cancérologie de l'AFU -Actualisation 2018-2020 : cancer de la prostate[END_REF] recommends a clinical examination of patients to look for cardiovascular and osteoporosis risk factors. In addition to bonedensity testing (BDT), biological examinations should include fasting blood glucose, vitamin D test and lipid assessment. During ADT follow-up, in addition to blood glucose, vitamin D and lipid examinations, blood PSA levels should be tested. However, a blood test for testosterone is not recommended as part of the ADT follow-up. The frequency of biological examinations is not clearly defined. The identification of one or several abnormalities following BDT and blood glucose, vitamin and lipid tests should lead to a specialist consultation (cardiologist, rheumatologist…) and, if necessary, to the introduction of specific treatments with a change in life-style factors.

In a 6-month cross-sectional French study using questionnaires administered to clinicians between 2012 and 2013 concerning the care and monitoring of 1100 patients treated with luteinizing hormone-releasing hormone (LHRH) agonist, Hennequin et al. reported that fasting blood glucose tests were prescribed to 41%, lipid assessments to 36%, BDT to 11 %, electrocardiograms to 25 %, and total blood testosterone levels to 5% [START_REF] Hennequin | Prostate cancer treated with androgen deprivation therapy: Care and monitoring in daily practice[END_REF] .

In order to examine whether the French CCAFU prostate guidelines (issued at the time of the study 5 ) were correctly followed by clinicians, including urologists, oncologists and radiotherapists, we used a nationwide cohort of French PCa patients, who were new users of ADT in 2011 and were followed up to 2013 [START_REF] Scailteux | Androgen deprivation therapy and cardiovascular risk: No meaningful difference between GnRH antagonist and agonists-a nationwide population-based cohort study based on 2010-2013 French Health Insurance data[END_REF] . Using the French "SNIIRAM" database, a comprehensive and representative claims database [START_REF] Bezin | The national healthcare system claims databases in France, SNIIRAM and EGB: Powerful tools for pharmacoepidemiology[END_REF] , PCa diagnosis over the study period was based on the International Classification Disease 10 th edition codes 'C61' or 'D40.0' for hospitalisation or on qualification for long-term disease status which provides 100% reimbursement of all disease-related claims. Biological examination reimbursements and prescribers were identified, as well as PCa specialist consultations before and after drug initiation (one year to 3 months before, and in the 6 months following). These reimbursements provide proof that the procedures have been carried out. Prescriptions that are not followed by the procedure do not result in reimbursement.

In 2011, 23 407 new ADT users were identified (median age, 75 years): 67 % were treated with LRHR agonist, 17 % with combined androgen blockade, 12 % with anti-androgen, and 4 % with LHRH antagonist. The most common baseline comorbidities included hypertension (66.5%), the use of lipid-lowering drugs (44.6%), and the use of anti-platelets (31.9%) and anti-diabetics (16.7%). In the 6 months before ADT initiation, 83% of patients had consulted a urologist and 25% an oncologist or a radiotherapist. In the 12 months after ADT initiation, 75% of patients saw a urologist and around 47% an oncologist or radiotherapist.

Figure 1 shows the proportions of patients undergoing specific procedures, blood examinations and BDT in the 3 months to one year before ADT initiation and in the 6 months following.

While the examination of blood testosterone levels is only required to confirm CRPC [START_REF] Rozet | Recommandations françaises du Comité de Cancérologie de l'AFU -Actualisation 2018-2020 : cancer de la prostate[END_REF] , testosterone level monitoring during androgen deprivation can be useful at earlier stages. Indeed, testosterone levels < 50 ng/dL under androgen deprivation confirm the success of the treatment administered and patient compliance. Nadir and spikes of blood testosterone have also been reported as strong prognosticators [START_REF] Klotz | Nadir testosterone within first year of androgen-deprivation therapy (ADT) predicts for time to castration-resistant progression: a secondary analysis of the PR-7 trial of intermittent versus continuous ADT[END_REF] . However, in our study, blood testosterone testing was only performed on fewer than 15% of patients after initiation of ADT.

Vitamin D, fasting blood glucose and lipid tests were mainly prescribed by general practitioners (GP) in the 3 months before and 6 months after ADT initiation (Figure 2). Regarding PSA in the 3 months before ADT initiation, we observed that tests were prescribed by urologists, other specialists and GPs in respectively 31.3%, 5.7% and 37.6% of the cases. Blood PSA and testosterone level assessments in the 6 months after ADT initiation were mainly prescribed by urologists (36.4%).

Although these tests are recommended by CCAFU, our results showed that, at the time of ADT initiation, blood examinations and BDT were not performed on all patients. One limitation was the use of the French "SNIIRAM" database, a healthcare reimbursement database [START_REF] Bezin | The national healthcare system claims databases in France, SNIIRAM and EGB: Powerful tools for pharmacoepidemiology[END_REF] . This database reflects the actual completion of prescribed examinations. However, the reason why some examinations were not performed remains unclear and could relate to failure to prescribe on the part of clinicians, and also to patient non-compliance. We also recognize that patients who died early or those with a short follow-up period may not have had time to undergo their examinations.

Regarding the prescriber specialty, the investigation of CV and fracture risk factors via vitamin D, fasting blood glucose and lipid tests was mainly initiated by GPs whereas blood PSA and testosterone level assessment seemed more likely to be prescribed by specialists.

The results of the study by Hennequin et al. using questionnaires [START_REF] Hennequin | Prostate cancer treated with androgen deprivation therapy: Care and monitoring in daily practice[END_REF] are consistent with those from our study, except for BDT: 11% of clinicians in their study reported having prescribed BDT whereas 1 % of the patients or less in our study actually underwent this assessment in the year before or in the six months after ADT initiation. This percentage is well below that in an American study showing that 15 to 20 % of patients underwent BDT within the 3-year period surrounding ADT initiation, which still appeared below the ideal testing rate [START_REF] Kirk | The implications of baseline bone-health assessment at initiation of androgen-deprivation therapy for prostate cancer[END_REF] .

In a review commentary on ADT cardio-metabolic toxicity, Khouri and Harrisson considered that the variety of backgrounds of the physicians who administer ADT predisposed to a heterogeneous approach to screening or monitoring [START_REF] Khouri | Look and You Will Find It: Practical Considerations for Improving Multidisciplinary Management of Androgen-Deprivation Therapy-Induced Cardiometabolic Toxicity[END_REF] . Our results suggest that the implementation of blood tests and BDT by urologists, radiotherapists and oncologists remains sub-optimal whatever the specialty they belong to. In a review on ADT adverse effects, Rhee et al. discussed Australian practice [START_REF] Rhee | Adverse effects of androgen-deprivation therapy in prostate cancer and their management[END_REF] . They indicated that the management of long-term adverse events under ADT should include a pro-active attitude to prevent reduce or address them. Thus, in addition to the educational package (provided in their review), a 'Men's Health Clinic' has been established in Austin, where a multidisciplinary team is responsible for monitoring and managing all patients who initiate ADT.

Overall, there is still room for improvement in terms of ADT monitoring by clinicians and in terms of information provided to GPs and patients regarding the expected adverse effects of this treatment.

The development of educational programs for patients and caregivers could be discussed, to improve the overall detection and management of ADT adverse events.

Figure 1 .Figure 2 .

 12 Figure 1. Prevalence of patients with specific care, blood examinations and bone-density testing (at least one reimbursement per considered variable) before and after ADT initiation.

Scailteux et al.

Funding: This study is part of an initial study that was funded by the national 'Programme Hospitalier de Recherche Clinique (PHRC)' 2013. Project number of: 13-0257.


Hager Jouili

Houda Bouazizi

Michel Rossignol

Gisèle Borderies

Elisabeth Jamet

Ezzeddine El Ferjani
email: ezzferjani@yahoo.fr

Ezzeddine El Ferjani

Partial purification and characterization of a copper-induced anionic peroxidase of sunflower roots

Keywords: Copper, Mass spectrometry, Peroxidase, Root, Sunflower

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Introduction

Peroxidases (EC. 1.11.1.7; donor: hydrogen peroxide oxidoreductase) belong to a large family of heme-containing enzymes which are distributed throughout the plant kingdom.

Plant peroxidases can be subdivided into three subgroups (acidic, neutral and cationic) according to their isoelectrophoretic mobilities. These enzymes catalyze the oxidation of a variety of electron donor substrate (e.g. phenols, aromatic amines) by hydrogen peroxide [START_REF] Passardi | Performing the paradoxical: how plant peroxidases modify the cell wall[END_REF]. Based on differences in primary structure, the plant peroxidase superfamily can be divided into three classes: classes I, II and III [START_REF] Welinder | The plant peroxidase superfamily[END_REF]. Class I peroxidases include intracellular enzymes in plants, bacteria and yeast. Class II peroxidases are extracellular peroxidases of fungi. Class III comprises classical plant secreted peroxidases.

Class III peroxidases have been found in crude plant extracts by staining for activity after separation by gel electrophoresis [START_REF] Hiraga | A large Family of class III Plant Peroxidases[END_REF]. These enzymes have approximately 300 amino acids. The majority are N-glycosylated and are predicted to be localized in cell walls or in vacuoles [START_REF] Hiraga | A large Family of class III Plant Peroxidases[END_REF]. The amino acid sequences were found to be highly variable inside the plant peroxidase superfamily, with less than 20 % identity in the most divergent cases. The evolution of that multigene family seems to be correlated with the increasing complexity of plant architecture and to the diversification of their biotopes [START_REF] Passardi | Performing the paradoxical: how plant peroxidases modify the cell wall[END_REF]. In some plants like Arabidopsis thaliana, a high duplication rate has led to a large multigene family of 73 members [START_REF] Welinder | Structural diversity and transcription of class III peroxidases from Arabidopsis thaliana[END_REF].

