Benjamin Marie
email: bmarie@mnhn.fr

Jaison Arivalagan

Lucrèce Mathéron

Gérard Bolbach

Sophie Berland

Arul Marie

Frédéric Marin

Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa

Keywords: biomaterials, biochemistry, evolution biomineralization, shell nacre, proteomics, bivalve, organic matrix proteins, calcium carbonate

The formation of the molluscan shell nacre is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins, glycoproteins and polysaccharides that is assembled and occluded within the mineral phase during the calcification process. The fine molecular basis that regulates nacre formation remains to be better characterized.

Noticeable advances in expressed tag sequencing of the freshwater mussels, such as Elliptio complanata and Villosa lienosa, provide a pre-requisite to further characterize the bivalve nacre proteins by a proteomic approach. In this study, we have identified a total of 48 different proteins from the nacre insoluble matrices, 31 of which are common of the two species. Few of these proteins, such as PIF, MSI60, CA, shematrin-like, Kunitz-like, LamG, chitin-binding containing proteins, together with A-, D-, G-, M-and Q-rich proteins, appear to be analogues, if not true homologs, of proteins previously described from the pearl oyster or the edible mussel nacre matrices, thus constituting a remarkable list of deeply-conserved nacre protein set. This work constitutes a comprehensive nacre proteomic study of nonpteriomorphid bivalves that concretely gives us the opportunity to describe the molecular basis of deeply conserved biomineralization toolkit among nacreous shell-bearing bivalves, with regard to proteins associated to other shell microstructures, to that of other mollusc classes (gastropods, cephalopods) and finally, to other lophotrochozoans (brachiopods).

Introduction (a) Mollusc shell biomineralization

The molluscan shell is a unique model for biomineralization and constitutes a remarkable example of a natural composite biomaterial synthesized at ambient temperature from polar to tropical environment. It is composed of one or two polymorphs of calcium carbonate (i.e. calcite and aragonite), generally assembled in superimposed CaCO3 layers of different microstructures [START_REF] Taylor | The shell structure and mineralogy of the Bivalvia, Part II, Lucinacea-Clavagellacea[END_REF]. These different shell layer textures are produced under the control of specific extracellular matrices that are secreted by different zones of the external mantle epithelium [START_REF] Marie | Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell[END_REF]. These 'cell-free' matrices, mainly constituted of proteins, glycoproteins and polysaccharides, surround nascent CaCO3 crystals and are incorporated into the shell crystalline architecture in growth. Although the organic matrix is quantitatively a minor component of the shell (0.1 to 5 wt-%), it is thought to play a central role in the formation and shaping of these biomineral structures [START_REF] Lowenstam | On Biomineralization[END_REF][START_REF] Suzuki | Mollusk shell structures and their formation mechanism[END_REF]. Indeed, these matrix components have been shown to be involved in the organization of a spatially prearranged scaffold, the nucleation, the orientation and growth of calcium carbonate crystals and the inhibition of their growth [START_REF] Mann | Biomineralization: Principles and Concepts[END_REF].

It has also been demonstrated that the nacre matrix controls the calcium carbonate polymorph [START_REF] Falini | Control of aragonite and calcite polymorphism by mollusk shell macromolecules[END_REF] and that it exhibits cell-signalling properties [START_REF] Berland | Nacre/bone interface changes in durable nacre endosseous implants in sheep[END_REF][START_REF] Marin | Molluscan shell proteins: primary structure, origin, and evolution[END_REF][START_REF] Marin | The formation and mineralization of mollusc shell[END_REF].

(b) Nacre

Because of its extraordinary toughness, commercial value in jewelry and remarkable biocompatibility properties, nacre, so called the mother of pearl, is by far the most studied molluscan shell microstructure. Moreover, many authors consider it as the reference model for understanding how molluscs control the fine deposition of calcium carbonate layers [START_REF] Addadi | Mollusk shell formation: a source of new concepts for understanding biomineralization processes[END_REF].

From a structural viewpoint, nacre is constituted by a regular superimposition of polygonal flat aragonitic tablets of about 0.5 μm thickness, embedded in a peripheral thin organic matrix [START_REF] Checa | Geometrical and crystallographic constrains determine the self-organisation of the shell microstructures in Unionidae (Bivalvia: Mollusca)[END_REF]. Nacre constitutes also a fascinating object for the investigation of evolutionary processes, as it appeared early in the fossil record, during the Cambrian times [START_REF] Kouchinsky | Shell microstructures in Early Cambrian molluscs[END_REF][START_REF] Vendrasco | New data on molluscs and their shell microstructures from the Middle Cambrian Gowers Formation, Australia[END_REF], and kept almost unchanged until now. At present it is distributed in four mollusc classes, comprising cephalopods, gastropods, monoplacophorans and bivalves. In this latter group, nacre is widespread in paleotaxodont, pteriomorphid, paleoheterodont and anomalodesmata clades [START_REF] Carter | Skeletal Biomineralization: Patterns, Processes and Evolutionary Trends[END_REF]. One key question consists in determining if similar shell nacre textures, present in these different taxa, are produced by homologous sets of protein assemblages, or not. Such information would provide us a better appreciation on the recruitment of nacre matrix proteins in the Cambrian, on the evolutionary constraints exerted on these proteins, and finally, on the open debate on the unique or multiple inventions of nacre among molluscs.

(c) Nacre proteins Since the initial description of the primary structure of a mollusc shell protein in 1996 [START_REF] Miyamoto | A carbonic anhydrase from the nacreous layer in oyster pearls[END_REF], numerous nacre associated-proteins have been described so far mainly from three principal models, the pearl oyster [2;16] and the edible mussel for bivalves [START_REF] Gao | Layer-by-layer proteomic analysis of Mytilus galloprovincialis shell[END_REF][START_REF] Liao | In-depth proteomic analysis of nacre, prism, and myostracum of Mytilus shell[END_REF], and the abalone for gastropods [START_REF] Marie | Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell[END_REF]. Comparison of their respective nacre biomineralization toolkits from the pearl mussel Pinctada maxima and the abalone Haliotis asinina underlined unexpectedly very few sequence similarities, suggesting either that the bivalves and gastropods acquired independently the capacity to synthesize nacre, or that these proteins present rapid evolution rate, especially in abalone, leading to functional conservation of respective nacre shell matrix protein (SMP) sets with only limited primary structure similarities [START_REF] Jackson | Parallel evolution of nacre building gene sets in molluscs[END_REF]. In bivalves, most of the published data for nacre proteins comes from the Pteriomorphia sub-class [2; [START_REF] Liu | In-depth proteomic analysis of shell proteins of Pinctada fucata[END_REF][START_REF] Gao | Layer-by-layer proteomic analysis of Mytilus galloprovincialis shell[END_REF][START_REF] Liao | In-depth proteomic analysis of nacre, prism, and myostracum of Mytilus shell[END_REF]; only a limited amount of molecular data has been published so far on the other nacre-bearing bivalve groups [START_REF] Marie | Proteomic analysis of the acid-soluble nacre matrix of the bivalve Unio pictorum: detection of novel carbonic anhydrase and putative protease inhibitor proteins[END_REF][START_REF] Ramos-Silva | Novel molluscan biomineralization proteins retrieved from proteomics: a case study with Upsalin[END_REF][START_REF] Berland | Proteomic and profile analysis of the proteins laced with aragonite and vaterite mussel Hyriopsis cumingii shell biominerals[END_REF]. To complete this patchy picture, we have developed a proteomic investigation on two nacreous bivalves that belong to the Palaeoheterodonta class, and to the Unionoida order, Elliptio complanata and Villosa lienosa [START_REF] Marie | Unveiling the evolution of bivalve nacre proteins by shell proteomics of Unionoids[END_REF]. Our molecular investigations are supported by the newly available large transcriptomic databases constructed in parallel from the two studied species [START_REF] Wang | Rapid development of molecular resources for a freshwater mussel, Villosa lienosa (Bivalvia: Unionidae), using an RNA-seq-based approach[END_REF][START_REF] Cornman | Transcriptomic analysis of the mussel Elliptio complanata identifies candidate stress-response genes and an abundance of novel or noncoding transcripts[END_REF], and are then providing strong support for the identification of SMP set of unionoid nacre. The present investigation gives us the opportunity to determine if bivalve nacres are made by molecular events that are controlled and conserved by the evolution processes, since the Phanerozoic times.

Our high-throughput high-output shotgun approach has allowed us to identify 48 different proteins from the nacre insoluble matrices, from which 31 are common to the two models.

While recent data suggest a rapid evolution of shell matrix proteins leading to their extreme diversity [START_REF] Jackson | Parallel evolution of nacre building gene sets in molluscs[END_REF], our work emphasizes on the contrary a deeply conserved biomineralization toolkit within nacre-bearing bivalves.

Results and Discussion

(a) Protein composition of unionoid nacre matrices In order to investigate the nacre protein set in both Elliptio complanata and Villosa lienosa, two closely related freshwater bivalves (Unionoida; Unionidae), the non-fractionated insoluble matrices derived from respective shell nacre powder were analysed using a XL Orbitrap nanoLC-MS/MS, after digestion with trypsin enzyme (Figure S1). For both samples, the peak list generated from the MS/MS spectra was directly interrogated against the unionoids EST database, comprising 195,192 nucleotide sequences using MASCOT proteomic search engine (Version 2.1). Using this approach, we were able to enumerate 48 different proteins from the two nacre matrices (Figure 1; Supplementary table S1). More than 80% of these proteins (40 on the 48 identified in total) have been identified with high confidence, because they are present in both species or are identified by more than one peptide. Interestingly, 31 of the identified proteins have been detected in both E. complanata and V. lienosa testifying of the similarities of the nacre matrix compositions of these two species. We noticed that all conceptually-translated expressed sequence tag (EST) sequences that match to the MS/MS peptides present a signal peptide, when sequence exhibits complete N-terminus, suggesting that these proteins are all secreted by the mantle epithelia through classical cellular secretion pathway. This list of nacre proteins represents among the largest of nacre SMP sets described so far, comparable to published SMP set from Pinctada and Mytilus models, which count 35 and 42 proteins, respectively [START_REF] Liu | In-depth proteomic analysis of shell proteins of Pinctada fucata[END_REF][START_REF] Gao | Layer-by-layer proteomic analysis of Mytilus galloprovincialis shell[END_REF][START_REF] Liao | In-depth proteomic analysis of nacre, prism, and myostracum of Mytilus shell[END_REF]. While the present results confirm the presence of previously described protein domains such as Pif, CA, Kunitz-like, WAP, M-rich, Q-rich, A-rich, or chitin-binding domains [21;23-24], in unionoid shell nacre matrix, they also contribute to consistently extend the list of proteins associated to nacre microstructure.

