Delphine Destoumieux-Garzón

Rafael Diego Rosa

Paulina Schmitt

Cairé Barreto

Jeremie Vidal-Dupiol

Guillaume Mitta

Yannick Gueguen

Evelyne Bachère

Antimicrobial peptides in marine invertebrate health and disease

Keywords: invertebrate immunity, polymicrobial disease, vibrio, immune suppression, abiotic stress, Invertebrate immunity, polymicrobial disease, vibrio, immune suppression, resistance, abiotic stress

Aquaculture contributes more than one-third of the animal protein from marine sources worldwide. A significant proportion of aquaculture products are derived from marine protostomes that are commonly referred to as "marine invertebrates". Among them, penaeid shrimp (Ecdysozosoa, Arthropoda) and bivalve molluscs (Lophotrochozoa, Mollusca) are economically important. Mass rearing of arthropods and molluscs causes problems with pathogens in aquatic ecosystems that are exploited by humans. Remarkably, species of corals (Cnidaria) living in non-exploited ecosystems also suffer from devastating infectious diseases that display intriguing similarities with those affecting farmed animals. Infectious diseases affecting wild and farmed animals that are present in marine environments are predicted to increase in the future. This paper summarizes the role of the main pathogens and their interaction with host immunity, with a specific focus on antimicrobial peptides (AMPs) and pathogen resistance against AMPs. We also provide a detailed review of penaeid shrimp AMPs and their role at the interface between the host and its resident/pathogenic microbiota. We also briefly describe the relevance of marine invertebrate AMPs in an applied context.

Economic relevance of marine invertebrates

Oceans and seas cover two-thirds of our planet; provide ecosystem services, such as fishing, aquaculture, carbon sequestration, regulation of water quality and nutrient storage; and support numerous recreational activities, all of which significantly contribute to employment and economic activity [START_REF]Millennium, Assessment & Ecosystem 2005 Ecosystems and Human Well-being: Synthesis[END_REF]. In 2012, aquaculture provided an unprecedented total of 66.6 million tons of seafood, including 175 species of "marine invertebrates", mainly crustaceans (Ecdysozosoa, Arthropoda) and molluscs (Lophotrochozoa, Mollusca) [2]. Farmed crustaceans accounted for 9.7% (6.4 million tons) of the seafood production by volume, but 22.4% (US$30.9 billion) by value. Shrimp is currently the largest single commodity in terms of value, with the main cultivated species being Litopenaeus vannamei and Penaeus monodon. In 2012, molluscs, such as oysters, mussels, scallops and clams, accounted for 20.5% (13.7 million tons) of seafood production. Molluscs are essentially produced for food, but they also contributed to 22,400 tons of non-food products, such as pearls and seashells for ornamental and decorative uses. However, disease outbreaks have heavily impacted this intensifying production over the past few years, and emerging infectious diseases are predicted to increase in the future in both wild and farmed animals as a result of climate change [START_REF] Burge | Climate change influences on marine infectious diseases: implications for management and society[END_REF].