In plants, peroxidase activity is presumed to be involved in the building and rigidification of cell walls [START_REF] Passardi | Performing the paradoxical: how plant peroxidases modify the cell wall[END_REF], auxin catabolism [START_REF] Delannoy | Les peroxydases végétales de la classe III[END_REF], and senescence [START_REF] Faivre-Rampant | Peroxidase activity, ethylene production, lignification and growth limitation in shoots of a non rooting mutant of tobacco[END_REF]. However, their physiological functions are not precisely known, as cells contain a large number of peroxidases each having a broad in vitro catalytic specificity [START_REF] Faivre-Rampant | Peroxidase activity, ethylene production, lignification and growth limitation in shoots of a non rooting mutant of tobacco[END_REF]. On the other hand, it has been suggested that genetic manipulations using antisense technology or screening of mutants is not a very powerful method for revealing functions of individual peroxidase genes because possible interference from other peroxidase genes may give complex phenotypes [START_REF] Hiraga | A large Family of class III Plant Peroxidases[END_REF].

Actually only two A. thaliana peroxidase mutants have yet been described [START_REF] Passardi | Two cell wall associated peroxidases of Arabidopsis influence root elongation[END_REF]. They are affected in root elongation. On the contrary, plants overexpressing one of these peroxidases have longer roots.

Increase in peroxidase activity was observed in several plants exposed to environmental injury such as biotic and abiotic stresses. It has thus been suggested that these enzymes play key roles in defense mechanisms [START_REF] Passardi | Peroxidases have more functions than a Swiss army knife[END_REF] that are induced in addition to the numerous mechanisms plant possess to reduce damages from exposure to metal ions [START_REF] Clemens | Molecular mechanisms of plant metal tolerance and homeostasis[END_REF]. In order to understand the role of peroxidases in defense mechanism in response to heavy metals, our laboratory has started the characterization of peroxidases induced by copper in sunflower roots. Actually, copper is known to be an essential micronutrient for higher plants. But, at high levels, it can induce many alterations in plant cells [START_REF] Vinit-Dunand | Effects of copper on growth and on photosynthesis of mature and expanding leaves in cucumber plants[END_REF]. It can catalyze the formation of some harmful free radicals, which cause an oxidative burst. This toxic effect can be alleviated by several antioxidative systems including peroxidases. In sunflower seedlings treated with 50 µM of CuSO 4 , 5 anionic peroxidases (A1-A5) were induced in a soluble fraction extracted from roots. They had a differential behavior under stress treatment. Here we report the partial purification, the identification and the characterization of A1. A1 was identified using liquid chromatography coupled to mass spectrometry (LC-MS/MS).

Results and Discussion

Growth inhibition and copper accumulation in roots under stress conditions

Forteen day-old sunflower seedlings were cultured on a 50 µM CuSO 4 for 5 days. This copper excess caused severe symptoms on sunflower roots (Fig. 1A). They became brownish, they showed less branching and reduced growth. Such symptoms were previously described for maize roots treated with nickel [START_REF] Baccouche | Nickel induced oxidative damage and antioxidant responses in Zea mays shoots[END_REF].

The length of main roots was measured and was found to be significantly shorter in Cutreated seedlings than in control, with a reduction of about 30% (Fig. 1B). In the same way, inhibition rate of dry and fresh mass production of Cu-treated roots was estimated at 76 % and 53 % respectively (Fig. 1C andD). All these observations suggest that copper treatment have a strong inhibitory effect on growth. Such effects were also observed on Cannabis sativa roots treated with copper [START_REF] Bona | Proteomic characterization of copper stress response in Cannabis sativa roots[END_REF]. The high toxicity of copper has been reported for several species including maize [START_REF] Mocquot | Copper toxicity in young maize (Zea mays L.) plants: Effects on growth, mineral and chlorophyll contents, and enzyme activities[END_REF], tomato [START_REF] Mazhoudi | Response of antioxidant enzymes to excess copper in tomato (Lycopersicon esculentum, Mill.)[END_REF], bean [START_REF] Gupta | Copper affects the enzymes of the ascorbate-glutathione cycle and its related metabolites in roots of Phaseolus vulgaris[END_REF] and cucumber [START_REF] Vinit-Dunand | Effects of copper on growth and on photosynthesis of mature and expanding leaves in cucumber plants[END_REF]. In some species, high Cu sensitivity of root growth is related to disturbances of mitosis [START_REF] Eleftheriou | Ultrastructural and morphological characteristics of cultivated wheat growing on copper-polluted fields[END_REF] or to damage of cell membranes [START_REF] Vos | Effect of copper on fatty acid composition and peroxidation of lipids on roots of tolerant and sensitive Silene cucubalus[END_REF]. After copper treatment of seedlings, Cu content of the roots was measured. It was first estimated at 3.7 mg.g -1 of dry mass (Fig. 1E). However, since copper ions could be adsorbed onto root cell walls and especially on pectins, this value could be overestimated [START_REF] Kartel | Evaluation of pectin binding of heavy metal ions in aqueous solutions[END_REF]. Thus, in order to eliminate adsorbed ions, roots were washed either with a chelating agent, i.e. EDTA, or with a competitor for adsorption, i.e. a CaCl 2 solution. In these experimental conditions, copper content of roots was found to be 0.78 and 1.50 mg.

g -1 of dry mass respectively (Fig. 1F). Actually, copper accumulation in roots can be considered as a mechanism of defense against metal toxicity by limiting their translocation to the photosynthetic tissues [START_REF] Zhang | Effects of zinc (zinc sulfate) on rhizobia-earleaf acacia (Acacia auriculaeformis) symbiotic association[END_REF].

Effect of copper excess on total peroxidase activity and PAGE analysis of anionic peroxidases

This important accumulation of Cu in roots was accompanied by the stimulation of soluble peroxidases. Total peroxidase activity was measured in roots at various times after transfer of seedlings in 50 µM of CuSO 4 , i.e. 0, 24, 48, 72 and 120 h. Same fractions were submitted to native PAGE (polyacrylamide gel electrophoresis) in order to separate anionic peroxidases. A significant increase in total peroxidase activity was observed 24 h after the beginning of the Cu treatment (Fig. 2A). This increase was continued during all the period of treatment. Such an increase in total peroxidase activity in response to copper treatment was already reported in leaves of Commoelina communis [START_REF] Wang | Responses of antioxidative enzymes to accumulation of copper in a copper hyperaccumulator of Commoelina communis[END_REF], in peanut roots [START_REF] Li | The acute effect of copper on the levels of indole-3-acetic acid and lignin in peanut roots[END_REF], in roots of Raphanus sativus [START_REF] Chen | Effect of copper on peroxidase activity and lignin content in Raphanus sativus[END_REF], and in wheat roots [START_REF] Sgherri | Antioxidative enzymes in two wheat cultivars, differently sensitive to drought and subjected to subsymptomatic copper doses[END_REF]. On the contrary, proteomic analysis of Cannabis sativa roots showed that a peroxidase disappeared in response to an excess of copper [START_REF] Bona | Proteomic characterization of copper stress response in Cannabis sativa roots[END_REF]. Qualitative analysis of anionic peroxidases during stress conditions by native PAGE is shown in Fig. 2B. It revealed the presence of 5 peroxidases, named A1 to A5. Copper treatment caused the stimulation of 4 peroxidases (A1, A3, A4 and A5) and the de novo induction of A2. While this stimulation decreased 72 h after beginning of treatment for A1, A2, A3 and A4, the stimulation of A5 continued. Such variations in the peroxidase pattern of sunflower leaves were described in response to iron deficiency [START_REF] Ranieri | Iron deficiency differently affects peroxidase isoforms in sunflower[END_REF]. Several anionic peroxidases associated to cell walls were found to disappear. The fact that several peroxidases exist in sunflower was expected since peroxidases usually belong to large multigene families [START_REF] Welinder | Structural diversity and transcription of class III peroxidases from Arabidopsis thaliana[END_REF]. Moreover, first data on systematic sequencing of sunflower expressed sequence tags (ESTs) indicate the presence of sequences showing homologies to 25 different peroxidases of A. thaliana (http://cgpdb.ucdavis.edu/).

Partial purification of A1

In this paper, we focused our study on A1, since its activity is transiently stimulated in response to copper treatment. In order to avoid difficulties associated with phenolic compounds present in sunflower roots, extracts were prepared after homogenization in phosphate buffer containing 10 % PVP (w/w). The resulting supernatant had a peroxidase specific activity of 58.45 nkat.mg -1 proteins (Table 1). Crude extract was desalted using size-exclusion chromatography on a Sephadex G25 column. Peroxidase activity was recovered in the void volume with a specific activity of 65.13 nkat.mg -1 proteins (Table 1).

Table 1. Yield of purification of A1 from sunflower roots at the three steps of the procedure. Enzymatic activity was assayed with guaïacol as a substrate.

Next step of the purification consisted in anionic exchange chromatography on a Fast

Flow Q Sepharose column. The column was equilibrated with 100 mM Tris-HCl buffer (pH 7.5). A linear gradient of NaCl (0-0.4 M) was applied. Two peaks of peroxidase activity were obtained: peak 1 at 0.04 M NaCl, peak 2 at 0.16 M NaCl (Fig. 3A). Specific activity in peak 1 was 2196.05 nkat.mg -1 proteins, corresponding to a purification factor of 38 (Table 1). Analysis by native PAGE showed that fractions corresponding to peak 1 contained A1, whereas fractions corresponding to peak 2 contained A4 and A5 (Fig. 3B).