The proteomic analysis of these two unionoid nacre matrices reveals a diversity of SMP structures that can be broadly categorized into 5 main protein groups: ECM (ExtraCellular Matrix), RLCD-containing (Repetitive-Low Complexity Domain), enzyme, immunity-related, and 'uncharacterized'. Although our protein list may not be exhaustive -i.e. other minor protein matrix components remain to be characterized -we assume that it contains most of the abundant nacre SMPs: our approach indeed can identify most of the analogs/homologs of the main nacre SMP described in pteriomorphid models (Supplementary table S1). Many of these proteins, such as Pif, MSI60, CA, shematrin-like, Kunitz-like, LamG, chitin-binding containing proteins, together with A-, D-, G-, M-and Q-rich proteins, appear to be analogues, if not true homologs, of proteins previously described from the pearl oyster or the edible mussel nacre matrices, and thus, constitute a remarkable set of deeply-conserved nacre proteins. Indeed, although only genetic information on the gene synteny would reliably support an hypothesis of homology relation between two proteins/genes of two organisms, we assume because of sequence of high sequence identities and also because of the specificity of some SMP domain architecture, that some of these proteins retrieved in these unionoid organisms would be true homologs of those observed in the pteriomorphid models.

(b) Extracellular matrix-related (ECM) proteins Among the different unionoid nacre proteins that exhibits ECM protein signature (comprising collagen, fibronectin, laminin, chitin-binding and other protein-and polysaccharideinteracting domains), we identified different Pif-like isoforms, various chitin-binding containing proteins, a Ca-binding protein, a calcium-dependent (C-type) lectin, a mytilin-3 ortholog, together with previously unreported proteins for nacre such as a collagen-alpha-like, a MSP130-like, DMP-like, and a ANF_receptor (Figure 2; Supplementary table S1).

Pif-like and Pif-related proteins. The unionoid nacre matrix exhibits different forms of Piflike proteins. Pif-177 (C7G0B5) was first described in the nacre of the pearl oyster [START_REF] Suzuki | An acidic matrix protein, Pif, is a key macromolecule for nacre formation[END_REF], and homologous forms, BMSP-220 and BMSP-like, were subsequently detected in the shell matrices of the blue mussel Mytilus galloprovincialis, and of the giant limpet Lottia gigantea, respectively [START_REF] Suzuki | Identification and characterisation of a calcium carbonate-binding protein, blue mussel shell protein (BMSP), from the nacreous layer[END_REF][START_REF] Marie | The shell-forming proteome of Lottia gigantea reveals both deep conservations and lineagespecific novelties[END_REF]. We have identified here at least 2 different Pif-like isoforms, called Piflike 1 and 2, that exhibit both, from N-to C-terminus, the following succession of domains: one VWA, three CBD2, one RLCD and one LamG. This domain arrangement is characteristic of the Pif-177/BMSP-200/BMSP-like family [START_REF] Suzuki | An acidic matrix protein, Pif, is a key macromolecule for nacre formation[END_REF][START_REF] Suzuki | Identification and characterisation of a calcium carbonate-binding protein, blue mussel shell protein (BMSP), from the nacreous layer[END_REF][START_REF] Marie | The shell-forming proteome of Lottia gigantea reveals both deep conservations and lineagespecific novelties[END_REF]. Considering that the protein scores obtained for the Mascot identification of Pif-like 1 is among the highest obtained here, this supports the idea that the Pif/BSMP protein members are present in very high amount in the nacre matrix and that Pif/Pif-like proteins must certainly play a key function in mollusc shell formation [START_REF] Suzuki | An acidic matrix protein, Pif, is a key macromolecule for nacre formation[END_REF][START_REF] Suzuki | Identification and characterisation of a calcium carbonate-binding protein, blue mussel shell protein (BMSP), from the nacreous layer[END_REF][START_REF] Marie | The shell-forming proteome of Lottia gigantea reveals both deep conservations and lineagespecific novelties[END_REF].

Interestingly, three other proteins exhibit a domain organisation that partially resembles that of Pif-like proteins: UNP-2 (1 VWA, 2 long repeats and 1 LamG domains), UNP-3 (1 VWA, 16 CCP and 3 CBD domains), and UNP-5 (2 CBD2 and 1 LamG domains). Furthermore, various unionoid nacre proteins, called UNP-1, UNP-4, UNP-5, UNP-6 and UNP-7, bear characteristic chitin-or polysaccharide-binding domains (CBD2, Chitin_binding_3 or lectin C-type). The presence of these various VWA and chitin-binding domains suggest that these proteins might participate in structuring the chitin/protein nacre scaffold. In addition, we identified other ECM-related proteins, such as DMP-like, MSP130-like, mytilin3-like, ANFreceptor containing or Ca-binding proteins, for which homologs have previously been described from other proteins associated with biomineralizations [START_REF] Marie | Molecular evolution of mollusc shell proteins: insights from proteomic analysis of the edible mussel Mytilus[END_REF][START_REF] Marie | The calcifying shell matrix of the Pacific oyster Crassostrea gigas: proteomic identification of novel proteins[END_REF][START_REF] Xiao | Crystal structure of the Golgi casein kinase[END_REF]. The involvement of these proteins in biomineralization processes is discussed below.

(c) Repeated low-complexity domain (RLCD) containing proteins Our proteomic investigation also highlights the presence of a large set of 16 RLCD-containing proteins (supplementary table S1) for which similar proteins (that are genuine orthologous for some of them) were previously observed in Pinctada nacre, namely MSI60-like, shematrin, G-,Q-, Y-, D-and M-rich proteins [START_REF] Marie | Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell[END_REF]. In addition, we identified new features such as a byssal protein-like, the homologs of which have been detected some years ago in the byssus of the blue mussel [START_REF] Sagert | Hyperunstable matrix protein in the byssus of Mytilus galloprovincialis[END_REF]. MIS60-like. This protein fragment presents high sequence similarities with MSI60, presenting characteristic repeats of G-rich and poly-A blocks (45% identity and E-value 3.1E-55), and initially described as a framework protein in the shell of Pinctada fucata [START_REF] Sudo | Structures of mollusc shell framework proteins[END_REF]. MSI60 is a nacre-specific insoluble protein that exhibits poly-A and poly-G blocks giving MSI60 a certain similarity with hydrophobic spider and worm silk fibroins. More recently, another protein (has_CL10Contig2) with global similarities but no direct homology to MSI60 sequence has also been identified in the shell nacre of the abalone Haliotis asinina [START_REF] Marie | Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell[END_REF]. This suggests once more that such G-and A-rich proteins with hydrophobic silk-fibroin-like domains are key-players in nacre formation, in both bivalves and gastropods.

Shematrin-like. Only one protein corresponding to a shematrin-like protein has been identified

in the two unionoids according to one peptide that gives a heterologous match with the EST sequence UTP_var00312 available from the brook floater Alasmidonta vericosa, another unionoid bivalve. This protein presents strong sequence similarity (72% identity and E-value 5E-155) with silkmapin a nacre protein from the unoinoid Hyriopsis cumingii [START_REF] Liu | Silkmapin of Hyriopsis cumingii, a novel silk-like shell matrix protein involved in nacre formation[END_REF], that present sequence similarity with shematrin proteins previously retrieved from Pinctada shell.

The overall primary structure of shematrins is characterized by a signal peptide, G-G-Y-repeat motif, together with other more variable G-and Y-rich repeated low-complexity domains (RLCDs) and KKKY N-termini [START_REF] Yano | Shematrin: a family of glycine-rich structural proteins in the shell of the pearl oyster[END_REF], that are also observed in this novel unionoid putative member. Shematrin-likes constitute one of the main SMP family, found in both nacre and prismatic layer of the shell of Pinctada spp., and their corresponding transcripts are highly expressed in the mantle [2;20]. Surprisingly, their homologous sequences lack in the TSA databases of E. complanata and V. lienosa. We assume that the matching peptide observed in the two unionoid models may correspond to a genuine shematrin-like protein, similar to the one derived from A. vericosa EST. The apparent absence of shematrin-like transcripts in the E. complanata and V. lienosa databases call for explanations at two levels: a) Restricted temporal expression [START_REF] Miyazaki | Daily oscillation of gene expression associated with nacreous layer formation[END_REF], outside the 'window' of tissue sampling. Indeed, previous investigations have highlighted that key SMPs could be underrepresented or absent from a mantle EST database for such a reason [START_REF] Marie | The shell-forming proteome of Lottia gigantea reveals both deep conservations and lineagespecific novelties[END_REF]. b) Spatial expression of the shematrin transcripts restricted to the mantle; their absence in the transcriptomes of E. complanata and V. lienosa may be simply due to tissue sampling for EST construction, which was not particularly focused on the mantle [25;26].

Byssal protein-like, N-, T-, Q-, G-and M-rich proteins.

Our proteomic investigation has detected in the shell nacre of V. lienosa a sequence, which exhibits global similarities with a byssal protein-like from the mussel Mytilus galloprovincialis (33% identity). This protein exhibits repeat domains that are enriched in G, D, N and Y residues (Supplementary figure S2; supplementary table S1). Byssus is a silk-like acellular thread that is secreted by the foot of various bivalves, and that firmly anchors the organism to its substratum owing to strong adhesive distal plaques. A very recent proteomic investigation [START_REF] Qin | In-depth proteomic analysis of the byssus from marine mussel Mytilus coruscus[END_REF] has shown that several proteins constituting the byssal acellular structures (tyrosinase, A2M, EGF and VWA containing proteins) are common with those of the shell layers, claiming for similarities of the formation and the evolution of these two structures.