Infectious diseases affecting marine invertebrates

The health status of marine invertebrates is intimately related to the microbial communities that are present in the aquatic environment, which include both commensals and opportunistic pathogens. While microorganisms hosted by invertebrates help maintain homeostasis, under stressful conditions, some can become highly virulent and severely damage their host [START_REF] Schmitt | The Antimicrobial Defense of the Pacific Oyster, Crassostrea gigas. How Diversity may Compensate for Scarcity in the Regulation of Resident/Pathogenic Microflora[END_REF]. In coastal environments and lagoons, marine invertebrates are exposed to multiple abiotic stresses, which can be of anthropogenic origin. Thermal stress, high density [START_REF] Bruno | Thermal stress and coral cover as drivers of coral disease outbreaks[END_REF], and nutrient rich environments [START_REF] Vega Thurber | Chronic nutrient enrichment increases prevalence and severity of coral disease and bleaching[END_REF] are factors favouring infectious diseases in the wild as well as in intensive farming. Remarkably, while marine invertebrates are incredibly diverse in terms of phylogeny and ecological niche, disease patterns are repeatedly found across species. These diseases include temperature-dependent vibrioses [START_REF] Ben-Haim | Vibrio coralliilyticus sp. nov., a temperature-dependent pathogen of the coral Pocillopora damicornis[END_REF][START_REF] Fukui | Environmental determinants correlated to Vibrio harveyi-mediated death of marine gastropods[END_REF] and polymicrobial diseases [START_REF] Petton | Crassostrea gigas mortality in France: the usual suspect, a herpes virus, may not be the killer in this polymicrobial opportunistic disease[END_REF][START_REF] Barneah | Characterization of black band disease in Red Sea stony corals[END_REF]. Differences are observed in the susceptibility of the animals, from larvae to juveniles and adults, and in the diversity of the pathogens (vibrios, viruses, etc.) that affect each developmental stage. Relevant examples of infectious diseases are presented below within three different phyla of marine invertebrates (Arthropoda, Mollusca and Cnidaria) that are directly/indirectly and intensively/extensively exploited by humans in a diversity of ecosystems.

Arthropods. Diseases in marine arthropods are dominated by those described in penaeid shrimp aquaculture, which is characterized by intensive cultural practices favouring disease development. Twenty viruses are known to infect penaeid shrimp. Two types of viruses cause major viral diseases, namely, DNA viruses, such as the monodon baculovirus (MBV) [START_REF] Lightner | A baculovirus-caused disease of penaeid shrimp, Penaeus monodon[END_REF], the white-spot syndrome virus (WSSV) [START_REF] Lo | White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods[END_REF], the hepatopancreatic parvovirus (HPV) and the infectious hypodermal and hematopoietic virus (IHHNV) [START_REF] Lightner | Epizootiology, distribution and the impact on international trade of two penaeid shrimp viruses in the Americas[END_REF], and RNA viruses, such as the yellow-head virus (YHV), the Taura syndrome virus (TSV) [START_REF] Dhar | Genomic organization, biology, and diagnosis of Taura syndrome virus and yellowhead virus of penaeid shrimp[END_REF] and the infectious myonecrosis virus (IMNV) [START_REF] Poulos | Purification and characterization of infectious myonecrosis virus of penaeid shrimp[END_REF]. WSSV is the most severe threat for farmed adult shrimp worldwide and is one of the best studied crustacean viruses [START_REF] Sánchez-Paz | White spot syndrome virus: An overview on an emergent concern[END_REF]. Bacterial infections, particularly vibrioses, are a major concern for the production of shrimp larvae and juveniles. V. harveyi, and V. vulnificus are associated with larvae mortality [START_REF] Bachère | Antiinfectious immune effectors in marine invertebrates: potential tools for disease control in larviculture[END_REF], whereas V. damsela, V. alginolyticus, V. parahaemolyticus, V. penaeicida and V. nichripulchritudo cause disease outbreaks in shrimp nurseries or grow-out ponds [START_REF] Bachère | Antiinfectious immune effectors in marine invertebrates: potential tools for disease control in larviculture[END_REF]. In 2010, a new shrimp disease that affects postlarvae has emerged from Asia [START_REF] Lee | The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin[END_REF]. This acute hepatopancreatic necrosis disease (AHPND) is caused by a highly virulent strain of V. parahaemolyticus, which has acquired a virulence plasmid encoding a pore forming bacterial toxin that is as toxic as the insecticidal Bacillus Cry toxin [START_REF] Lee | The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin[END_REF].