The fraction containing A1 was further analysed by SDS-PAGE followed by silver staining (Fig. 3C). Only a few bands were visible, showing the efficiency of this purification step. peptides (YYVDLMNR and MGQLSVLTGTQGEIR) allowed getting a complete sequence. These sequences were used to find homologous plant sequences with the BLAST software. Best hits were the A. thaliana AtPrx12 (At1g71695) and the Nicotiana tabacum NtPER9_6 peroxidases (Fig. 4). Search for functional domains using

Step of purification

InterProScan indicated that such protein are bona fide peroxidases containing functional domains PF00141 (peroxidase), PS50873 (Plant heme peroxidase family profile), PS00435 (Peroxidases proximal heme-ligand signature) and PS00436 (Peroxidases active site signature). Amino acids important for protein structure such as Cys, or for active site such as Arg, Asn, Pro, His, and Asp are well-conserved (Fig. 4). All these proteins have a C-terminal extension that is assumed to be cleaved to get full activity [START_REF] Welinder | Structural diversity and transcription of class III peroxidases from Arabidopsis thaliana[END_REF].

Table 2. Sequences of the 3 peptides obtained through LC-MS/MS and showing identity to amino acid sequences of Helianthus peroxidases.

Sequences are written using the one-letter code. Database searching was performed using the Mascot software and a Helianthus EST database as described in Material and methods. In those conditions, individual ion scores are significant when higher than 48. Mascot search results are given in Table 1 (supplementary data). When Mascot search was performed against the Viridiplantae database, it should be mentioned that an additional peptide was found in the 47 kDa band (NLVTLLNK) (Table 2 supplementary data). This peptide had a significant Mascot score of 45 (score higher than 43 was required in these conditions). Such a sequence is found in a H. annuus protein (AAL77103) that is predicted to be secreted by both PSORT and TargetP. This protein contains two functional domains: FAD-binding domain (PF01565) and Berberine and berberine-like domain (PF08031). It is a good candidate to be a protein homologous to berberine-brige oxido-reductases that were described in many cell wall proteomes [START_REF] Jamet | Cell wall proteins: a new insight through proteomics[END_REF].

Sequences of peptides

The precise roles of such proteins in plant cell walls are not known.

This analysis allowed the identification of two proteins, among which a peroxidase probably contributing to the reaction of sunflower roots to cupric stress. This peroxidase is predicted to be secreted. It is the first time that a sunflower peroxidase can be precisely identified using LC-MS/MS. It will allow further characterization of the protein as well as study of the fine regulation of gene expression in response to cupric stress.

Characterization of the enzymatic activity of A1

To further characterize the enzymatic activity of A1, it was tested in different experimental conditions of pH, temperature and H 2 O 2 concentration (data not shown). At 25°C, A1 showed an optimum of activity at pH 8. In the same way, it has been reported that African oil palm tree anionic peroxidases showed a optimum of activity at alkali pH as well as a high stability [START_REF] Sakharov | Extremely high stability of African oil palm tree peroxidases[END_REF]. At pH 6.5, A1 had the highest enzymatic activity at 40°C.

Also, it has been demonstrated that corn root peroxidases presented a high activity at high temperature, that is around 50 ºC [START_REF] Mika | Properties of guaïacol peroxidase activities isolated from corn root plasma membranes[END_REF]. Concerning the substrate specificity (Table 3), A1 had an affinity 10 times higher for coniferyl alcohol than for guaïacol. No reaction was observed using indole acetic acid (IAA). Coniferyl alcohol is one of the first precursors for lignin biosynthesis in plants [START_REF] Sato | Separation and characterization of the isoenzymes of wall-bound peroxidase from cultured Zinnia cells during tracheary element differentiation[END_REF]. Thus the possible involvement of A1 in lignification process could be suggested. However, this assumption should be checked in vivo. Finally, A1 activity was decreased by 30% already at 20 mM H 2 O 2 in the incubation medium as compared to standard conditions (10 mM H 2 O 2 ). Increasing H 2 O 2 concentration up to 50 mM resulted in a further decrease of 30% of activity. This was observed in previous works showing the inactivation of peroxidases by hydrogen peroxide [START_REF] Sakharov | Extremely high stability of African oil palm tree peroxidases[END_REF]. These modulations of peroxidase activities in response to variations in temperature, pH, or H 2 O 2 concentration could be considered as regulatory processes acting under physiological modifications to favour some biochemical processes. It has been suggested

10 20 30 40 50 60 | | | | | | AtPrx12 MTKAYSTRVLTFLILISLMAVTLNLFPTVEAKKRSRDAPIVKGLSWNFYQKACPKVENII NtPER9_6 --MAS-ITLSSFAASL-LLILSVNFYQTEAQGT----QPIVKGLSWTFYDSICPNAESII H_ann_1 MFMAFFFKASSIPTLI-FLLLSCNLIRISETQT----PAPAPGLSYDFYRTTCPRLERII H_ann_2 MFMAFFFKASSIPTLI-FLLLSCNLIRISETQT----PAPAPGLSYDFYRTTCPRLETII H_par_1 ------------------------------------------------------------ H_arg_1 ------------------------------------------------------------ 70 80 90 100 110 120 | | | | | | AtPrx12 RKELKKVFKRDIGLAAAILRIHFHDCFVQGCEASVLLAGSASGPGEQSSIPNLTLRQQAF NtPER9_6 RSRLQQVFRQDIGQAAGLLRLHFHDCFVQGCDGSVLLDGSASGPSEKDAPPNLTLRQQAF H_ann_1 RQNLQSVFASDIGQAAGLLRLHFHDCFVQGCDGSVLLDGSASGPSEKDAPPNLSLRAQAF H_ann_2 RQNLQSVFASDIGQAAGLLRLHFHDCFVQGCDGSVLLDGSASGPSEKDAPPNLSLRAQAF H_par_1 -----------------------------------------GGPSEKDAPPNLSLRAQAF H_arg_1 ------------------------------------------------------------ 130 140 150 160 170 180 | | | | | | AtPrx12 VVINNLRALVQKKCGQVVSCSDILALAARDSVVLSGGPDYAVPLGRRDSLAFASQETTLN NtPER9_6 RIIEDLRRRVHRDCGRVVSCADITAIAARDSVFLSGGPDYDLPLGRRDGLNFATRNETLA H_ann_1 VIIENLRRLVHNACNRTVSCSDITALAARDAVFLSGGPNYSIPLGRRDGLTFATRNVTLA H_ann_2 VIIENLRRLVHNACNRTVSCSDITALAARDAVFLSGGPNYSIPLGRRDGLTFATRNVTLA H_par_1 VIIENLRRLVHNACNRTVSCSDITALAARDAVFLSGGHNYSIPLGRRDGLTFATRNVTLA H_arg_1 ------------------------------------------------------------ 190 200 210 220 230 240 | | | | | | AtPrx12 NLPPPFFNASQLIADFANRNLNITDLVALSGGHTIGIAHCPSFTDRLYPNQDPTMNQFFA NtPER9_6 NLPPPSFNASAILTSLATKNFTPTDVVALSGGHTIGIGHCTSFTERLYPNQDPSMDKTFA H_ann_1 NLPPPSANTTTLLNSLGQKGFTPTDVVALSGGHTIGIAHCTSFTGRLYPTRDPTMDQTFF H_ann_3 NLPPPSANTTTLLNSLGQKGFTPTDVVALSGGHTIGIAHCTSFTGRLYPTRDPTMDQTFF H_par_1 NLPPPSANTTTLLNSLGQKGFTPTDVVALCGGHTIGIAHCTSFTGRLYPTRDPTMDQTFF H_arg_1 ----------------------------------------------------------FF 250 260 270 280 290 300 | | | | | | AtPrx12 NSLKRTCPTANSSNTQVNDIRSPDVFDNKYYVDLMNRQGLFTSDQDLFVDKRTRGIVESF NtPER9_6 NNLKNTCPTSNSTNTTVLDIRSPNKFDNKYYVDLMNRQGLFTSDQDLYTDRRTRGIVTSF H_ann_1 NNLRTTCPTTNSTNTTFMDIRSPNVFDNKYYVDLMNRQGLFTSDQDLYTDSRTRSIVTNF H_ann_2 NNLRTTCPTTNSTNTTFMDIRSPNVLDNRYYVDLMNRQGLFTSDQDLYTDSRT------- H_par_1 NNVRTTCPTTNSTNTTFMDIQSPNVFDNRYYVDLMNRQGLFTSDQD-------------- H_arg_1 NNVRTTCPPTNSTNPTFMDIRSPDGFDNRYYVDLMNRQGLFTSDQALYADSRTRSIVTNF 310 320 330 340 350 360 | | | | | | AtPrx12 AIDQQLFFDYFTVAMIKMGQMSVLTGTQGEIRSNCSARNTQS---F-MSVLEEGIEEAIS NtPER9_6 AINESLFFEEFVNSMIKMGQLNVLTGTQGEIRANCSVRNSANYNLLLSTSVAEEQQRTWS H_ann_1 CGYQTLFYKNLWMQ---------------------------------------------- H_ann_2 ------------------------------------------------------------ H_par_1 ------------------------------------------------------------ H_arg_1 AVNQTLFYQNFVNAMIKMGQLSVLTGTQGEIRSNCFRRNSN--DLFMFPAMESAQDRKLA AtPrx12 MI NtPER9_6 EI H_ann_1 -- H_ann_2 -- H_par_1 -- H_arg_1 SY
that small variations in pH values could represent efficient in vivo regulatory ways to shift optimal conditions from one peroxidase to another and thereby favor one of the different processes connected to cell wall metabolism and preferentially catalyzed by specialized peroxidases [START_REF] De Marco | Isolation of tobacco isoperoxidases accumulated in cell-suspension culture medium and characterization of activities related to cell wall metabolism[END_REF]. Thus, peroxidase activities could be regulated during development or in response to stress factors [START_REF] Mohan | Developmental and tissue-specific expression of a tomato anionic peroxidase (tap1) gene by a minimal promoter, with wound and pathogen induction by an additional 5′-flanking region[END_REF].