In addition, mollusc matrix proteins rich in Q have also been found in bivalves: for example MPN88 was previously characterized from the pearl oyster Pinctada margaritifera [START_REF] Berland | Coupling proteomics and transcriptomics for identification of novel variant forms of mollusc shell proteins: a study with P. margaritifera[END_REF]. In the shell of the gastropod Lottia, at least 4 different matrix proteins present similar features [START_REF] Marie | The shell-forming proteome of Lottia gigantea reveals both deep conservations and lineagespecific novelties[END_REF]. Interestingly, vertebrate teeth contain various Q-rich proteins belonging to the secreted calcium-binding phosphoprotein family, which are believed to interact with calcium ions and regulate the tooth mineralization process [START_REF] Kawasaki | Biomineralization in humans: making the hard choices in life[END_REF]. In parallel, it has been proposed that the numerous Q residues of various silk-like fibroins, which also present numerous A-and G-rich domains, are implicated in the tensile mechanical properties of these proteins [START_REF] Catesy | Conservation and convergence of spider silk fibroin sequences[END_REF]. Few proteins with M-and G-rich composition were identified among the RLCD-containing shell proteins listed in this study. Proteins with similar enrichments in M and G residues have also been recently described in the matrix of Pinctada [START_REF] Marie | MRNP34, a novel methionine-rich protein from the pearl oysters[END_REF], of Haliotis [START_REF] Marie | Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell[END_REF] and of Lottia [START_REF] Marie | The shell-forming proteome of Lottia gigantea reveals both deep conservations and lineagespecific novelties[END_REF].

However, the functional characterization of these characteristic domains in mollusc SMPs remains to be done.

In the present case, in spite of overall sequence similarities observed within these several RLCD protein categories identified in different models, it remains difficult to clearly outline whether they are truly homolog considering limitation of standard Blastp tools for lowcomplexity sequences, the fast rate of evolution of these domains [41;43] and that fortuitous low-complexity sequence similarities can be obtained between non-homologous SMPs that have evolved completely independently.

(d) Other bivalve shell-conserved proteins Carbonic anhydrase. Carbonic anhydrase (CA) is also a ubiquitous enzyme that is essential for calcium carbonate biomineral formation. It has been found in the organic matrices of various metazoan skeletons [START_REF] Tambutté | Characterization and role of carbonic anhydrase in the calcification process of the azooxanthellate coral Tubastrea aurea[END_REF][START_REF] Jackson | Sponge paleogenomics reveals an ancient role for carbonic anhydrase in skeletogenesis[END_REF][START_REF] Mann | Proteomic analysis of the acid-soluble organic matrix of the chicken calcified eggshell layer[END_REF]. In molluscs, shell-specific form of CA, exhibiting G, N and D-rich LCDs, have been isolated first from the shell matrix of the pearl oysters Pinctada sp. [START_REF] Miyamoto | A carbonic anhydrase from the nacreous layer in oyster pearls[END_REF]. In our study, the CAs detected in the two unionoid nacres do not possess similar supernumerary RLC domains, suggesting that matrix-associated CAs do not necessarily require such domains for being occluded in calcium carbonate microstructure or for exhibiting affinity with calcium carbonate mineral surfaces. At last, our results confirm the presence of CA in a non-pteriomorph bivalve shell as previously suggested [21;23]. Taken together, our findings reemphasize the central role played by extracellular CAs in CaCO3 biomineralization processes.

Chitin-interacting enzymes. Among the SMPs, we identified two enzymes, namely chitinase 3 and chitobiase, which both exhibit glyco_hydro_18 and glyco_hydro_20 domains. These domains belong to the glycoside hydrolases family that hydrolyses the glycosidic bond between carbohydrates or between a carbohydrate and other components. Indeed, chitin is known to represent a major polysaccharide in mollusc shells. It is involved in constituting a 3dimensional framework, in association with silk-like and acidic proteins in which nacre tablet crystals grow [START_REF] Addadi | Mollusk shell formation: a source of new concepts for understanding biomineralization processes[END_REF]. The identification of chitin-interacting enzymes, coupled with the detection of several chitin-binding domains in bivalve nacre SMPs strongly suggests an important role of chitin-cleaving enzymes in the fine molecular rearrangement of the chitin framework following the secretion of the nacre macromolecular ingredients at the vicinity of nascent growing mineral tablets.

Tyrosinase. The first mention of the involvement of a tyrosinase in bivalve shell formation concerned the deposition of the periostracum and of the prismatic layer in the pearl oyster [START_REF] Nagai | Tyrosinase localization in mollusc shells[END_REF] In particular, it was shown by in situ hybridization that the zone of the highest expression of tyrosinase was the mantle edge from which these two layers are secreted. Recently, this enzyme has also been detected in lesser amount in nacre shell structure [START_REF] Liu | In-depth proteomic analysis of shell proteins of Pinctada fucata[END_REF]. Tyrosinase is an oxidase that controls the production of protein melanogenesis via cross-linking by hydroxylation of the phenol groups present in tyrosine residues and their conversion to quinones. Biomineral-associated tyrosinases contribute to periostracum tanning [START_REF] Nagai | Tyrosinase localization in mollusc shells[END_REF]. They may be also involved in the maturation and hardening of the biomineral-hydrogel (that precedes crystallization) in prismatic as well as in nacreous shell layers [START_REF] Addadi | Mollusk shell formation: a source of new concepts for understanding biomineralization processes[END_REF].

Thioredoxin (TRX) and superoxide dismutase (SOD). Two enzymes involved in antioxidant

processes, TRX and SOD, were also detected in the shell nacre of V. lienosa. Besides the identification of various isoforms of peroxidases in Lottia shell matrix [START_REF] Marie | The shell-forming proteome of Lottia gigantea reveals both deep conservations and lineagespecific novelties[END_REF], there are to date only a couple of examples of such redox enzymes in mollusc shell matrices [17;48]. In the present study, TRX and SOD were detected only via a limited number of peptides, suggesting that they may be minor component of the nacre matrix. Their putative function in shell biomineralization remains enigmatic. We cannot exclude the possibility that they are contaminants: SOD, for example, constitutes the main hemolymph protein, in charge of oxygen transport in bivalves [START_REF] Oliveri | Biochemical and proteomic characterisation of haemolymph serum reveals the origin of the alkali-labile phosphate (ALP) in mussel (Mytilus galloprovincialis)[END_REF]; its accidental occlusion in shell calcified tissues is not an unlikely scenario.

Immunity-related proteins. Finally, we detected, in both Elliptio and Villosa's nacre matrices, four proteins with potential immunity-related functions. They exhibit indeed Kunitz-like (KU), or whey acidic protein (WAP) or macroglobulin protease inhibitor domains (Figure 1; supplementary table S1). The presence of such protease inhibitors in an acellular biomineral is, at first sight, puzzling. However our finding does not constitute a single observation, since the presence of such immunity-associated domains in shell proteins has been already noticed in different metazoan calcium carbonate biominerals: the shells of abalones [19;50], of pearl oysters [2;51-52], of the giant limpet [START_REF] Marie | The shell-forming proteome of Lottia gigantea reveals both deep conservations and lineagespecific novelties[END_REF] and, at last, the chicken egg shells [START_REF] Mann | Proteomic analysis of the acid-soluble organic matrix of the chicken calcified eggshell layer[END_REF]. One has proposed that these molecules are involved either in immunological defence mechanisms, or in the regulation of the matrix maturation, by controlling cleavage processes of other matrix proteins [START_REF] Arivalagan | Shell matrix proteins of the clam, Mya truncata: Roles beyond shell formation through proteomic study[END_REF][START_REF] Dombre | Eggsepins: The chicken and/or the egg dilemma[END_REF].

(e) Inherited set of nacre and shell matrix proteins in bivalves One of the most striking results of our study was the detection, in Elliptio and Villosa's shell matrices, of various proteins that exhibit high sequence homologies with SMPs extracted from the nacreous shell layers of Pinctada and/or Mytilus bivalves. This constitutes a very consistent evidence of the existence of a set of ancient, i.e. Palaeozoic, nacre proteins shared between the various nacre-forming bivalve taxa (Figure 3). More than two decades ago, when molluscan nacre matrices were solely analyzed for their amino acid compositions after hydrolysis of the bulk, the similarity of their compositions characteristically marked by high contents in glycine, alanine and aspartate residues, suggested identical molecular mechanisms for controlling the deposition of nacre tablets in the different nacre-bearing molluscs [START_REF] Keith | Comparative analysis of macromolecules in mollusc shells[END_REF]. But, since the publication of the first full primary structure of a nacre protein, in 1996 [START_REF] Miyamoto | A carbonic anhydrase from the nacreous layer in oyster pearls[END_REF], the increasing number of discovered SMPs has point out an extraordinary diversity of nacre-associated proteins among the different taxa [8;9]. This suggested on the contrary the use of different protein toolkits for nacre deposition. This view seemed to prevail, in particular with the finding that the bivalve Pinctada and the gastropod Haliotis possess absolutely dissimilar sets of shell-building genes [19;20]. This abrupt conclusion was however counterbalanced later by one of our studies that clearly demonstrated that the nacre of the edible mussel Mytilus and the non-nacreous shell microstructure of the limpet Lottia, contains number of proteins that are similar -if not homologous -to that of Pinctada's nacre [2;29;30]. The present set of data supports and reinforces the "deep origin scenario" by revealing a shared protein toolkit in the nacre of bivalve species that belong to three clades, Mytiloida, Pterioida (both belonging to Pteriomorphia subclass) and Unionoida (the main order of the Palaeoheterodonta subclass) that diverged in the Palaeozoic times [START_REF] Giribet | Bivalvia In Phylogeny and Evolution of the Mollusca[END_REF].

The list of shared nacre proteins between the three main bivalve models comprises: MSI60, Pif, CA, macroglobulin, VWA-, LamG-, CBD2-and Kunitz-like-containing proteins, along with various RLCD protein exhibiting A-, D-, G-, Q-and M-rich domains. This short list potentially represents the inherited ancestral toolkit -or at least a part of it -required for nacre formation. Other proteins appear to be shared by only two species out of three, and are probably related with taxon specificity adaptations. One has to notice that some of the proteins listed on figure 3 have been also identified in the prismatic layers of the three bivalves, suggesting that their molecular functions might not be exclusively restricted to nacre deposition.