Molluscs from the shellfish industry are affected by a variety of infectious diseases whose importance largely depends on the degree of exploitation and ecosystem health. The most significant epizootic events are caused by bacteria from the Vibrio genus [START_REF] Romalde | New Vibrio species associated to molluscan microbiota: a review[END_REF][START_REF] Travers | Bacterial diseases in marine bivalves[END_REF], viruses from the Malacoherpesviridae family [START_REF] Segarra | Detection and description of a particular Ostreid herpesvirus 1 genotype associated with massive mortality outbreaks of Pacific oysters, Crassostrea gigas, in France in 2008[END_REF] and protozoans from the Perkinsus, Marteilia, Bonamia and Haplosporidium genera [START_REF] Bachère | Antiinfectious immune effectors in marine invertebrates: potential tools for disease control in larviculture[END_REF]. Some of these microorganisms can affect a broad range of mollusc species at all life stages around the world, while others are highly species-specific. The epizootic events that they cause are frequently devastating. Over the past few decades, abnormal mortalities of juvenile C. gigas oysters have affected the US, Japan, Australia and Western Europe. Those mortalities of complex aetiology are due to a temperature-dependent polymicrobial disease that involves pathogenic Vibrio strains of the Splendidus clade and the ostreid herpes virus OsHV-1 µvar [START_REF] Petton | Crassostrea gigas mortality in France: the usual suspect, a herpes virus, may not be the killer in this polymicrobial opportunistic disease[END_REF][START_REF] Bachère | Antiinfectious immune effectors in marine invertebrates: potential tools for disease control in larviculture[END_REF][START_REF] Lemire | Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters[END_REF]. Vibrios also cause diseases at other oyster developmental stages. Vibrio aestuarianus is responsible for mortality of adult oysters [START_REF] Goudenège | A single regulatory gene is sufficient to alter Vibrio aestuarianus pathogenicity in oysters[END_REF], whereas V. tubiashi causes a necrotic disease in hatcheries [24].

Cnidarians, which live in coastal marine systems and lagoons, also suffer from infectious diseases. Among the known pathogens, all of the classical agents (Eubacteria, cyanobacteria, fungi and viruses) have been described [START_REF] Bourne | Microbial disease and the coral holobiont[END_REF][START_REF] Soffer | Potential role of viruses in white plague coral disease[END_REF]. Most diseases were observed in corals. One of the most famous coral diseases is type I White Band Disease (WBD). Described for the first time in the 1970s, WBD caused the loss of up to 95% of the Acroporid found throughout the great Caribbean area [START_REF] Aronson | White-band disease and the changing face of Caribbean coral reefs[END_REF]. However, as with many coral diseases, the causative agents of type I WBD are unknown and do not fully satisfy the Koch postulate or correspond to microbial consortia [START_REF] Bourne | Microbial disease and the coral holobiont[END_REF]. Another well-known example is Black Band Disease, which is caused by cyanobacteria assemblages and other unidentified heterotrophic microbes [START_REF] Barneah | Characterization of black band disease in Red Sea stony corals[END_REF][START_REF] Sekar | Microbial communities in the surface mucopolysaccharide layer and the black band microbial mat of black banddiseased Siderastrea siderea[END_REF]. Many of the known coral pathogens belong to the Vibrio genus. While V. harveyi/charcariae is the causative agent of type II WBD and other "White Syndroms" [START_REF] Luna | Bacteria associated with the rapid tissue necrosis of stony corals[END_REF][START_REF] Luna | Vibrio harveyi as a causative agent of the White Syndrome in tropical stony corals[END_REF], V. shiloi and V. coralliilyticus are responsible for bacteria-induced bleaching in corals and tissue lysis in several other cnidarians [START_REF] Ganz | Defensins: antimicrobial peptides of innate immunity[END_REF][START_REF] Bachère | The new insights into the oyster antimicrobial defense: Cellular, molecular and genetic view[END_REF][START_REF] Augustin | Activity of the novel peptide arminin against multiresistant human pathogens shows the considerable potential of phylogenetically ancient organisms as drug sources[END_REF][START_REF] Vidal-Dupiol | Innate Immune Responses of a Scleractinian Coral to Vibriosis[END_REF].

Antimicrobial peptides (AMPs) in marine invertebrate immunity, a focus on penaeid shrimp.

The economic consequences of infectious diseases affecting farmed bivalve molluscs and arthropods (crustaceans) have motivated a substantial research effort, which has considerably enriched our knowledge of the immune system of protostomes. Similarly, coral bleaching events are a threat for society and have inspired studies on the interaction of the immune system of cnidarians with its resident microbiota and pathogens. To date, AMPs are among the best described immune effectors of marine invertebrates.