Conclusion

We showed that cupric stress caused the stimulation of total peroxidase activity in 14 day-old sunflower roots. Several anionic peroxidases seemed to contribute to this increase of peroxidase activity. The A1 peroxidase was identified through de novo sequencing of 3 peptides thanks to mass spectrometry analysis. It showed high level of identity to the AtPrx12 A. thaliana and the NtPER9_6 N. tabacum peroxidases. In order to fully understand the role of peroxidases in defense mechanisms against cupric stress, our work is now in progress to purify and characterize the other peroxidases (A2-A5).

Materials and methods

Plant material

Sunflower (Helianthus annuus L. var. Dorra RM) seedlings were grown for 14 days in nutrient solution. Cu treatment was performed as previously described [START_REF] Mazhoudi | Response of antioxidant enzymes to excess copper in tomato (Lycopersicon esculentum, Mill.)[END_REF]. Roots were collected from 19 day-old plants with and without treatments with 50 µM of CuSO 4 .

Metal analysis

For copper analysis, roots were subdivided in 3 samples which were washed for 10 min with either distilled water, EDTA (100 mM, pH 8.0) or CaCl 2 (100 mM). Roots were dried and subsequently digested in 65 % nitric acid (1 mL/0.1 g of dry matter). The digested material was resuspended in distilled water. Cu content was determined using an atomic absorption spectrophotometer (Perkin Elmer 2380 Atomic Spectrometer, Waltham, MA, USA).

Peroxidase extraction and partial purification

Sunflower roots (20 g) were homogenized in a mortar at 0ºC with 50 mM phosphate buffer (KH 2 PO 4 /K 2 HPO 4 ), pH 7.0 containing 0.2 mM EDTA and 10 % (w/w) PVP.

Homogenates were centrifuged at 10 000 g for 15 min. Supernatant was used as crude extract for further purification and measurement of total peroxidase activity. Peroxidase activity was determined spectrophotometrically in the presence of H 2 O 2 using guaïacol as an electron donor [START_REF] Fielding | A biochemical and cytochemical study of peroxidase activity in roots of Pisum sativum[END_REF].

Crude extract was desalted through size-exclusion chromatography (G25 Sephadex, GE Healthcare Life Sciences, Saclay, France) using the phosphate buffer described above.

Resulting fractions were loaded on a 1 mL Fast Flow Q-Sepharose (HiTrap Q FF, GE Healthcare Life Sciences, Saclay, France) equilibrated with 100 mM Tris-HCl, pH 7.5.

Anionic peroxidases were eluted with a linear gradient of NaCl (0-0.4 M) at a flow rate of 0.5 mL/min. Protein quantification was performed using the method of Bradford [START_REF] Bradford | A rapid and sensitive method for the quantification of mg quantities of protein[END_REF] using bovine serum albumine (BSA) as a standard. Peroxidase activity was detected in eluted fractions using guaïacol as a substrate. To be monitored on a native PAGE, fractions were dialyzed against 50 mM Tris-HCl (pH 7.5) buffer. Aliquots were also monitored by SDS-PAGE.

In vitro tests of A1 activity

The enzymatic activity was measured in a total volume of 2 mL. The effect of temperature on A1 was studied after heating of buffer solution (50 mM phosphate buffer, pH 6.5; 10 mM H 2 O 2 , 9 mM guaiacol) at desired temperature for 15 min. Twenty µL (16 µg proteins) of peak 1 fractions (see Fig. 3) were added and the oxidation of guiacol was monitored by the increase of absorbance at 470 nm.

pH effect on A1 was studied in the same buffer at different pH from 3 to 9. A1 was incubated at 25 °C, then 20 µL (16 µg proteins) of peak 1 fractions (see Fig. 3) were added to each sample and their activity were measured as described previously.

Assays towards coniferyl alcohol and IAA were performed according to [START_REF] Sato | Separation and characterization of the isoenzymes of wall-bound peroxidase from cultured Zinnia cells during tracheary element differentiation[END_REF] and [START_REF] De Forchetti | Effect of dicarboxylic acids on the peroxidase-IAA oxidase isozymes of soybean callus[END_REF].

Twenty µL (16 µg proteins) of peak 1 fractions (see Fig. 3) were used for each test.

Identification of the A1 peroxidase by mass spectrometry

After SDS-PAGE, the silver-stained band of 47 kDa was cut into 3 pieces of 1x1 mm.

Gel pieces were washed in 25 mM ammonium bicarbonate, acetonitrile (ACN) 50%.

After ACN removal, gel pieces were dried at room temperature, covered with a trypsin Mass accuracy of the mono-isotopic peptide precursor and peptide fragments was set to 0.5 Da and 0.4 Da respectively. Oxidation of Met (+16) and deamidation (+1) of Asp or Gln were considered as differential modifications. Two missed trypsin cleavages were allowed. Only peptides with an identification confidence higher than 95% were considered. All MS/MS spectra corresponding to these peptides were manually checked.

Bioinformatic analysis of nucleic acid and protein sequences

The presence of signal peptides was predicted using PSORT (http://psort.ims.utokyo.ac.jp/form.html), and TargetP (http://www.cbs.dtu.dk/services/TargetP/).

Functional annotation was done using InterProScan (http://www.ebi.ac.uk/InterProScan/).

Fig. 1 :

 1 Fig. 1: Effect of cupric stress on root morphology and level of accumulation of copper in roots (A) Morphologic effect of cupric stress on 14 day-old sunflower roots treated with 50 µM of CuSO 4 during 5 days (Cu) compared to untreated roots (control). (B) Length of main root. (C) Dry mass of roots. (D) Fresh mass of roots. (E) Copper content of 14-day-old sunflower roots grown in control nutrient medium (open bars) or supplemented with 50 μM CuSO 4 for 5 days (black bars). (F) Estimation of copper content in treated sunflower roots after washing in different solutions: distilled water alone (DW), EDTA (100 mM, pH 8.0) or CaCl 2 (100 mM). The values are the means of 5 independent experiments. Vertical bars indicate standard errors.

Fig. 2 :

 2 Fig. 2: Peroxidase activity in sunflower roots in response to copper treatment. (A) Guaïacol peroxidase activity in soluble fraction extracted from control (open diamonds) and Cu-treated (black squares) roots at various times after beginning of Cu treatment. Values are the means of 5 independent experiments. Vertical bars indicate standard errors. (B) Anionic peroxidase patterns in soluble root extracts of 14 day-old sunflower seedlings grown in control nutrient medium (control) or supplemented with 50 M CuSO 4 for 24 h to 120 h (Cu). Peroxidases (A1-A5) were separated by anionic PAGE (10 % acrylamide) and their enzymatic activity was revealed with guaïacol as a substrate as described in Material and methods. Fifty µg of proteins were loaded on each lane.

Fig. 3 : 2 2. 4 .

 324 Fig. 3: Partial purification of the A1 peroxidase. (A) Anionic exchange chromatography on Fast Flow Q-Sepharose. Elution of proteins was carried out with a linear gradient of NaCl (0-0.4 M) indicated by a dotted line. Enzymatic activity was detected using guaïacol as a substrate (black continuous line). (B) Detection of peroxidases in peaks 1 and 2 after anionic PAGE (10% acrylamide) using guaïacol as a substrate. (C) Analysis of peak 1 by SDS-PAGE and staining with silver nitrate. The band of 47 kDa which was further analyzed by LC-MS/MS is indicated with an arrow.

Fig. 4 :

 4 Fig. 4: Amino acid sequences of fragments of Helianthus peroxidases comprising peptides identified by LC-MS/MS.

(

  PROMEGA, Charbonnières, France) solution at 10 ng/µL in 25 mM NH 4 HCO 3 , and incubated overnight at 38°C. The supernatant was collected. An H 2 O/ACN/HCOOH (59:40:1) solution was added onto gel slices. Extraction of peptides was performed under rotary shaking for 15 min at room temperature. Extraction step was repeated with the H 2 O/ACN/HCOOH (74:25:1) solution. Supernatants were pooled and concentrated in a vacuum centrifuge to a final volume of 5 µL. Twenty µL of an H 2 O/ACN/HCOOH (98:2:0.1) solution was added before LC-MS/MS analysis. Peptide mixture was analyzed by on-line nano-HPLC (LC Packings, Amsterdam, The Netherlands) coupled to a nanospray Q-TRAP mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA, USA) as described previously [34]. Data were searched by Mascot through Analyst 1.4 interface (Applied Biosystems/MDS Sciex, Foster City, Ca, USA) against the subset Viridiplantae of NCBI nr database and against an Expressed Sequence Tag (EST) library of the Helianthus genus downloaded on 1/07/2007 (http://www.ncbi.nlm.nih.gov/). The search parameters were as follows.