By identifying nacre proteins from two non-pteriomorph species and reporting a deeply conserved nacre protein set in bivalves this work adds new evidence that that the calcifying matrix of molluscs is comprised of both deep conservations and lineage-specific protein novelties. The comparison of these bivalve-conserved protein sets to those previously detected in gastropod nacre is confirming the global dissimilarity of the nacre SMP sets of bivalves and of gastropods (at least those of the abalone) [START_REF] Marie | Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell[END_REF]. Bivalve and gastropod exhibit different kinds of nacres (respectively, sheet and columnar nacres), which homology still constitute an actual debate that still oppose parcimony principles [START_REF] Carter | Skeletal Biomineralization: Patterns, Processes and Evolutionary Trends[END_REF], crystallographic and microstructural textures [11;57], along with paleontological and molecular evidences [12;13;20].

Figure 4 summarizes all available information on the occurrence of protein domains observed from shell proteome of various mollusc models, comprising nacre and prismatic shell layers of bivalves (Unionoids, Pinctada and Mytilus), cross-lamellar layers of gastropod (Lottia and Cepaea), and finally, fibrous layers of brachiopods, taken as an outgroup. This figure shows that several proteinaceous domains, which appear to be shared between the different nacre models, are also detected in bivalve prismatic layers or in the shell of cross-lamellar gastropods. This clearly suggests that these domains are not specifically involved in nacre formation, but that they display general function in shell formation.

The brachiopod shell proteomes, retrieved from the CaCO3 shell of Magellania venosa [START_REF] Jackson | The Magellania venosa biomineralizing proteome: a window into Brachiopod shell evolution[END_REF] and the CaPO3 one of Lingula anatina [START_REF] Luo | The Lingula genome provides insights into brachiopod evolution and the origin of phosphate biomineralization[END_REF], respectively, exhibit many similarities with those of molluscs in their respective protein and functional domain compositions. The list of shared domains comprises potentially key contributors of shell formation processes, such as D-, Aand G-rich, VWA, EGF, CCP, peroxidase, tyrosinase, chitinase, CA, KU, A2M and CBD2. Furthermore, Luo and co-workers [START_REF] Luo | The Lingula genome provides insights into brachiopod evolution and the origin of phosphate biomineralization[END_REF] notice also that genes shared between Lingula and molluscs, such as calcium-dependent protein kinase and chitin synthase, exhibit high expression in larvae and mantle, indicating that they may also be involved in brachiopod shell formation. Taken together, these results indicate that a conserved molecular machinery exists for shell biomineralization in brachiopods and molluscs. This assumption needs however a further validation via functional analyses.

(h) Are metazoan SMPs deeply conserved?

The appealing scenario of an ancient and common origin of the biomineralization within the metazoan has been proposed at various occasions [3;60]. This scenario is based on the supposed existence of a shared ancestral metazoan toolkit that was implemented before the main metazoan radiations. But to date, the data that support this view still remain extremely tenuous. For example, no one ever evidenced a universal molecular marker of the skeletal matrices of all calcifying metazoans. Many skeletal matrix proteins belong to families that are not necessarily specific of biomineralization processes, suggesting that many proteins could have been coopted secondarily for calcification purpose. For example, although CA has been observed in various biomineralization models, comprising egg-shell, echinoderm skeleton, mollusc shell or sponge spicules, it constitutes a broad and ubiquitous protein family presenting various members in all organisms, calcifying or not; their phylogenetic analysis shows a very complex evolutionary history, indicating that they could have been recruited several times, in independent manners, in biomineralization structure within the different metazoan clades [START_REF] Roy | The volution of metazoan α-carbonic anhydrases and their roles in calcium carbonate[END_REF].

At the opposite of the CA example, MSP-130 represents a remarkable illustration of a conserved molecular function that may be strictly restricted to biocalcification, but present in distinct metazoan lineages. MSP-130 is a protein, initially identified in the sea urchin Strongylocentrotus purpuratus [START_REF] Leaf | Antibodies to a fusion protein identify a cDNA clone encoding msp130, a primary mesenchymespecific cell surface protein of the sea urchin embryo[END_REF], which expression is specific to primary mesenchyme cells [START_REF] Harkey | Differential expression of the msp130 gene among skeletal lineage cells in the sea urchin embryo: a three dimensional in situ hybridization analysis[END_REF] -the cells that initiate the skeletal tissues in echinoderms -and that represents one of the main constituent of the calcifying matrix of the different skeletal structures, including the teeth, the spicules, the test or the spines [START_REF] Mann | Proteomic analysis of sea urchin (Strongylocentrotus purpuratus) spicule matrix[END_REF]. So far, no other function than those related to biomineralization has been described for this protein. Interestingly, during our previous investigation of the shell proteins of the Pacific oyster Crassostrea gigas, we detected a protein, called gigasin-3, which partial sequence exhibits noticeable similarity with S. purpuratus MSP-130 [START_REF] Marie | The calcifying shell matrix of the Pacific oyster Crassostrea gigas: proteomic identification of novel proteins[END_REF]. Even more recently, the msp130 gene was also identified in a calcified tube-forming polychaete [START_REF] Szabo | Another biomineralising protostome with an msp130 gene and conservation of msp130 gene structure across Bilateria[END_REF]. With the present study, the recognition in nacre matrix of two unionoids of a similar MSP-130-like protein firmly supports the idea that this biomineralization-specific protein or protein family is shared between different metazoan calcifying lineages, reintroducing the scenario of common ancestral mechanism for metazoan biomineralization processes.

In addition, we have detected that the two unionoid proteomes contain both a dentin-matrix protein-like (DMP-like), exhibiting a remarkable FAM20C domain (Supplementary figure S3). Indeed, FAM20C is a kinase that phosphorylates the majority of the phosphoproteome in the extracellular matrices of bones and teeth, constituting a key player of vertebrate biomineralization process [START_REF] Tagliabracci | A single Kinase generates the majority of the secreted phosphoproteome[END_REF]. Other recent investigations have revealed the presence of FAM20C containing protein in the shell matrix of the giant limpet Lottia gigantea [START_REF] Mann | The Lottia gigantea shell matrix proteome: re-analysis including MaxQaunt iBAQ quantification and phosphoproteome analysis[END_REF], and of the brachiopod Magellania venosa [START_REF] Jackson | The Magellania venosa biomineralizing proteome: a window into Brachiopod shell evolution[END_REF]. The presence of such key-protein, regulating the biomineralization formation of vertebrates, in the shell matrices of molluscs and brachiopods strongly reinforces the idea of common molecular machinery, supporting the scenario of a conserved biomineralization toolkit between different metazoan lineages.

Conclusion

Our study presents proteomic results obtained from the nacre matrices of two unionoid (Paleoheterodonta) bivalves. Our approach is robust and powerful since it allows annotating genes encoding proteins that are undoubtedly shell matrix constituents, therefore, putatively important players in the nacre assembling process. It excludes genes that encode mantlesecreted proteins -recognizable by their signal peptide signature -but which are not occluded within the calcified tissue, i.e. proteins, which are not constituents of the shell organic matrix.

We are fully conscious that some of these proteins, not detected by our approach, may be important players for calcification as well [START_REF] Marin | Organic matrices in metazoan calcium carbonate skeletons: composition, functions, evolution[END_REF].

Our study has macro-evolutionary implications. Nacre is supposed to represent an ancestral shell microstructure, shared by different orders within the bivalve class. Because it remained almost unchanged until now, from a microstructural viewpoint, this suggests that at least a part of the molecular toolkit used for constructing nacre must have been kept conserved within these nacre-forming clades. Our proteomic approach focused on the unionoid bivalves has allowed detecting several genuine sequence homologies with known sequences of nacre proteins of pteriomorph bivalves, but also with other SMPs from gastropod and brachiopod models, unveiling a part of a deeply conserved shared legacy.

Material and methods

(a) Nacre matrix extraction

Fresh Villosa lienosa (6-8 cm in length) and Elliptio complanata (8-10 cm in length) shells were collected from Chewacla Creek near Auburn, Alabama (USA), in April 2012, and from Cree Lake near Cochrane, Ontario (Canada), in spring 2011, respectively. Among the paleoheterodont bivalves, both are representatives of unionoid order, the shell microstructures of which are characterized by the association of aragonitic prisms and nacre. Superficial organic contaminants as well as the periostracum were removed by incubating intact shells in NaOCl (5%, v/v) for 24-48 h [START_REF] Marie | Proteomics of CaCO3 biomineralassociated proteins: How to properly address their analysis[END_REF]. Shells were then thoroughly rinsed with water. The external prismatic layer was mechanically removed and the shell nacre broken into 1-mm large fragments before being ground to fine powder (> 200 µm) that was subsequently decalcified in cold acetic acid overnight (10%; 4 °C). The insoluble nacre matrix was then collected by centrifugation (15,000 g; 10 min; 4 °C), and the pellet rinsed six times with MilliQ water by series of resuspension-centrifugation before being freeze-dried and weighed.

(b) Peptide digest proteomic analyses

In-solution digestions of insoluble nacre matrix samples were performed. One mg of the matrices was reduced with 100 μL of 10 mM dithiothreitol (Sigma-Aldrich, France) in 500 mM triethylammonium bicarbonate pH 8.0 for 30 min at 57° C. Fifteen μL of iodoacetamine (50 mM, final concentration) was added and alkylation was performed for 30 min at room temperature in the dark, then digestion was performed by adding 10 μg of trypsin (T6567, proteomics grade, Sigma). The samples were incubated overnight at 37 °C, then centrifuged (30 min, 14,000 g) and 5 μL of the acidified supernatants was injected into the tandem mass spectrometer with an electrospray source coupled to a liquid chromatography system analysis (nanoLC-ESI-MS/MS). The protein digest concentration was prior estimated using a micro-BCA kit (Sigma-Aldrich, USA), with BSA as protein standard.