Diversity and specificity of AMPs in the immune response of marine invertebrates

Similar to AMPs from other phyla [START_REF] Schmitt | An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes[END_REF], most of the AMPs characterized in marine arthropods, including molluscs and cnidarians, are cationic and hydrophobic and target essential components of microbial cell walls and membranes, which determines their spectrum of activity [START_REF] Schmitt | Insight into invertebrate defensin mechanism of action: oyster defensins inhibit peptidoglycan biosynthesis by binding to lipid II[END_REF]. A high diversity of mechanisms of action has been reported for AMPs from marine invertebrates and is described in detail for some families. For example, mollusc defensins, which are essentially active against Gram-positive bacteria, bind to lipid II, the precursor of peptidoglycan [START_REF] Gonzalez | Evidence of a bactericidal permeability increasing protein in an invertebrate , the Crassostrea gigas Cg-BPI[END_REF]. Arthropod antilipopolysaccharide factors (ALFs) and mollusc bactericidal/permeability-increasing protein (BPI), which are essentially active against Gram-negative bacteria, bind to lipopolysaccharide (LPS) [START_REF] Tanaka | Limulus anti-LPS factor: an anticoagulant which inhibits the endotoxin mediated activation of Limulus coagulation system[END_REF][START_REF] Muta | Primary structure of antilipopolysaccharide factor from American horseshoe crab, Limulus polyphemus[END_REF][START_REF] Petit | A hemocyanin-derived antimicrobial peptide from the penaeid shrimp adopts an alpha-helical structure that specifically permeabilizes fungal membranes[END_REF]. Finally, crustacean PvHCt, which is strictly antifungal, permeabilizes the fungal plasma membrane [START_REF] König | The diversity and evolution of anuran skin peptides[END_REF]. Those AMP families have been generated through different patterns of diversification (gene duplication, gene copy number variation, recombination and allelic polymorphisms) [START_REF] Schmitt | Insight into invertebrate defensin mechanism of action: oyster defensins inhibit peptidoglycan biosynthesis by binding to lipid II[END_REF] due to multiple evolutionary drivers and have given rise to functional divergence. Some families appear to have evolved within specific phyla or species [START_REF] Ganz | Defensins: antimicrobial peptides of innate immunity[END_REF], whereas others are found in a diversity of phyla [START_REF] Bachère | The new insights into the oyster antimicrobial defense: Cellular, molecular and genetic view[END_REF]. Most AMP families from molluscs, including defensins, big-defensins and BPI (for recent review see [START_REF] Augustin | Activity of the novel peptide arminin against multiresistant human pathogens shows the considerable potential of phylogenetically ancient organisms as drug sources[END_REF]), are also found in other protostomes (Ecdysozoa) and/or in deuterostomes (Mammalia). In contrast, in cnidarians, taxonomically restricted AMPs have been described, such as arminins, which are among the most highly expressed genes in Hydra [START_REF] Vidal-Dupiol | Innate Immune Responses of a Scleractinian Coral to Vibriosis[END_REF], or damicornin in the coral Pocillopora damicornis [START_REF] Saito | A novel big defensin identified in horseshoe crab hemocytes: isolation, amino acid sequence, and antibacterial activity[END_REF]. In marine arthropods, the pioneering studies in Chelicerata (horseshoe crabs) have identified both taxonomically restricted (tachyplesins, ALFs) and more widely distributed AMPs (big defensins) [START_REF] Muta | Primary structure of antilipopolysaccharide factor from American horseshoe crab, Limulus polyphemus[END_REF][START_REF] Nakamura | Tachyplesin, a Class of Antimicrobial Peptide from the Hemocytes of the horseshoe crab (Tachypleus tridentatus). Isolation and chemical structure[END_REF][START_REF] Rosa | Antimicrobial peptides in crustaceans[END_REF]. Finally, in decapod crustaceans, highly diverse AMPs were characterized that are specific to penaeid shrimp (penaeidins, stylicins), crustaceans (crustins) or arthropods (ALFs) [START_REF] Smith | Antimicrobial proteins: From old proteins, new tricks[END_REF][START_REF] Bachère | Insights into the antimicrobial defense of marine invertebrates: The penaeid shrimps and the oyster Crassostrea gigas[END_REF]. The best known AMPs from crustaceans were characterized in penaeid shrimp, and many of them are composed of structural domains that have distinct biological functions [START_REF] Smith | Antimicrobial proteins: From old proteins, new tricks[END_REF][START_REF] Li | Signaling pathways regulating innate immune responses in shrimp[END_REF]. A description of the major AMP families in shrimp is provided below. Unlike in insects, there is still little knowledge about the molecular regulation of AMPs in marine invertebrates. In shrimp, which is one of the best described organisms, some AMP families are controlled by NF-κB signalling pathways. Alternatively, mature AMPs can be stored in immune cells and are released upon challenge (see below).