Table 2 ,

 2 Table 1 supplementary data). Several ESTs covering the N-terminus region of Helianthus peroxidases and containing 2

peptides (AQAFVIIENLR and YYVDLMNR) could be found among which: H. paradoxus CF083921, H. annuus DY926191 and H. annuus DH918041. One EST covering the C-terminus of a H. argophyllus peroxidase (CF096535) and containing 2

  AAK52084) are indicated. Peptides identified by LC-MS/MS are underlined. Note that closely related peptides are present in the A. thaliana and N. tabacum peroxidase sequences. Predicted signal peptides are in italics. Conserved Cys residues forming disulfide bridges are in bold. Active site residues are in bold underlined.

	H_ann_1 and H_ann_1 derive from H. annuus ESTs DY926191 and DH918041 respectively. H_par_1
	and H_arg_1 derive from H. paradoxus CF083921 and H. argophyllus CF096535 ESTs respectively. For
	comparison, two closely related sequences from A. thaliana (At1g71695, AtPrx12, NP_177313) and N.
	tabacum (Nt PER9_6,

Acknowledgements

The authors thank the Ministry of Higher Education and Scientific Research, Tunisia, Université Paul Sabatier-Toulouse III, and CNRS, France, for supporting this project.

LC-MS/MS analyses were performed on Plate-forme de Protéomique de la Génopole Toulouse Midi-Pyrénées. The authors wish to thank Prof. Philippe Behra (ENSIACET-INP Toulouse, France) for stimulating discussions. They are grateful to Mr Bechir Azib for skilful technical assistance.

Search for protein sequences homologous to Helianthus peroxidases was performed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/).


Andrés Fernando Cardozo Perez

Eric Manoury

Carine Julcour-Lebigue

Jean-François Blanco

Henri Delmas

Florence Gayet

Rinaldo Poli

Andrés F Cardozo

Carine Julcour

Preparation of Phosphine-Functionalized Polystyrene Stars by Metal Catalyzed Controlled Radical Copolymerization and Their Application to Hydroformylation Catalysis

published or not. The documents may come

Preparation of phosphine-functionalized polystyrene

stars by metal catalyzed controlled radical copolymerization and their application to hydroformylation catalysis

Introduction

Polymer science and the desire to fabricate advanced materials with specific functions have made a giant step forward with the development of controlled radical polymerization techniques that combine functionality tolerance with precise control of architecture, topology, composition, molecular weight, mole fraction and location of specific functional groups. [1][2][3][4][5][6] A particularly successful protocol for controlled radical polymerization is Atom Transfer Radical Polymerization (ATRP). 7,8 This is a metal-catalyzed process whereby multiple controlled insertions of monomers (M) into the halogen-capped chain end occur by reversible activation of the dormant chain (Pn-X). It involves a halogen atom transfer process to the catalyst, a complex of a transition metal Mt supported by a given coordination sphere L (Mt x /L) that changes its formal oxidation state from x to x+1 during the activation process, and deactivation by the reverse process (Scheme 1). 4,5 The position of the ATRP equilibrium is in great favour of the dormant species over the active radicals, thereby greatly reducing the incidence of bimolecular terminations. Scheme 1. Mechanism of the ATRP process.

Phosphines have so far received little attention as functional groups in the controlled radical polymerization approach, in contrast with the wide interest on phosphine functionalized polymers for catalytic applications. 9,10 For instance, rhodium catalysts anchored on polymer-grafted phosphine ligands were first described by Manassen 11 and then developed in the 70's by 40 Grubbs,12,13 Čapka 14 and Pittman 15 as hydrogenation, hydrosilylation and hydroformylation catalysts. Many other polymer-supported phosphine ligands have since been reported, mostly containing modified triphenylphosphine. 9,[16][17][18][19][20] The strategies used to obtain polymeric ligands can be divided into 45 two families, the first one involving incorporation of the phosphine function by chemical modification of a pre-existing phosphine-free polymer 16,21,22 and the second one involving the use of a phosphine-containing comonomers during the polymerization. Notably, both linear and cross-linked triphenylphosphine-50 containing polystyrenes have been obtained by copolymerization of 4-diphenylphosphinostyrene (or styryldiphenylphosphine, SDPP) and regular styrene (S) by anionic or free radical methods, possibly in the presence of p-divinylbenzene or 1,4-bis(4vinylphenoxy)-butane as a cross-linking comonomer. These 55 polymers are characterized, however, by broad molecular weight distributions and uncontrolled architectures. 23,24 Living/controlled polymerization techniques allow exerting control over these parameters but examples involving phosphine containing monomers are limited. The anionic living polymeri-60 zation of SDPP has provided homopolymers with narrow MW distribution, subsequently extended into diblock copolymer structures by polymerization of regular styrene. 25 Terashima, Sawamoto et al. have demonstrated the power of ATRP with a ruthenium catalyst to produce polymers incorporating SDPP.

65

More specifically, core-shell polymers with the phosphine functions confined in the cross-linked nanogel core were produced starting from a halogen-terminated polyacrylate macroinitiator in the presence of SDPP and a cross-linker (ethylene glycol dimethyl dimethacrylate) as comonomers. 26-28 70 These polymers have subsequently been used as macroligands to catalyze a variety of reactions. [29][30][31] In our own laboratory, we wish to develop macroligands where the phosphine functions are contained on flexible linear arms rather than in a cross-linked core, thereby being potentially more accessible to substrates and reagents in catalysis. We have recently reported that the copper-catalyzed atom transfer radical copolymerization of S and SDPP yields well controlled linear polymers when the amount of SDPP in the feed is up to 25%. [START_REF] Cardozo | ChemCatChem[END_REF] In the present contribution, we extend this approach to the synthesis of well-defined phosphine-functionalized polystyrene stars. The potential of the resulting macroligands in catalysis is shown using the hydroformylation of 1-octene as a benchmark reaction.

Experimental Section

General

All glassware was dried at 125°C overnight. The polymerization reactions were carried out under argon in round bottom Schlenk tubes connected to three-way stopcocks. All solvents (toluene, 99.7%, Aldrich; anisole, 99.0%, Fluka; dichloromethane, >99.5%, Aldrich; and THF, >99.9%, Aldrich) were dried using conventional methods and then distilled under an argon atmosphere prior to use. All the purification operations concerning the SDPP-containing polymers were equally 20 conducted under argon.

Styrene (99%) and divinylbenzene (80%) (Sigma-Aldrich) were dried on CaH2 (99%, Aldrich), then distilled under reduced pressure and kept under an inert atmosphere at -25ºC prior to use. The deuterated solvents for the NMR analyses were purchased from Eurisotop. 4-Styryldiphenylphosphine (97%, Aldrich), PPh3 (99%, Aldrich), CuBr (99.999%, Aldrich), CuBr2 (99.0%, Fluka), CuCl (99.999%, Aldrich), K2CO3 (99%, Aldrich), tris(2pyridylmethyl)amine (97%, Aldrich), AIBN (> 98%, ACROS), [Rh(acac)(CO)2] (99%, Alfa Aesar), 4-(dimethylamino)pyridine 30 (99%, Fluka), pentamethyldiethylenetriamine (99%, Aldrich), 1bromo ehylbenzene (97%, Aldrich), methyl 2-bromopropionate (99,0%, Fluka), ethyl 2-bromoisobutyrate (98%, Aldrich), triethylamine (96 %, Aldrich), 2-bromoisobutyroyl bromide (98%, Aldrich), nBu3SnH (97%, ACROS), tin bis(2-35 ethylhexanoate) (96%, Alfa Aesar), methanol (98%, Aldrich), hexane (>95%, Aldrich), 1-octene (99+%, ACROS), n-nonanal (> 97%, Alfa Aesar) and dodecane (99%, Aldrich) were used as received. Boltorn TM H30 was dried under vacuum for 24 h at room temperature and analyzed by 1 H NMR according to 40 established protocols, [START_REF] Burgath | [END_REF][34][35] confirming the specifications provided by the commercial source (Perstorp Chemicals; Mn = 3600 g/mol, NOH = 32). N,N,N',N',N",N"-Hexamethyltris(2-aminoethyl)amine (Me6TREN) was prepared according to a literature protocol. 36 The macroinitiator (CH3)2C(COOEt)-(Sx-co-SDPPy)-Br (x ~ 40, y ~ 12; Mn,SEC = 7520 g/mol; Đ = 1.38) was prepared as described in our previous contribution. [START_REF] Cardozo | ChemCatChem[END_REF] Carbon monoxide and dihydrogen were obtained from Linde Gas. Syngas was prepared by introducing equimolar amounts of CO and H2 into a monitored gas reservoir feeding the autoclave reactor at constant pressure.

Instrumentation.

The 1 H NMR spectra were recorded at 25.0 ºC on Bruker Avance 300 (5 mm TXO or QNP probes) or Bruker Avance 400 (5 mm Atma BBFO probe) spectrometers operating at 300 and 400 MHz, respectively. The chemical shifts were calculated 55 relative to the residual protonated solvent peaks as internal standards and reported in ppm with positive values upfield from SiMe4. The 31 P{ 1 H} NMR spectra were obtained at 25.0°C on a Bruker Avance 300 spectrometer operating at 121.5 MHz and the chemical shifts are given in ppm relative to the signal of a 85% 60 H3PO4 solution in D2O, which was used as external standard. The SDPP conversion during the polymerization was followed by 31 P{ 1 H} NMR using a 40 s relaxation delay to insure the complete recovery of the magnetization vector between consecutive scans and the relative areas of the free and 65 polymerized SDPP resonances were quantified by deconvolution of the signal with help of the MestRe-C program using Lorentzian lineshapes. The amount of phosphorus in the purified polymer was measured by integration of the 31 P{ 1 H} NMR resonance using a 50 s relaxation delay between pulses, against a 70 known amount of Ph2EtP=O as internal standard.