Protein digests were analyzed using a nano-LC (Ultimate 3000, Dionex) coupled to an ESI-LTQ-Orbitrap (LTQ Orbitrap XL, Thermo Scientific) mass spectrometer [START_REF] Arivalagan | Insight from the shell proteome: biomineralization to adaptation[END_REF]. Above 10 µg of AIM digest protein digests solubilized in 10% ACN with 0.1% formic acid were injected in triplicates by the autosampler and concentrated on a trapping column (Pepmap, C18, 300 µm x 5 mm, 5 µm 100 Å, Dionex) with water containing 10% ACN with 0.1% formic acid (solvent A). After 5 min, the peptides eluted onto a separation column (Pepmap, C18, 75 µm x 500 mm, 2 µm 100 Å, Dionex) equilibrated with solvent A. Peptides were separated with a 2hlinear gradient of 80% ACN + 0.1% formic acid (solvent B) at flow rate of 200 nL.min -1 . The LTQ-Orbitrap mass spectrometer is outfitted with a nano-ESI electrospray source. Spectra of multiple charged precursor ions (+2 to +4) were collected from m/z 300-2000 at a resolution of 30,000 in the profile mode followed by data dependent CID and/or HCD spectra of the ten most intense ions. A dynamic exclusion window of 60 s was used to discriminate against previously analyzed ions.

(c) Bioinformatics

The MS/MS data were used for database searches using an in house version of the MASCOT search engine (Matrix Science, London, UK; version 2.1) against 138,345 and 46,051 TSA nucleotide sequences derived from E. complanata and V. lienosa EST libraries [25;26], respectively (plus few hundreds Alasmidonta spp. sequences), downloaded in December 2015 from the NCBI server (http://www.ncbi.nlm.nih.gov). LC-MS/MS data generated by each shell sample and protein band were searched separately, using carbamido-methylation as a fixed modification and methionine oxidation as variable modification. The peptide MS tolerance was set to 10 ppm and the MS/MS tolerance was set to 20 mDa. Proteins with at least two different peptides or identified in both species were considered to be fully valid.

Protein identification was performed using BLAST searches against the UniProtKB/Swiss-Prot protein sequence database (www.uniprot.org), retrieving only hit with e-value < 7E-8, and without filtering low complexity region. Signal peptides were predicted using SignalP 4.0 (http://www.cbs.dtu.dk/services/SignalP) and conserved domains from database models were predicted using external source database SMART (http://smart.embl-heidelberg.de). 3-D protein structures were predicted according to sequence homologies using the Phyre 2.0 online tool (http://www.sbg.bio.ic.ac.uk/phyre2) [START_REF] Kelley | The Phyre2 web portal for protein modelling, prediction and analysis[END_REF]. Data accessibility. Supplementary table S1 containing the complete list and description of all proteins identified in E. complanata and V. Lienosa nacre matrices by shotgun proteomics has been uploaded as electronic supplementary materials.

Abbreviations: A2M, alpha-2 macroglobulin; APase, alkaline phosphatase; ANF, extracellular ligand binding; BMSP, blue mussel shell protein; C1q, complement complex protein 1; CA, carbonic anhydrase; CBD, peritrophin-A chitin-binding domain; CCP/SUSHI, complement control protein; Chit_bind, chitin-binding domains; CLECT, C-type lectin;

Copamox, copper amine oxidase; DMP, dentin matrix protein; EGF, epidermal growth factor; FAM20C, family with sequence similarity 20 -member C; FN3, fibronectin 3; IGF-BP, insulin growth factor-binding protein; KU, Kunitz-like domain; LamG, laminin G; MSP, mesenchym-specific protein; MSI60, matrix silk-like insoluble protein 60; PIF/Pif177, Pinctada fucata 177-kDa protein, RLCD, repetitive low-complexity domain; SMP, shell matrix protein; SCP, Cysteine-rich secretory protein; SOD, superoxide dismutase; TIMP, tissue inhibitor of metalloprotease; TRX, thioredoxin; UNP, uncharacterized nacre protein; VWA, von Willebrand factor type A; WAP, whey acidic protein; ZP, zona pellucida.

Figure 1 .

 1 Figure 1. Identification of the nacre matrix proteins from Elliptio complanata and Villosa lienosa shells using MS/MS analyses. Venn diagram of 41 and 38 proteins identified in both nacre matrices. The hashtag symbol (#) beside some protein names represents cases where the identification is not 100% certain, since it is based only on one peptide and only in one of the two species.

Figure 2 .

 2 Figure 2. Schematic representation of the primary structure of unionoid extracellular matrix (ECM) nacre proteins retrieved from Elliptio complanata and/or Villosa lienosa. Each complete protein sequence possesses a signal peptide indicated in red. Typical ECM domains are indicated as boxes on the protein structure. The list of ECM protein domains comprises VWA, CBD2, LamG, Chitin_binding_3, EFh, CLECT, ANF_receptor and FAM20C/DUF1193, together with few RLCDs or repeated domains. The "?" symbol indicates missing sequence information.

Figure 3 .

 3 Figure 3. Nacre SMPs from bivalve models. In total, 48, 35 and 42 various matrix proteins have already been detected in the nacre of the three bivalve models that belong to the Unionidae, Pteriidae and Mytilidae families, respectively. Names correspond to proteins and/or proteinaceous domains identified in the different nacre SMPs. p and m exponents indicate that the protein was also detected in the prismatic shell layer of either Pinctada spp.or Mytilus spp., respectively[2;15;17;18].

Figure 4 .

 4 Figure 4. Comparison of SMP domain distribution in the shells of various lophotrochozoan models, focused on molluscs and brachiopods. The domains are sorted according to main categories detected in SMPs. * indicates that only genuine abundant SMPs presenting high confidence for their specific identification in the shell matrix, and not being potential considered as contaminant have been mentioned here.

  Fig. 1

  750

Acknowledgments. We are especially grateful to Dr. James Stoeckel and Dr. Emily Awad for kindly providing us the Villosa lienosa and Elliptio complanata shells.

Author's contribution. B.M., G.B., S.B. & F.M. conception, design and coordination; J.A., A.M., L.M. preparation of the sample, analysis and interpretation of the data. All author contribute to the preparation and the revision of the manuscript.

Competing interests. The authors have no competing financial interests.

Funding. Jaison Arivalagan is supported by a funding from the Marie Curie European Union Seventh Framework Programme under grant agreement no. 605051.


André Pontes Da Costa

Dr Danilo Rosa Nunes

Mickaël Tharaud

Prof Julie G Oble
email: julie.oble@upmc.fr

Giovanni Poli

Jutta Rieger
email: jutta.rieger@upmc.fr

Dr A P Da Costa

Dr J Poli

Rieger

Pd(0)-Nanoparticles Embedded in Core-Shell Nanogels as Recoverable Catalysts for the Mizoroki-Heck Reaction

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

Introduction

Palladium nanoparticles (NPs) are ubiquitous catalysts in C-C coupling reactions, including when the pre-catalyst is a soluble palladium complex. [1] In the past decades, a special interest has been directed to understand the mode of formation of these nanoparticles during a reaction, and their catalytic mode of action. Furthermore, some efforts have been undertaken to stabilize these NPs in the reaction medium, in order to avoid the generation of inactive palladium black. The most common approach to perform C-C coupling reactions without added dative ligands makes use of tetraalkylammonium salts as stabilizers of in situ generated NPs, known as "Jeffery's ligandless conditions". [2] The use of very low amounts of palladium catalyst without stabilizers, frequently referred to as "homeopathic palladium loading", is also a valuable alternative to molecular catalysts, since no palladium black is formed at very low catalyst concentrations. [3] Immobilization of metal NPs in solid supports such as silica or carbon aerogels allowed obtaining recoverable catalysts for cross-coupling reactions, [4] including the Mizoroki-Heck reaction. [5] Polymers have also been used to stabilize NPs and help catalyst recycling. [6] In recent years, soluble polymers, i.e. linear polymers, or more sophisticated structures, such as dendrimers and hyperbranched polymers have attracted attention as catalyst matrix or stabilizers, mainly due to their easy manipulation in the solid state, allowing catalyst recovery, as well as the quasi-homogenous behavior of the catalysis. [7] However, only few examples addressed the stabilization of Pd(0) NPs by soluble polymers behaving as quasihomogeneous supports for C-C coupling reactions. In this respect, Biffis et al. have reported the generation of palladium nanoparticles stabilized by polyacrylamide-based swellable polymeric 3D architectures, and proved their efficiency as catalysts in both Mizoroki-Heck as well as Suzuki coupling reactions. [8] More recently, nanostructured polymeric architectures have also been used as metal supports and stabilizers for applications in catalysis. The first examples have involved the postfunctionalization of polymer NPs, usually polystyrene solid cores surrounded by a soluble cross-linked polymeric shell, able to stabilize metallic NPs. [9] The advent and development of controlled polymerization techniques provided an easy access to a wide range of well-defined nanostructured polymers such as star-polymers, cross-linked micelles or core-shell nanogels. [10] These new polymeric architectures, being rationally designable and able to exhibit responsive behavior, are usually classified as smart nanomaterials. [11] However, their use as catalyst supports started to grow only recently. Thus, well-defined core-shell structures incorporating late transition metals have been synthesized and proved to be active catalysts in industrially relevant transformations such as the polymerization of methyl methacrylate (Ru) [12] and olefin hydroformylation under biphasic conditions (Rh). [13] Nevertheless, the use of nanostructured soluble polymers as supports for quasi-homogeneous catalysis is still in its infancy and further exploration of this domain is a hot topic, expected to lead to the discovery of innovative catalytic systems.

Among the polymeric core-shell architectures, core-shell nanogels, i.e. nanometric globular-shaped polymers structured in a swollen core and surrounded by a stabilizing thin polymer shell, have recently attracted increasing interest. Thanks to the use of heterogeneous polymerization processes, combined with controlled polymerization techniques, their synthesis is now mastered. [14, 15b] In the last years, we have developed an easy synthetic route to tailor-made core-shell nanogels by using reversible additionfragmentation chain-transfer (RAFT)-mediated aqueous dispersion polymerization of acrylates or acrylamides. [15] Based on this former work, we describe here the synthesis of core-shell nanogels rationally designed to stabilize Pd(0) NPs through its poly(N,N-diethylacrylamide-co-N,N-dimethylaminoethyl acrylate) core, and the use of this hybrid nanogel as catalyst in the Mizoroki-Heck reaction, along with its recycling. Moreover, three-phase tests were performed in order to establish the homogenous/heterogeneous character of this hybrid catalyst.