Gene-encoded AMPs from penaeid shrimp

Penaeidins

Penaeidins were the first AMPs characterized in shrimp [START_REF] Destoumieux | Penaeidins , a New Family of Antimicrobial Peptides Isolated from the Shrimp Penaeus vannamei (Decapoda)[END_REF]. Those peptides, which are restricted to species of penaeid shrimp, are abundant in the circulating immune cells (the haemocytes) of L. vannamei [START_REF] Destoumieux | Penaeidins , a New Family of Antimicrobial Peptides Isolated from the Shrimp Penaeus vannamei (Decapoda)[END_REF]. They are cationic peptides (4.7-7.2 kDa; pI ~9) composed of an unconstrained N-terminal proline/arginine-rich domain followed by a C-terminal domain that contains an amphipathic helix and two coils stabilized by three disulphide bonds (Table 1) [START_REF] Destoumieux | Penaeidins , a New Family of Antimicrobial Peptides Isolated from the Shrimp Penaeus vannamei (Decapoda)[END_REF][START_REF] Yang | Solution structure of the recombinant penaeidin-3, a shrimp antimicrobial peptide[END_REF]. Penaeidins can carry post-translational modifications, such as a N-terminal pyroglutamic acid and an amidated Cterminus. AMPs from this diverse family fall into 4 subgroups (PEN1/2, PEN3, PEN4 and PEN5, Table 1), whose specific sequence signature and biochemical features have been used to standardize their nomenclature after the name of the shrimp species and the penaeidin subgroup [START_REF] Gueguen | PenBase, the shrimp antimicrobial peptide penaeidin database: Sequence-based classification and recommended nomenclature[END_REF]. Each subgroup is encoded by distinct genomic sequences [START_REF] Gueguen | PenBase, the shrimp antimicrobial peptide penaeidin database: Sequence-based classification and recommended nomenclature[END_REF][START_REF] Kang | Characterization and expression of a new subfamily member of penaeidin antimicrobial peptides (penaeidin 5) from Fenneropenaeus chinensis[END_REF]. While the PEN3 gene is widely distributed among species of penaeid shrimp, PEN2, PEN4 and PEN5 genes are restricted to a given species of shrimp [START_REF] Tassanakajon | Cationic Antimicrobial Peptides in Penaeid Shrimp[END_REF]. PEN genes are highly and constitutively expressed in haemocytes of healthy individuals [START_REF] Cuthbertson | Diversity of the penaeidin antimicrobial peptides in two shrimp species[END_REF] [START_REF] Destoumieux | Penaeidins, antimicrobial peptides with chitin-binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge[END_REF]. Penaeidins, which are stored in haemocyte granules, are released in response to microbial challenge [START_REF] Destoumieux | Penaeidins, antimicrobial peptides with chitin-binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge[END_REF]. Penaeidin sequence diversity translates into diverse biological activities [START_REF] Muñoz | Expression and distribution of penaeidin antimicrobial peptides are regulated by haemocyte reactions in microbial challenged shrimp[END_REF]. Most penaeidins from subgroups PEN1/2, PEN3 and PEN4 are active against Gram-positive bacteria and filamentous fungi, but not Gram-negative bacteria. In contrast, a PEN5 member (Fenchi PEN5) from Fenneropenaeus chinensis is active against Gram-negative bacteria [START_REF] Kang | Characterization and expression of a new subfamily member of penaeidin antimicrobial peptides (penaeidin 5) from Fenneropenaeus chinensis[END_REF]. Penmon PEN5 from P. monodon also participates in the shrimp antiviral defence against WSSV [START_REF] Cuthbertson | Diversity in penaeidin antimicrobial peptide form and function[END_REF]. Although little is known about the mechanism of action of penaeidins, the function of the proline/arginine-rich domain was investigated in different penaeidins. This domain was devoid of antimicrobial activity in PEN3 [START_REF] Destoumieux | Penaeidins, antimicrobial peptides with chitin-binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge[END_REF], but possessed both antifungal and antibacterial activity in PEN4 [START_REF] Woramongkolchai | The possible role of penaeidin5 from the black tiger shrimp, Penaeus monodon, in protection against viral infection[END_REF]. Interestingly, this domain was also reported to behave as a cytokine by attracting haemocytes towards sites of injury [START_REF] Cuthbertson | A new class (penaeidin class 4) of antimicrobial peptides from the Atlantic white shrimp (Litopenaeus setiferus) exhibits target specificity and an independent proline-richdomain function[END_REF]. In addition, the cysteine-rich domain of PEN3 was proposed to mediate penaeidin antifungal activity by binding to chitin [START_REF] Destoumieux | Penaeidins, antimicrobial peptides with chitin-binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge[END_REF]. The ability of penaeidins to bind to the chitin exoskeleton suggests that penaeidins could play a role in wound healing and/or moulting in shrimp [START_REF] Li | Signaling pathways regulating innate immune responses in shrimp[END_REF].