The size exclusion chromatographic (SEC) analyses were carried out at 35°C on an instrument equipped with a 5 m gel PL pre-column (50 x 7.5 mm) and a 5 m mixed gel PL-D separation column (300 x 7,5 mm) from Polymer Laboratories, using THF 75 (1 mL/min) pre-filtered through 200 nm membranes as mobile phase. The samples were prepared by dissolution in THF at concentrations of 4 mg/mL, then immersed in an ultrasound bath for 1-2 min, followed by standing for at least 4 h for the linear polymers (24 h for the star polymers) and filtration through 200 80 nm Teflon filters. Molar masses were measured with a multiangle light scattering detector (MALLS ; minidawn Tristar Wyatt Technology Corporation), coupled to a differential refractometer (RI2000 Sopares). An operating protocol of total recovery of the injected mass was employed for the variable composition 85 copolymers, allowing the simultaneous determination of the dn/dc parameter. This method gave a dn/dc value in agreement with the literature (0.184) for the PS homopolymer. [START_REF]Polymer Handbook[END_REF] The SEC traces of the star polymers prepared by the convergent route were deconvoluted as the sum of two Gaussian functions.

90

The gas chromatographic analyses were carried out on a Thermo Fisher Trace GC 2000 chromatograph equipped with a CB-CP WAX 52 capillary column (25 m x 0,25 mm; 0.2 μm film thickness) and a flame ionization detector, using helium as carrier gas.

95 Dynamic light scattering measurements were recorded at 25°C on a Malvern Instruments Nano-ZS Zetasizer for the cross-linked polymers using toluene solutions at concentrations of ca. 4 mg/mL. The dissolution was accomplished with help of an ultrasound bath for 1 min, followed by standing for 24 h and 100 filtration through 450 nm filters. The hydrodynamic radius was obtained from the measured diffusion coefficient through the Stokes-Einstein equation.

Convergent preparation of poly(S-co-SDPP) stars. (a) ATRP conditions 105

In a Schlenk tube equipped with a magnetic stirrer bar was prepared a solution of CuBr (22 mg, 0.15 mmol), CuBr2 (4 mg, 0.02 mmol) and PMDETA (40 L, 0.17 mmol) in toluene (5 mL). This was then mixed with a separate solution containing the (CH3)2C(COOEt)-(S40-co-SDPP12)-Br macroinitiator (0.574 g, 110 0.076 mmol) and DVB (0.3 mL, 1.7 mmol) in 4 mL of toluene. The mixture was then immersed into an oil bath thermostated at 90°C for 60 h, then cooled to room temperature, diluted with 50mL of toluene and filtered through an activated neutral alumina column. The polymer was precipitated by addition of cold hexane (400 mL) and the mother liquor was removed by filter-cannula. The polymer was dried under vacuum until complete removal of the residual solvent (ca. 2 days) to yield a white powder (0.47 g, 64% yield). Mn,SEC = 130600 g/mol (dn/dc = 0.117), Đ = 1.52.

Cross-linking yield = 41%.

Additional experiments carried out with a DVB/styrene mixture, or using CuCl in place of CuBr, or other macroinitiators having shorter or longer chains yielded lower cross-linking yields. Experiments carried out with EGDMA as cross-linker yielded a macrogel.

(b) ARGET-ATRP conditions

In a Schlenk tube equipped with a magnetic stirrer bar was prepared a solution of CuBr2 (0.5 mg, 0,002 mmol), TPMA (6 mg, 0.02 mmol) and DVB (210 L, 1.2 mmol) in toluene (2 mL).

This was then mixed with a separate solution containing the (CH3)2C(COOEt)-(S40-co-SDPP12)-Br macroinitiator (0.625 g, 0.08 mmol) in 3 mL of toluene. A solution of tin bis(2ethylhexanoate) (11 L, 14 mg, 0.032 mmol) in toluene (1 mL) was prepared separately. A 70 L sample of this solution was 20 added to the mixture before immersion into an oil bath thermostated at 90°C. Additional aliquots of the tin reagent solution were added at later times (120 L after 1 h, 310 L after 2h30' and 500 L after 5 h). After a total of 64 h, the reaction was stopped by cooling to room temperature and the polymer was recovered and dried by the same procedure described above in part (a) to yield a white powder (0.205 g, 25% yield). Mn,SEC = 1.04•10 6 g/mol (dn/dc = 0.028), Đ = 1;32. Cross-linking yield = 81%.

Divergent preparation of poly(S-co-SDPP) stars. (a) Preparation of the H30Br macroinitiator

This procedure follows a protocol that was previously described for a closely related multifunctional microgel (Boltorn TM H40). [START_REF] Kreutzer | [END_REF] In a Schlenk tube was prepared a solution of Boltorn TM H30 (2.25 g, 0.625 mmol) in THF (70 mL). After stirring overnight to insure complete dissolution, a solution of 4-(dimethylamino)pyridine (4.85 g, 39.3 mmol) and Et3N (3.52 mL, 25.0 mmol) in THF (20 mL) was added. The reaction vessel was equipped with a dropping funnel and placed in an ice bath. A solution of 2-bromoisobutyroyl bromide (~10 mL, 75.0 mmol) in 40 10 mL of THF was placed in the dropping funnel and then added dropwise over 45 min to the Boltorn TM H30 solution. After the end of the addition, the reaction mixture was stirred for 2 h in the ice bath and then for additional 96 h at room temperature. The precipitated salts were filtered off and the solvent was partially evaporated to ca. 20 mL under reduced pressure. The functionalized Boltorn TM product (H30Br) was precipitated by adding the mixture to 150 mL of cold methanol. The viscous coagulated product was washed three times with methanol, letting the mixture settle overnight each time. The viscous material was mental Figure S1).

(b) Preparation of the poly(S-co-SDPP) stars from the H30Br core

Only the preparation of the B-3 product will be described in detail. In a Schlenk tube, CuBr (258 mg, 1.76 mmol), (56 mg, 60 0.25 mmol), TPMA (597 mg, 2.01 mmol) and anisole (4 mL) were added to styrene (24 mL, 201.5 mmol). After stirring for 5 min at room temperature, a solution containing the H30Br macroinitiator (527 mg, 0.063 mmol) and SDPP (11.62 g, 40.3 mmol) in toluene (33 mL) was added to reactor through a Teflon 65 cannula. The reactor was then immersed into a preheated thermostatic oil bath at 80°C. Samples were periodically withdrawn with an argon purged syringe, diluted with toluene and filtered through an activated neutral alumina column. Three separate fractions of each sample were used respectively for the 70 gas chromatographic determination of the styrene conversion, for the 31 P NMR analysis of the SDPP conversion and for the polymer analysis by SEC. The reaction was stopped after 29.5 h and the mixture was diluted with 15 mL of toluene and filtered through an activated neutral alumina column. The polymer was 75 precipitated in 400 mL of cold hexane and separated from the mother liquor by filtration through a teflon filter cannula, then dried under vacuum (ca. 3 days) to yield a white powder (1.25 g, 14% yield). Mn,SEC = 490600 g/mol (dn/dc =0.059), Đ = 1.13.

The same reaction was repeated to produce the following 80 polymers (the reaction conditions and the amount of reagents were the same unless otherwise stated): B-1. Reaction time = 25.5 h; 1.7 g (15 % yield). Mn,SEC = 387200 g/mol (dn/dc = 0,067), Đ = 1,10. DH DLS = 94.2 nm in toluene. 85 B-2. Reaction time = 24.5 h, CuBr (86 mg, 0.59 mmol), CuBr2 (19 mg, 0.08 mmol), SDPP (1.937 g, 6.73 mmol), TPMA (199 mg, 0.67 mmol), styrene (8.0 mL, 67.3 mmol), anisole (1.0 mL), H30Br (176 mg, 0.021 mmol) in 11 mL of toluene. 5.7 g (17% yield). Mn = 235800 g/mol (dn/dc = 0.081), Đ = 1.43.

90

Dehalogenation of polymer B-3

In a Schlenk tube equipped with a magnetic stirrer bar was prepared a solution of B-3 (600 mg) and nBu3SnH (0.11 mL, 0.39 mmol) in 15 mL of toluene. After homogeneization, AIBN (1 mg, 0.007 mmol) was added and the reaction vessel was immersed 95 into a preheated thermostatic oil bath at 60°C. After 26 h of reaction and cooling, the polymer was recovered by precipitation in 300 mL of cold hexane. The polymer (556 mg) was thus recovered after filtration and vacuum drying at 50°C for 24 h. Mn,SEC = 465800 g/mol (dn/dc = 0.083), Ð = 1.35, DH DLS = 21 nm 100 in toluene.