Results and Discussion

Hybrid nanogel synthesis and characterization

The core-shell nanogels were prepared according to a previously established procedure using RAFT radical polymerization in aqueous dispersion. [15] This heterogeneous process allows the synthesis of nanometric monodisperse coreshell particles in which the core can be easily crosslinked through the use of small amounts of a bifunctional monomer, without the formation of large ill-defined gel structures (Scheme 1). [14b] Scheme 1. Approach to core-shell nanogel architecture.

Poly(N,N-dimethylacrylamide) was selected as stabilizing shell constituent for its good solubility in a large range of solvents. [15a, 15c,16] Poly(N,N-diethylacrylamide) (PDEAAm), being also soluble in a large range of solvents, except in warm water, appeared to be the ideal candidate for the preparation of the core matrix via an aqueous heterogeneous polymerization process at moderate temperature. [15a,15b,16] In order to provide amines required for Pd coordination, N,N-dimethylaminoethyl acrylate (DMAEA) was introduced in the core. In this way, the nanogel NG was obtained in water at 70°C by RAFT dispersion polymerization, as depicted in Scheme 2 DEAAm (85 mol%), DMAEA (10 mol%) and N,Nmethylenebis(acrylamide) (MBA, 5 mol%) as cross-linker, in the presence of the stabilizing macromolecular RAFT agent based on poly(N,N-dimethylacrylamide) -PDMAAm-macroRAFT, Mn LS = 14.2 kg mol -1 and Ð = 1.3. The polymerization occurred smoothly and the reaction medium became cloudy after about 15 min, a typical signal of particle nucleation initiation in dispersion polymerization systems. After 1h, the monomer conversion was complete and a stable colloidal aqueous dispersion was formed. For comparison, linear PDMAAm-block-P(DEAAm-co-DMAEA) chains were prepared by the same synthetic protocol but in the absence of cross-linker, see Supporting Information Table S2 andS3.

Size exclusion chromatography (SEC), dynamic light scattering (DLS) and transmission electron microscopy (TEM) assessed the formation of well-defined nanogels (NG).

Scheme 2. Synthesis of the core-shell nanogel, PDMAAm-block-P(DEAAmco-DMAEA-co-MBA): NG. Reaction conditions: i) DEAAm 85 mol%, DMAEA 10 mol%, MBA 5 mol%, V-50, H2O, 70°C, 1h. Mn(PDMAAm-macroRAFT) LS = 14.2 kg mol -1 , Ð = 1.3.

The size exclusion chromatograms (Figure 1) evidence the complete extension of the stabilizing PDMAAm chains with the formation of high molar mass monodisperse NG, demonstrating the great efficiency of the transfer reaction. The number-average molar mass of NG was determined to be Mn LS = 13,400 kg mol -1 with low dispersity (Ð = 1.8), obtained by coupling the SEC to a static light scattering detector combined with a differential refractive index detector. Based on the observed numberaverage molar mass of NG and the number-average molar mass of linear chains (Mn LS = 55.5 kg mol -1 , Ð = 1.4), Figure 1, we could estimate the number of polymer chains (Nagg) in one NG particle to be 240, which is in good agreement with the results obtained in the synthesis of other nanogels by a similar strategy. [15c] The reported reactivity ratios of DMAEA and DMAAm (rDMAEA/DMAAm = 0.29, rDMAAm/DMAEA = 1.46), [17] reveal that DMAEA is less reactive than DMAAm, and therefore inserted preferentially at the end of the polymerization. Considering that the nanogel grows from the shell (macroRAFT) towards the core, a larger concentration of the DMAEA units deeply embedded in the core of the nanogels is expected. Transmission electron microscopy (TEM) images of the dried, collapsed nanogel showed spherical particles, below 30 nm in diameter (Figure 2A). The size of the nanogels in their swollen state was then determined by DLS at 20°C (Figure 2B). Their z-average hydrodynamic diameter was 67 nm in water, and 82 nm in N,N-dimethylacetamide (DMAc). The observed difference in size for the dried and the swollen NG unambiguously confirms the low degree of crosslinking of the polymeric structures, possibly allowing the easy diffusion of small molecules, such as substrates/reactants, inside these highly swollen structures. Embedding of Pd(0)NPs in NG was obtained through addition of a Pd(II) salt, which is expected to be coordinated by the nitrogen functions of DMAEA units, followed by metal reduction by ethanol (Scheme 3); a method that has been already reported for this task. [8b] Accordingly, a solution of NG and palladium acetate in dichloromethane was initially stirred at room temperature for 24 h under argon atmosphere, which allowed diffusion of the metal salt into the NG. The color of the resulting solution turned from orange to dark red, indicating that coordination of the palladium atoms by the amino groups (from DMAEA) in the NG core occurred successfully. After isolation by precipitation, the Pd(II)-containing NGs were redissolved in dichloromethane/ethanol mixture (5:1) and the solution was heated to 40°C for 24 h. The solution turned brown, revealing the reduction of the metal from (II) to (0) oxidation state and formation of the NPs. [18] After evaporation to dryness, the solid was purified by dialysis in water and lyophilized, affording a brown powder.

The integrity of the NG structure after reaction and the formation of Pd particles were verified by DLS after dissolution in water. No large aggregates were detected, and the PdNP@NG showed z-average hydrodynamic diameter of 104 nm at 20 °C (Supporting Information, Figure S4), i.e. slightly higher average diameters than the empty nanogels, which is consistent with the incorporation of Pd nanoparticles. TEM was used to determine the size of Pd-NPs (Figure 3A). Very small Pd particles were formed with an average diameter of Dn = 2.9 ± 0.6 nm. No bigger NPs or aggregates were observed.

The hybrid NG (PdNP@NG) was further characterized by thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), and inductively coupled plasma mass spectroscopy (ICP-MS), in order to determine the percentage and oxidation state of the Pd species. The loading of palladium introduced was determined to be 1.4 wt% by TGA (from the weight difference between the empty NG and the hybrid NG PdNP@NG at 650°C, see Supporting Information, Figure S3). This value was confirmed by ICP-MS (1 wt%) and XPS (1.3 wt%) (via integration of all peaks). This value is only slightly below the maximum theoretic loading (1.6 wt% based on the amount of palladium introduced in the reaction), indicating that only few Pd atoms were lost during the PdNP@NG preparation, i.e. that most of the Pd was successfully supported in the NG. XPS (Figure 3B) revealed that the majority of the Pd atoms were successfully reduced to Pd(0), with only a few atoms in a higher oxidation state, possibly corresponding to surface atoms of the Pd(0) nanoparticles which are stabilized by polymeric chains of nanogel. According to the literature, [19] we assume that the nanoparticles are stabilized by the amine function of DMAEA as well as by the amide function of DEAAm units in the nanogel core. As to the catalyst loading, we assume a 1.4 wt% value, as from the TGA analysis. Indeed, this technique, in contrast to ICP-MS analysis, does not involve sample digestion, and, in contrast to XPS analysis, is not surface-sensitive. In any case, as this analysis gave the highest experimental value amongst the techniques applied, assumption of this value as the most reliable one, would, in the worst case scenario, make us underestimate the amount of palladium actually present in the catalytic reactions. S5 andS6). Scheme 3. Embedding of Pd(0) NPs in core-shell NG (PdNP@NG). Reaction conditions: i) Pd(OAc)2, DCM, rt, 24h. ii) EtOH, DCM, 40°C, 24h.

Application of PdNP@NG as catalyst in the Mizoroki-Heck reaction

To evaluate the potential of such a novel nanostructured system as catalyst in the frame of homogeneous supported catalysis, we selected the C-C Mizoroki-Heck coupling between 4-bromo-acetophenone and n-butylacrylate as a benchmark reaction (Scheme 4). [3b] Scheme 4. The Mizoroki-Heck reaction between 4-bromo-acetophenone and n-butyl acrylate. Reaction conditions: i) 4-bromoacetophenone (0.25 mmol), n-butyl acrylate (0.425 mmol), NaOAc (0.3 mmol), 0.1 mol% Pd (either PdNP@NG or Pd(OAc)2) and DMAc (0.60 mL), under argon atmosphere, 120°C.

The evolution of the reaction was investigated first (Figure 4), (round dots representing spectroscopic NMR yields of the cinnamate product). Full conversion was reached in about 2h of reaction in DMAc at 120°C. The same reaction was next repeated using a "homeopathic palladium loading" [3b] as catalyst instead of PdNP@NG. The reaction kinetics with this catalytic system was comparable to that using PdNP@NG (Figure 4, hollow squares). It thus appears that the complex structure of the core-shell NGs does not affect the catalytic performance of the palladium NPs when comparing to non-supported catalytic systems. These encouraging results prompted us to further explore the scope of the Mizoroki-Heck reaction catalyzed by this hybrid PdNP@NG. In this respect, the reactivity of several bromo-and iodo-arenes with n-butylacrylate was tested. All the reactions were conducted in the same conditions as the benchmark reaction, using 4-bromo-acetophenone (Scheme 4), and the products were isolated by flash chromatography and characterized by 1 H and 13 C NMR. The results are summarized in Table 1. The hybrid PdNP@NG was able to catalyze the coupling of n-butyl acrylate with bromo-arenes activated by an electron-withdrawing group in para position (entries 1 to 4). However, no reaction was observed when more electron-rich halo-arenes were used as substrates, such as chloro-benzene, bromo-benzene and 4-methoxy-bromo-benzene (entries 5, 6 and 9). In contrast, when using more reactive iodo-arenes as starting materials, the electronic nature of the substituent (hydrogen, electron-withdrawing or electron-donating group) in para or meta position did not influence the reaction yields (entries 10 to 17). Finally, low yields were obtained with bromoas well as iodo-arenes ortho-substituted with carbonyl groups (entries 7, 20 and 21), whereas a trifluoromethyl (entry 8), a methoxy (entry 18), or a methyl group (entry 19) in ortho position, allowed almost quantitative yield (> 96%). We attribute the lack of reactivity associated to the ortho-carbonyl substituted substrates to the coordinating nature of the functional group rather than to sterical hindrance. [20] Indeed, a coordinating group in the ortho position, typically a carbonyl group, might hamper either the olefin coordination step or the formation of the cis isomer that could lead to the carbopalladation step, thus lowering the yield.

Table 1. Application of the hybrid PdNP@NG in the Mizoroki-Heck reaction. [a,b] Entry X R Yield (%) [b] 1 Br 4-(C=O)CH3 92 [c,d] Thus, the catalytic activity of the hybrid NG PdNP@NG in the Mizoroki-Heck reaction followed the same trend as that of the most common homeopathic ligand-free homogeneous catalysts [3b] and of Pd NPs supported by P(DMAAm-co-DMAEA), [8b] while showing a better performance than Pd NPs stabilized in polymeric solid supports, [21] although this last example represents a different class of hybrid material. [START_REF]For recent examples[END_REF] We then turned our attention to other olefin partners in similar reaction conditions using 4-bromo-acetophenone (Scheme 5). The hybrid PdNP@NG was moderately active in the Mizoroki-Heck reaction between 4-bromo-acetophenone and acrylonitrile (20% NMR yield in 2h at 120°C). In contrast, the reaction between 4-bromo-acetophenone and styrene occurred smoothly to full conversion in 2h and was selective towards the C-C coupling in the  position. This selectivity suggests the involvement of a neutral mechanism, ruling out a cationic palladium intermediate. [23] Intriguingly, the reaction between 4bromo-acetophenone and n-butyl methacrylate afforded the bisarylated product [START_REF]The E-configuration of the double bond was assigned with NOESY NMR experiment[END_REF] as the major product in 60% yield [START_REF]The yield was calculated by considering the use of two equivalents of 4bromo-acetophenone[END_REF] (Scheme 5). Although such bis-arylated products have already been reported as side products of the Mizoroki-Heck reaction with methacrylates, optimization of their formation has never been addressed. [26] In our case, the selectivity offered by the hybrid NG catalyst PdNP@NG in forming such bis-arylated product appears of synthetic interest.

Hybrid nanogel recycling

PdNP@NG recycling was investigated next. Accordingly, the model Mizoroki-Heck reaction was scaled-up 10 times with respect to the amounts used previously, so as to dispose of enough PdNP@NG for isolation purposes (~20 mg) at the end of the cycle. The direct scale-up of the reaction afforded similar yields of isolated product as the small scale reaction. After the reaction, the hybrid NG was recovered by precipitation in diethyl ether, dried and then reused in a new cycle without any regeneration. The amount of the new starting material was maintained unchanged in the different recycling steps, independently of the catalyst mass loss during the recovery protocol (overall 10 wt% after 3 cycles). The results over 4 catalytic cycles are depicted in Figure 5. The catalytic activity turned out to be steady during the first 3 cycles (~ 90% isolated yield), reaching an accumulated turnover number (TON) of 2880 mol of product per mol of palladium. However, in the fourth cycle, the spectroscopic NMR yield dropped to 33%, indicating a clear erosion of the NG catalytic activity. To better understand the drop in activity, the hybrid NG recovered after the fourth catalytic cycle was analyzed by TEM, XPS and ICP-MS. The TEM image revealed the presence of large Pd aggregates, reaching up to several hundreds of nanometers (Figure 6A). Moreover, XPS analyses proved that most of the palladium (93%) recovered at this stage was oxidized to Pd(II) (Figure 6B and Table 2). S8 andS9).

Moreover, ICP-MS analysis of the isolated nanogel revealed an important loss (about 80 wt%) of palladium from the polymeric nanogel support. The nanogel recovered after the fourth run contained only 0.2 wt% Pd instead of the initial 1 wt%. Table 2 summarizes the palladium content and oxidation state of the hybrid nanogel before and after catalysis (determined by ICP-MS and XPS). It is noteworthy to mention that the integrity of the polymeric nanogel architecture was maintained in the course of the four catalytic cycles. By DLS no significant change in the PdNPs@NG size distribution was observed (see Supporting Information Figure S6). These combined analyses point out that palladium leaches out of the NG in the course of the reaction, and that large Pd aggregates are formed outside the nanogels. These Pd aggregates are hardly protected -as no stabilizing polymer chain is present -and thus more prone to oxidation. The size increase (up to hundreds of nanometers) could be caused by Ostwald ripening [27] in absence of stabilizer, and it confirms that the small nanoparticles initially formed by the reduction of Pd(OAc)2 with ethanol were generated in the confined NG core. It should be reminded that in the initial TEM micrographs no bigger particles or particles aggregates were observed. All in all, we attribute the activity drop to the combination of palladium loading reduction, virtual absence of Pd(0) species and decrease in surface area.

Entry

Pd content (wt%) Oxidation state (%) [a] ICP-MS XPS [b] Pd II Pd 0 1 Before catalysis 0.95 1.3 24 76 2 After 4 cycles (2 h each) 0.22 0.5 93 7 [a] Atomic percentage from the deconvolution of the Pd 3d XPS peak. [b] Wt% from the integration of all XPS peaks: C 1s, N 1s, O 1s and Pd 3d.

Finally, the amount of palladium leached into the organic phase during catalysis was assessed. To this end, after the first cycle and subsequent catalyst recovery by precipitation in diethyl ether, the supernatant was washed with water and evaporated to dryness, allowing to determinate the Pd contents in both the organic phase and the corresponding aqueous phase. ICP-MS revealed that the organic phase contained 77 ppm of palladium, which, considering 1.4 wt% of Pd in the PdNP@NG, corresponded to about 20% of the total palladium introduced in the catalytic reaction (Table 3). In contrast, the aqueous phase contained only traces of the metal (0.04 ppm that corresponds to 2 % of the total Pd introduced). Palladium Leaching test /three phase test Palladium leaching from the PdNP@NG to the organic solution [START_REF]solubility of the support under catalytic conditions[END_REF] could occur at different stages: either early in the catalytic reaction, meaning that most of the C-C coupling reaction would occur outside of the nanogel, or at the end of the catalytic reaction, after that most of the substrate is consumed, or even during the work-up, i.e. during the recovery of the NG by precipitation. In order to unambiguously determine when Pd leaching occurs, we decided to run some test reactions. We setup a typical three-phase test [START_REF] Rebek | [END_REF] using either a polymeric palladium scavenger or a polymeric substrate, which cannot diffuse to the core of the polymeric NG due to steric and entropic reasons. [START_REF]The polymeric scavengers can only sequester the palladium species that are leached out of the nanogel. Analogously, polymeric substrates can only undergo Mizoroki-Heck coupling if there are active palladium species outside the NG[END_REF] First, poly(4-vinylpyridine) (P4VP) was tested as a palladium scavenger. [31] Two reactions were run in parallel in which 1 mL of DMAc, in the presence or absence of P4VP, was added after 30 min reaction. [32] The kinetics is shown in Figure 7. The observed instantaneous inhibition of the reaction after the addition of P4VP, i.e. the palladium sequestrating polymer, indicates that after 30 min (at the latest) most of the active palladium species were outside of the nanogel.

Concerning the use of polymeric substrates, our initial approach was to use a PEGylated ester of 4-bromo-benzoic acid. However, this substrate gave no conversion, even when using soluble Pd(OAc)2 as catalyst. This result emphasizes the importance of a judicious choice of substrates for the threephase test, and, as already pointed out, [29d-f] alerts that the absence of catalysis is a necessary, but not a sufficient condition to prove a purely heterogeneous catalytic behavior. We next performed our test on the commercially available poly(ethylene glycol) methacrylate (Mn = 1.1 kg mol -1 ), which is expected to react only in the second step of the catalytic cycle (the carbopalladation step). In this case, the reaction was successful, affording, after 2h at 120°C, 80% yield of products with 60% selectivity toward the bis-arylated product (Scheme 6). Scheme 6. Mizoroki-Heck reaction with polymeric methacrylate substrate. Reaction conditions: i) 4-bromoacetophenone (0.25 mmol), PEG-methacrylate (0.425 mmol), NaOAc (0.3 mmol), PdNp@NG (0.1 mol% Pd) and DMAc (0.60 mL), 120°C, 2 h. PEG-methacrylate Mn = 1.1 kg mol -1 .

These results clearly indicate that palladium is leaching out of the nanogel in an early stage of the reaction, and that these soluble palladium species (outside the nanogel) are responsible for most, if not all, the catalytic activity of the hybrid nanogel. We estimate the amount of palladium leached out by the end of the reaction to be about 20% of the total palladium introduced into the reaction. This figure is in agreement with the 20% of palladium found in the organic phase, by ICP-MS, and with the total amount of palladium lost in the course of 4 catalytic cycles (80%), as shown in Tables 2 and3, respectively. The extent of palladium leaching is in line with the observed behavior in Pd NPs stabilized in zeolites, or dispersed in ionic liquids during the Mizoroki-Heck reaction. [33] This scenario is also in agreement with NG activity being of the same order of magnitude as those of soluble/homogeneous systems, but much better than Pd nanoparticles supported in polymeric solid, supports where leaching was shown to be irrelevant. [21] With this in mind, we propose a plausible reaction mechanism for this PdNP@NG catalyzed transformation (Scheme 7). Oxidative addition of the halo-arene to Pd(0) generates the corresponding σ-aryl-palladium complex A (ArPdX), expected to be soluble in the reaction media and stable enough to diffuse outside the nanogel. Subsequent alkene coordination to complex A followed by syn carbopalladation leads to complex B, which, after syn-β-hydride elimination, affords the arylated alkene and hydridopalladium(II) complex C (HPdX). Finally, reversible reductive elimination of C regenerates the active Pd(0) complex, thereby closing the catalytic cycle. The base shifts this equilibrium toward the Pd(0) catalyst by scavenging the generated hydrogen halide. During catalyst turnover, the initially tiny Pd NPs become larger and larger Pd aggregates with decreasing oxidative addition power. The proposed catalyst life-cycle is in agreement with the most commonly accepted reactivity of palladium nanoparticles as catalyst in the Mizoroki-Heck reaction in organic solvents. [34] Scheme 7. Proposed catalyst life-cycle of the hybrid PdNP@NG.