1-octene hydroformylation

The Rh(CO)2(acac) precatalyst (53.2 mg, 0.2 mmol) and the desired quantity of ligand (PPh3 or functionalized polymer) were introduced into a Schlenk tube. Then, a separate mixture 105 containing 1-octene (16 mL, 102 mmol) dodecane (inert reference, 2 mL) and toluene (84 mL) was added through a teflon cannula and the resulting mixture was stirred for 20 min. It was then transferred into a Hastelloy C276 autoclave equipped with a gas inducing stirrer. A first sample was withdrawn, then the 110 system was purged three times with 15 bar of dinitrogen, then four times with 15 bar of syngas. The reactor was subsequently heated under low syngas pressure (2 bar) and slow stirring speed (140 rpm, well under self gas induction) to hinder significant gas dissolution and the start of the reaction. When the desired

4 | Journal Name, [year],
[vol], 00-00 reaction temperature (90°C) was achieved, stirring was stopped and the autoclave was pressurized and constantly fed with syngas at the desired pressure (20 bar). A sample was withdrawn to evaluate the amount of products formed during the heating procedure. Then, the data acquisition was started and the stirring speed was set to 1200 rpm. Both temperature and pressure of the reactor and the gas ballast were recorded on-line on a computer at a rate of 1 Hz. The instantaneous reaction rate was measured from the syngas consumption. Samples were withdrawn periodically for the kinetic monitoring by GC/FID (after 10, 20, 40 and 60 min for PPh3, every 30 min until 2 h and then every hour for the macroligands). For the gas-chromatographic analyses, a precise quantity of anisole (internal standard) was added before dilution with diethyl ether and injection into the GC/FID. The identification of the reaction components (1-octene, n-nonanal, 2-methyloctanal) was confirmed by GC/MS analysis.

Results and Discussion

Multiarm star polymers may be accessed by two complementary, convergent (or "arm first") and divergent (or "core first") approaches. In addition to using well defined multifunctional molecular systems as terminating agents (for the arm first approach) or initiators (for the core first approach), it is also possible to use diolefins as cross-linkers yielding microgel cores, with the advantage of simpler operating procedures and a more moderate cost but with the inconvenience of yielding polymers with a variable number of arms. 39

(a) Convergent polymer synthesis

Since we have recently synthesized linear P(S-co-SDPP)-Br polymers by ATRP, [START_REF] Cardozo | ChemCatChem[END_REF] we first attempted the synthesis of star polymers from these macroinitiators by the convergent approach and a diolefin cross-linker. Cross-linking indeed took place when using 1,4-divinylbenzene (DVB) to form well defined stars (Scheme 2) as shown by the polymer SEC analysis, but SEC also revealed that a large fraction of the linear macroinitiators was not incorporated into the star product. This phenomenon is well known in the convergent synthesis of star polymers 39 and is not exclusively caused by the loss of bromide functionality from the linear macroinitiators: the degree of arm chain end functionality revealed by quantitative NMR analysis (>90%) [START_REF] Cardozo | ChemCatChem[END_REF] is indeed much larger than the cross-linking yield, at best 41% (see Experimental Section and Supplemental Figure S1). A number of attempts to optimize the cross-linking yield (use of a DVB-styrene mixture, or EGDMA as cross-linker) did not give better results. A final attempt involved the use of ARGET (Activator ReGenerated by Electron Transfer) 40 ATRP conditions to accomplish the crosslinking reaction with DVB, since recent reports have demonstrated the advantage of this approach for the convergent synthesis of star polymers. 41 Indeed, the cross-linking yield was significantly improved, but it still remained unacceptably low (19% of free arms remaining, see the SEC trace in Figure S2).

Therefore, the convergent synthetic strategy was abandoned and we turned to the divergent synthesis from a preformed microgel core.

Scheme 2 55 (b) Divergent polymer synthesis Among various possible multifunctional initiators, we have focused our attention on Boltorn TM , a commercially available dendridic polyester obtained by polycondensation of 2,2bis(hydroxymethyl)propionic acid on a central polyol core that 60 offers a large number of hydroxo functionalities. For our purposes we have selected Boltorn TM H30 that contains 32 such functional units on average. Therefore, its use as macroinitiator leads to stars with an average of 32 arms. Before divergent arm growth, Boltorn TM H30 was converted to a suitable macroinitiator 65 for ATRP (henceforth abbreviated as H30Br) by reaction with 2bromoisobutyroyl bromide, see Scheme 3. The same transformation was previously described for Boltorn TM H40. [START_REF] Kreutzer | [END_REF] The completeness of the transformation was confirmed by the 1 H NMR analysis (Figure S4). The 2-bromoisobutyrate function 70 introduced on H30Br is a suitable initiator for the ATRP of a variety of monomers, including styrenics. It has been used to functionalize a number of substrates (silicon wafers, cellulose nanocrystals, PEO, …) for the initiation of styrene polymerization. [42][43][44] The macroinitiator H30Br was then used to 75 grow the copolymer arms as shown in Scheme 3.

Scheme 3

We selected the CuBr/TPMA couple in toluene at 80°C as the catalytic system and operating conditions for the subsequent star 80 growth (conditions that were optimized in preliminary experiments on the corresponding star PS homopolymers), which resulted in very slow but well controlled polymerization (the results are collected in Table 1). It is important to point out that, as shown by previously reported controlled experiments, the 85 phosphine groups do not attack the benzylic bromide chain ends under the polymerization conditions. [START_REF] Cardozo | ChemCatChem[END_REF] Three polymers were synthesized, two with a feed molar SDPP fraction fSDPP = 0.17 and one with fSDPP = 0.08. A low SDPP/S fraction in the feed was selected, because our previous work on the linear P(S-co-SDPP)-Br polymers has shown that good control is achieved only for low fSDPP values (≤ 25%). Given the very slow polymerization and in order to obtain stars of relatively small size, the polymerizations were stopped at low conversion. Use of a more active catalyst such as CuBr/Me6TREN or CuBr/PMDETA resulted in gelification even when operating under very dilute conditions. It is worth mentioning that for the synthesis of the linear P(S-co-SDPP)-Br copolymers, although the CuBr/TPMA could insure slower reactions and a better control, the CuBr/Me6TREN system was also able to yield products with well controlled molecular weights and low dispersities. The polymerization leading to B-2 was monitored in terms of kinetics and mass evolution with conversion. The results are shown in Figure 1. The evolution of the monomer conversion with time in the semilogarithmic plot of part (a) shows a relatively good fit to the first order law for both monomers. The 40 faster incorporation of SDPP is in line with the known reactivity ratios for the free radical copolymerization of these two monomers, (rSDPP = 1.43, rS = 0.52). 23 Since the reactions were stopped at relatively low conversions, the feed composition drift is limited and therefore the arms should not have a marked 45 compositional gradient. The molecular weight grows linearly with conversion, and the dispersity remains relatively constant around 1.4. These phenomena are characteristic of a controlled system. The SEC traces of these polymers show the uniform progression of the monomodal molecular weight distribution, see In addition to the SEC characterization, polymer B-1 has also been analyzed by dynamic light scattering, confirming the monomodal distribution and narrow size dispersion (see Figure S5), with an average hydrodynamic diameter of 22 nm. The 1 H 55 NMR spectrum of the B-1 copolymer, see Figure 2, clearly shows the aliphatic skeleton and aromatic substituent signals, and also the benzylic CHBr chain end resonance at the characteristic position (δ 4.5). The small and broad signal in the region between δ 3 and 4.5 is assigned to the protons of the central Boltorn TM

ARTICLE TYPE

[journal], [year], [vol], 00-00 | 6 [TPMA] = 0.032 M, CuBr/CuBr2/TPMA = 0.88:0.12:1; solvent = toluene ; T = 80 °C. b Average number of S (for x) and SDPP (for y) units in each arm (Scheme 3), calculated on the basis of the monomer conversions. c Not available.

In order to establish whether the residual benzylic bromide chain end functionalities interfere with the catalytic application of the macroligands, it was necessary to prepare a dehalogenated version. This was accomplished for polymer B-3, following an established protocol, 45 by reaction with tributyl tin hydride, as shown in Scheme 3, to give polymer B-3-H. The complete disappearance of the 1 H NMR resonance of the terminal CHBr functions at δ 4.5 (Figure 3) confirms the total elimination of the benzylic bromide chain ends, whereas the 31 P NMR spectrum indicates that the polymer-bonded -PPh2 units have been preserved intact (resonance at δ -6.2). The SEC analysis shows that a monomodal molecular weight distribution (Figure S7) with parameters close to those of the precursor B-3 polymer (Mn = 4.66•10 5 , Đ = 1.35).

(c) Hydroformylation of 1-octene

The polymers B-1, B-2, B-3 and B-3-H were then used as macroligands for Rh(acac)(CO)2 in the catalytic hydroformylation of 1-octene under homogeneous conditions, following the same protocol recently used by us for the same process with the corresponding linear polymers, P(S-co-SDPP)-Br, [START_REF] Cardozo | ChemCatChem[END_REF] and earlier for the catalyst based on PPh3. 46 The catalytic results are shown in Table 2. From previous work, it is known that the maximum linear/branched (or l/b) ratio is achieved with P/Rh ratio of 8 or higher, 46 whereas the catalytic activity is reduced in the presence of excess phosphine ligand. Indeed, the comparison of the results obtained with a P/Rh ratio of 4 and 8, carried out for the macroligand B-1 (runs 3 and 4) confirms these trends. The comparison with the hydroformylation results in the presence of 35 PPh3 (run 1) or linear copolymer CP-5 (run 2), [START_REF] Cardozo | ChemCatChem[END_REF] shows that increasing ligand complexity (on going from PPh3 to the linear copolymer to the star copolymer) the catalytic activity decreases whereas the l/b ratio increases reaching the maximum value of 5.6 for the copolymer B-1 (run 4). Copolymer B-2, containing a 40 lower density of phosphine functionalities, yields a lower l/b ratio than copolymer B-1 at similar conversions under identical operating conditions (cf. runs 4 and 5). A decrease of reaction rate for a polymer supported catalyst is generally explained by a reduced accessibility of the catalytic 50 sites to the olefin, a phenomenon that should be accentuated in bad solvents where the polymer prefers a less expanded conformation and the functionalities are therefore less accessible to the solvent and solute molecules. 47 The reduced mobility and/or accessibility of the phosphine functions in the more 55 complex star architecture is probably a key factor in the hydroformylation kinetics. The toluene solvent used in these catalytic experiments is actually a relatively good solvent for the PS-type polymer, but the Rh atom may act as intra-chain or interchain cross-linker, leading to an additional diffusional limitation. 60 This phenomenon should have a greater effect in the star polymers because of the possible inter-arm cross-linking.