Conclusions

In conclusion, we have shown that RAFT dispersion polymerization can be a useful approach for the preparation of well-defined core-shell NG functionalized with amino groups in the core. The rational design of the core afforded a NG that could be readily doped with Pd(0) NPs. The hybrid NG could be isolated a solid powder, and the Pd(0) NPs in its core showed remarkable stability for long periods (>1 year), even in the presence of air and moisture. Therefore, the NG reported in this work is a readily available and easy to handle Pd(0) NP reservoir.

The hybrid NG displayed good activities as catalyst in the Miroroki-Heck reaction between n-butyl acrylate and activated bromo-arene and iodo-arenes. Moreover, this hybrid catalyst could be recycled up to three times by precipitation, reaching an accumulated TON of 2880. However, the catalyst lost most of its activity after three cycles, which was attributed to palladium leaching out of the NG into the organic solution.

Palladium leaching seems to occur during the catalysis, probably after the oxidative addition step, which is expected to take place in the core of the NG. This leaching represents a loss of about 20% per cycle of total palladium introduced in the system.

We have thus demonstrated that core-shell NGs are promising catalyst supports, showing a quasi-homogeneous behavior while allowing catalyst recycling, although improvements in the NG design will be needed for future developments of this strategy in order to avoid palladium leaching into the organic phase during catalysis. Notably, one of the main advantages of using RAFT dispersion polymerization is the ability to fine tune the nanogel architecture. The coordinating/stabilizing nature of the core polymer and the cross-linking degree will be considered in future developments of this project.

Figure 1 .

 1 Figure 1. Size exclusion chromatography in DMF (LiBr, 1 wt%) of: a) the PDMAAm-macroRAFT (dotted red line), b) PDMAAm-block-P(DEAAm-co-DMAEA) linear polymer (dashed blue line), and c) PDMAAm-block-P(DEAAmco-DMAEA-co-MBA): NG (solid black line).

Figure 2 .

 2 Figure 2. Size and morphology of core-shell nanogel. (A) TEM image of a dried aqueous NG solution (0.01 wt%); (B) hydrodynamic diameter distribution obtained by DLS at 20°C of NG solutions (0.1 wt%) in water (solid line) and DMAc (dashed line).

Figure 3 .

 3 Figure 3. Size and oxidation state of palladium NPs: (A) TEM image, (B) Size distribution of the Pd NPs, and (C) XPS Pd 3d spectrum of the hybrid PdNP@NG (solid line) and its deconvolution into Pd(0) (dashed blue lined) and Pd(II) (dotted red line), full spectrum and assignment in the Supporting Information (Figure S5, TableS5 and S6).

Figure 4 .

 4 Figure 4. Kinetics of the reaction of 4-bromo-acetophenone with n-butyl acrylate using PdNP@NG catalyst () versus Pd(OAc)2 (homeopathic, ). Reaction conditions: 4-bromoacetophenone (0.25 mmol), n-butyl acrylate (0.425 mmol), NaOAc (0.3 mmol), 0.1 mol% Pd and DMAc (0.60 mL), under argon atmosphere, 120°C. Spectroscopic yields determined by 1 H NMR.

  [a] General reaction conditions: aryl halide (0.25 mmol), n-butyl acrylate (0.425 mmol), NaOAc (0.3 mmol), PdNP@NG (0.1 mol% Pd), DMAc (0.60 mL), 120°C, 2h.[b] Isolated yield after column chromatography.[c] Reaction conditions: 4-bromoacetophenone (2.5 mmol), n-butyl acrylate (4.25 mmol), NaOAc (3 mmol), DMAc (6 mL), PdNP@NG (0.1 mol% Pd), 120°C, 2h.[d] TOF = 1167 h -1 at 30 min (58 % spectroscopic yield)[e] No reaction.[f] Average of 2 runs. [g] TOF = 1100 h -1 at 30 min (55 % spectroscopic yield).

Scheme 5 .

 5 Scheme 5. Mizoroki-Heck reaction between 4-bromo-acetophenone and different olefins. Reaction conditions: i) 4-bromo-acetophenone (0.25 mmol), alkene (0.425 mmol), NaOAc (0.3 mmol), PdNP@NG (0.1 mol% Pd), and DMAc (0.60 mL), 2h, 120°C.

Figure 5 .

 5 Figure 5. Spectroscopic NMR yield (solid white bar), isolated yield (diagonally striped bar) and accumulated turnover number (black dots) for the recycling of the hybrid PdNP@NG. Reaction conditions: Reaction conditions per cycle: 4bromoacetophenone (2.5 mmol), n-butyl acrylate (4.25 mmol), NaOAc (3 mmol), DMAc (6 mL), 120°C, 2h, PdNP@NG (0.1 mol% Pd) was added in the first cycle and recycled thereafter.

Figure 6 .

 6 Figure 6. (A) TEM image and (B) XPS Pd 3d spectrum XPS Spectra of the hybrid PdNP@NG after 4 catalytic cycles (solid black line) and its deconvolution into Pd(0) (dashed blue lined) and Pd(II) (dotted red line), full spectrum in the supporting information (Figure S7, TableS8 and S9).

Figure 7 .

 7 Figure 7. Effect of the addition of poly(4-vinylpyridine) (Mw = 50 kg mol -1 ) to the Mizoroki-Heck reaction catalyzed by PdNP@NG. Reaction conditions: 4bromoacetophenone (0.25 mmol), n-butyl acrylate (0.425 mmol), NaOAc (0.3 mmol), PdNP@NG (0.1 mol% Pd) and DMAc (4 mL), after 30 minutes reaction 1 mL of DMAc is added, with or without P4VP (50 mg). Spectroscopic yields determined by 1 H NMR.

Table 2 .

 2 Palladium content and oxidation state in the hybrid nanogel before and after catalysis.

Table 3 .

 3 Palladium leaching out of the nanogel.

	Entry	Phase		Pd content
			ppm	% total Pd
	1	Organic	76.6	19
	2	Aqueous	0.04	2

Acknowledgements

The authors would like to acknowledge Labex Michem for funding the NACRE project as well as a post-doc fellowship (A.P.C.). CNRS, UPMC and Horizon 2020 European Research Area Network, ERANet-LAC (ref. ELAC2014/BEE-0341) project CelluloseSynThech are also acknowledged for financial support. ICP-MS analyses were supported by IPGP multidisciplinary program PARI, and by Region Île-de-France SESAME Grant no. 12015908. UPMC collaborators, Omar Khaled (IPCM) for the HRMS analyses, Patricia Beaunier (LRS) and Jean-Michel Guigner (IMPMC) for TEM analyses and Christophe Calers (LRS) for XPS analysis are thanked. The authors would like to acknowledge Dr. François Stoffelbach for valuable discussions. Keywords: Core-Shell Nanogel • RAFT-Polymerization • Palladium • Mizoroki-Heck Reaction • Recycling

Experimental Section

Materials N,N-Diethylacrylamide (DEAAm) [35] and the macromolecular RAFT agent (PDMAAm-macroRAFT, Mn LS = 14.2 kg.mol -1 , Ð = 1.3) [16] were synthesized according to published procedures. N,N-Dimethylacrylamide (DMAAm), N,N-diethylacrylamide (DEAAm) and N,N-dimethylaminoethyl acrylate (DMAEA) were distilled prior to polymerization. All other reagents and solvents were obtained from commercial sources and used as received.

Synthesis of NG

The PDMAAm-macroRAFT (590 mg, 41.5 mol), DEAAm (1.48 g, 11.6 mmol), DMAEA (196 mg, 1.4 mmol), MBA (105 mg, 0.68 mmol) and 1,3,5-trioxane (123 mg, 1.4 mmol) were in dissolved 45 mL of distilled water, in a septum-sealed flask. The mixture was purged with argon in an ice-bath for 30 min. After heating to 70°C, 1 mL (5.5 mol) of an aqueous V-50 stock solution (5.5 mM) was added under inert atmosphere. The reaction was nearly complete after 30 min and it was stopped after 1h by cooling in an ice bath and opening to air. Conversion was determined by the integration of olefinic protons using 1,3,5-trioxane as reference. The reaction was dialyzed against distilled water and the final solution freezedried affording the core-shell nanogel NG as a white powder (1.5 g).

Synthesis of PdNP@NG

A solution of NG (1.0 g) and Pd(OAc)2 (33 mg, 0.15 mmol) in 40 mL of dichloromethane was purged with argon and stirred under inert atmosphere. After 24h the reaction mixture was poured into 150 ml of diethyl ether, the precipitate washed with diethyl ether (3 x 50 mL) and dried under vacuum. The dark red powder was dissolved in 30 mL of dichloromethane and purged with argon. Then 6 mL of ethanol were added the reaction mixture was heated to 40°C under inert atmosphere. After 24h the reaction mixture was poured into 150 ml of diethyl ether and the precipitate washed with diethyl ether (3 x 50 mL) and dried under vacuum. The brown solid was dissolved in 20 mL of distilled water and dialyzed against distilled water. The final solution freeze-dried affording the PdNP@NG as a brown powder (0.6 g).

General Mizoroki-Heck reaction

The arene (0.25 mmol) and sodium acetate (25 mg, 1.2 eq, 0.3 mmol) were added to a solution of alkene (1.7 eq, 0.425 mmol) in N,Ndimethylacetamide (DMAc) (0.3 mL). Then 0.3 mL (0.25 µmol of Pd, 0.1 mol%) of a stock solution of PdNP@NG in DMAc (7 mg/mL) were added and the mixture purged with argon for 15 min. The reaction was stirred at 120°C under inert atmosphere for 2h. The crude product was purified by Combi-flash chromatography in silica gel using a gradient from pentane to pentane/diethyl ether (4:1) as eluent. The fractions were evaporated to dryness and dried under vacuum.

Catalyst recycling

Catalyst recycling experiments were run using 4-bromoacetophenone and n-butyl acrylate as substrates following the above protocol scaled-up ten times. The catalyst was recovered after 2h reaction time by pouring the reaction mixture into cold diethyl ether (100 mL). The recovered solid was dissolved in 10 mL of distilled water, dialyzed against distilled water, freeze-dried and used as recovered in the following catalytic cycle.

Full instrumental details and product characterization are given in the supporting information.