From the point of view of selectivity, an improvement of the l/b ratio for polymer supported phosphine is well known in the literature. [START_REF] Pittman | [END_REF][49][50][51][52] For instance, the l/b ratio for the hydroformylation 65 of propene at 85°C approximately doubles from 3 to 6 on going from PPh3 to polypropylene-supported phosphine. 50 The major effect has been observed, as detailed in our previous contribution, [START_REF] Cardozo | ChemCatChem[END_REF] on going from PPh3 (run 1) to the linear copolymer (run 2). There is, however, a minor but definite further increase for the l/b ratio upon going from the linear to the star polymer when comparing the results obtained with the same P/Rh ratio (cf. run 2 and runs 4 and 5). The polymer B-2 (run 5), containing ca. half the density of -PPh2 functionalities relative to B-1, gives a somewhat lower l/b ratio. In our previous study on the linear polymers, longer chains were shown to increase the l/b ratio for equivalent phosphine density. [START_REF] Cardozo | ChemCatChem[END_REF] For the present star polymers, on the other hand, the l/b ratio decreases when going from the lower MW polymer B-1 to the higher MW polymer B-3 (cf. runs 3 and 6).

We have also carried out a catalytic test with the debrominated macroligand B-3-H (run 7) under the same conditions as run 6, namely with a P/Rh ratio of 4. A comparison of the two runs shows that the catalysis is faster with B-3-H than with B-3, whereas the selectivity is identical for the two runs. This observation is at variance with the behavior of the corresponding linear polymers, where the presence of the Br chain end was found to be totally neutral in terms of both rate and selectivity. [START_REF] Cardozo | ChemCatChem[END_REF] Hence, the terminal C-Br functionalities do not appear to chemically inhibit the catalysis. A possible interpretation of this 20 difference is based on the different average conformation for the two polymeric architectures, resulting in a steric hindrance of the 32 Br atoms in the star polymer toward access of the olefin substrate to the catalytic sites, whereas the microstructure of the sites, which control the stereoselecivity, would not be affected.

The linear polymers will tend to organize as random coils that will be essentially independent (no entanglements) in dilute solution in a good solvent, as is the case here (1 M solution in toluene). Under these conditions, the presence of only one Br atom at the chain end will not significantly perturb the substrate 30 access to the catalytic centers distributed in the center of the coil. For the star polymers, on the other hand, each of the 32 arms will propel radially away from the core and therefore place the Br atoms on or near the polymeric particle surface. Incidentally, this different macromolecular organization of linear and star polymers may also be responsible for the different behavior with respect to the trend of the l/b ratio with the molecular weight (chain length for the linear polymers or arm length for the star polymers).

Thus, several factors are at play (density of phosphine functionalities, polymer size, architecture, P/Rh ratio). 40 Understanding how each one of them independently acts on the l/b selectivity requires more extensive investigations that are outside the scope of the present preliminary application of our star polymers to catalysis. The important point, however, is that controlled radical polymerization and specifically ATRP under the conditions outlined in the present contribution has the possibility to access polymeric materials where each of these parameters can be individually changed by design. Thus, tailored polymeric macroligands with an optimized structure for each specific application may be developed.

Conclusions

In this paper, we have taken the atom transfer radical copolymerization of styrene and styryldiphenylphosphine one step further, going from linear copolymers to star polymers with controlled size and phosphine density. The convergent method 55 consisting of cross-linking pre-made chain-end functionalized macroinitiators with a diolefin (divinylbenzene) did not lead to a satisfactory cross-linking yield, although star polymer with controlled size could be obtained. The divergent method starting from a multifunctional initiator, in the example shown here a 60 hyperbranched polyester of the Boltorn TM family with ca. 32 external OH groups, leads to well defined stars with small molecular weight distributions. Their preliminary application to a catalytic process of industrial interest, the hydroformylation of higher olefins, exemplified in the present case by the model 1-65 octene substrate, shows the expected performance for a polymersupported phosphine and also demonstrates, in comparison with the previously reported performance of the linear polymers, [START_REF] Cardozo | ChemCatChem[END_REF] that the polymer architecture has an effect on the catalytic performance in addition to polymer size, phosphine density, and

Acknowledgement

We are grateful to the Agence Nationale de la Recherche (programme BLANC, project BIPHASNANOCAT, Grant No. ANR-11-BS07-025-01), to the Centre National de la Recherche 80 Scientifique, and to the Institut Universitaire de France, for financial support. AFC thanks the PRES Toulouse for a Ph.D. fellowship. We thank Professor Sergio Castillon (Université Rovira i Virgili, Tarragone) for lending us a generous amount of Boltorn TM H30.

Figure 1 .

 1 Figure 1. First-order kinetic plot of the monomer conversion (a) and evolution with conversion of Đ (b) and Mn,SEC (c) for the star copolymer B-2.Polymers B-1 and B-3 were obtained under the same operating 20

35

 35

50

 50

Figure S6.

  Figure S6.In addition to the SEC characterization, polymer B-1 has also been analyzed by dynamic light scattering, confirming the monomodal distribution and narrow size dispersion (see FigureS5), with an average hydrodynamic diameter of 22 nm. The 1 H

  Figure 2. 1 H NMR spectrum of polymer B-3 in CD2Cl2. The sharp starred resonance (δ 1.56) is due to the solvent water impurity.

Figure 3

 3 Figure 3. 1 H NMR spectrum of polymer B-3-H in CDCl3. The starred resonances is due to the solvent water impurity.

Table 2 .a

 2 Homogeneous hydroformylation of 1-octene in toluene solution. a Run Ligand P/Rh t/h Residual octene/% b l/b Initial rate, r0•10 4 (mol l -1 s -1 Conditions: [Rh(acac)(CO)2] = 2.0•10 -3 M, [1-octene] = 1.0 M, T = 90 45 ºC, Psyngas = 20 bar (CO/H2 = 1). b From GC/FID analysis. c From syngas consumption. d CP-5 = (CH3)2C(COOEt)-(SDPPx-s-Sy)-Br with x = 103 and y = 26; data from ref. 32. e Not available.

70P/

  Rh ratio. The present results open several new perspectives in terms of polymer structure optimization, application to other catalytic reactions, catalyst recovery and recycling by ultrafiltration, and development of other polymers with different grafted ligands for an even greater number of catalytic 75 applications.

85

  Notes and referencesa CNRS, LCC (Laboratoire de Chimie de Coordination), 205 route de Narbonne, BP 44099, F-31077 Toulouse Cedex 4, France CNRS. Fax: +33-561553003; Tel: +33-561333173; E-mail: rinaldo.poli@lcctoulouse.fr.

Table 1 .

 1 H30-[P(S-co-SDPP)-Br]32 stars synthesized by ATRP. a

	Product S/SDPP/Br fSDPP t/h	S conv/% SDPP conv/%	Mn,SEC/g•mol -1	Mth/g•mol -1	Đ	dn/dc	x b	y b
	B-1	100:20:1	0.17 25.5	17	21	3.87•10 5	1.04•10 5	1.10	0.067	17.0	4.2
	B-2	100:10:1	0.08 24.5	13	32	2.36•10 5	0.81•10 5	1.43	0.081	13.0	3.2
	B-3	100:20:1	0.17 29.5	-c	23	4.91•10 5	-	1.13	0.059
	a Conditions: [H30Br] = 0.01 M;

  90 b CNRS, LGC (Laboratoire de Génie Chimique), 4 Allée Emile Monso BP 84234, 31030 Toulouse Cedex 4, France c Université de Toulouse, UPS, INPT, F-31077 Toulouse Cedex 4, France d Institut Universitaire de France, 103, bd Saint-Michel, 75005 Paris, France. 95 † Electronic Supplementary Information (ESI) available: GPC characterization of some of the isolated star polymers. See DOI: 10.1039/b000000x/ 1. K. Matyjaszewski, Y. Gnanou and L. Leibler, Macromolecular Engineering: Precise Synthesis, Materials Properties, Applications, 100 Wiley-VCH Verlag GmbH, 2007. 2. C. J. Hawker, A. W. Bosman and E. Harth, Chem. Rev., 2001, 101, 3661-3688. 3. W. A. Braunecker and K. Matyjaszewski, Progr. Polym. Sci., 2007, 32, 93-146.

	105

| Journal Name, [year], [vol], 00-00

dried under vacuum at 35°C for 3 days, yielding the product in the form of an expanded foam, which turned into a viscous resin after a few days (1.85 g, 35% yield). Mn,SEC = 17800 g/mol; dn/dc = 0.068 ; Đ = 1.46. The 1 H NMR of the product confirmed the complete esterification of the Boltorn TM OH groups (see supple-

Journal Name, [year], [vol], 00-00 | 7


Marc Vandeputte

Mathieu Besson

François Allal

Florence Phocas

Edwige Quillet

Selective breeding to reduce the ecological footprint of aquaculture

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SELECTIVE BREEDING TO REDUCE THE ECOLOGICAL FOOTPRINT OF AQUACULTURE

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