Anti-lipopolysaccharide factors (ALFs)

ALFs form a diverse family of AMPs that are composed of both cationic and anionic polypeptides. First identified in horseshoe crabs and later in penaeid shrimps [START_REF] Li | Prolinerich domain of penaeidin molecule exhibits autocrine feature by attracting penaeidinpositive granulocytes toward the wound-induced inflammatory site[END_REF] and other crustaceans [START_REF] Tassanakajon | Cationic Antimicrobial Peptides in Penaeid Shrimp[END_REF], ALFs were named "anti-LPS factors" for their immunomodulatory function. ALFs are able to inhibit the LPS-mediated activation of the limulid coagulation system [START_REF] Muta | Primary structure of antilipopolysaccharide factor from American horseshoe crab, Limulus polyphemus[END_REF][START_REF] Petit | A hemocyanin-derived antimicrobial peptide from the penaeid shrimp adopts an alpha-helical structure that specifically permeabilizes fungal membranes[END_REF]. ALFs contain a hydrophobic N-terminal region with two conserved cysteine residues. The disulphidebond delimits a β-hairpin structure that is referred to as the LPS-binding domain [START_REF] Gross | Immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopancreas in the Pacific White Shrimp, Litopenaeus vannamei, and the Atlantic White Shrimp, L. setiferus[END_REF]. Most ALFs bind to Lipid A from Gram-negative bacteria, but they can also interact with lipoteichoic acid (LTA) from Gram-positive bacteria [START_REF] Hoess | Crystal structure of an endotoxin-neutralizing protein fromothe horseshoe crab, Limulus anti-LPS factor[END_REF] and βglucan from fungi [START_REF] Somboonwiwat | Localization of antilipopolysaccharide factor (ALF Pm 3) in tissues of the black tiger shrimp, Penaeus monodon, and characterization of its binding properties[END_REF]. The known 3D-structures of ALFs consist of three α-helices (one at the N-terminus and two at the C-terminus) packed against a four-stranded β-sheet (Table 1) [START_REF] Gross | Immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopancreas in the Pacific White Shrimp, Litopenaeus vannamei, and the Atlantic White Shrimp, L. setiferus[END_REF][START_REF] Sun | An antilipopolysaccharide factor from red swamp cray fish, Procambarus clarkii, exhibited antimicrobial activities in vitro and in vivo[END_REF]. In shrimp, ALFs form a large and diverse family of five groups, namely, ALF-A (anionic and cationic polypeptides of 11.4-11.5 kDa), ALF-B (highly cationic polypeptides of 10.6-11.2 kDa), ALF-C (cationic polypeptides of 11-11.3 kDa), ALF-D (highly anionic polypeptides of 10.7-10.8 kDa) and ALF-E (anionic and cationic polypeptides of 11.4-12.5 kDa) (Table 1) [START_REF] Yang | NMR structure of rALF-Pm3, an antilipopolysaccharide factor from shrimp: model of the possible lipid A-binding site[END_REF][START_REF] Rosa | Functional Divergence in Shrimp Anti-Lipopolysaccharide Factors (ALFs): From Recognition of Cell Wall Components to Antimicrobial Activity[END_REF]. ALFs are encoded by separate genes and are transcribed at basal levels in healthy individuals [START_REF] Yang | NMR structure of rALF-Pm3, an antilipopolysaccharide factor from shrimp: model of the possible lipid A-binding site[END_REF]. In L. vannamei, ALF genes are differentially expressed in response to a fungal infection. Although ALF-A gene expression remains stable, the other ALF genes are inducible [START_REF] Yang | NMR structure of rALF-Pm3, an antilipopolysaccharide factor from shrimp: model of the possible lipid A-binding site[END_REF]. While ALFs can be detected in diverse shrimp tissues, results for ALFPm3 strongly suggest that the expression of ALF-B is restricted to haemocytes, which infiltrate shrimp tissues [START_REF] Hoess | Crystal structure of an endotoxin-neutralizing protein fromothe horseshoe crab, Limulus anti-LPS factor[END_REF]. ALFs are potent and broad spectrum AMPs. For example, cationic Group B ALFs are active against Gram-positive, Gram-negative bacteria, yeast, filamentous fungi and some enveloped viruses [START_REF] Smith | Antimicrobial proteins: From old proteins, new tricks[END_REF]. In contrast, anionic Group D ALFs have impaired LPS-binding properties and display very low antimicrobial activity in vitro [START_REF] Yang | NMR structure of rALF-Pm3, an antilipopolysaccharide factor from shrimp: model of the possible lipid A-binding site[END_REF].

Crustins

Crustins are antimicrobial polypeptides (6-22 kDa; pI 4-8) containing a Whey Acidic Protein (WAP) domain [START_REF] Jiang | A new group of antilipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response[END_REF]. This WAP domain, which is also found in some mammalian proteins, supports different biological functions, including antiprotease activities [START_REF] Tassanakajon | Sequence diversity and evolution of antimicrobial peptides in invertebrates[END_REF]. Crustins are composed of four members (Type I to IV) that are widely distributed across crustaceans [START_REF] Jiang | A new group of antilipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response[END_REF], which differ by their N-terminal sequence. While the sequence of Type I crustins begins with a cysteine-rich domain, Type II crustins (subtypes IIa and IIb) harbour a glycine-rich hydrophobic domain at the Nterminal position ahead of the cysteine-rich domain that is also found in Type I crustins (Table 1). Comparatively, Type III crustins can contain (or not) a short proline/arginine-domain at the N-terminus, whereas Type IV crustins are composed of two WAP domains, but do not harbour any specific N-terminal sequence (Table 1) [START_REF] Jiang | A new group of antilipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response[END_REF]. Data on crustinPm1 strongly suggest that crustins are mainly expressed by haemocytes [START_REF] Ranganathan | The whey acidic protein family: a new signature motif and three-dimensional structure by comparative modeling[END_REF]. The diversity of crustin sequences supports diverse biological functions. While Type I and type II crustins are mainly antimicrobial, their spectrum of activity varies with the crustin type/sub-type. For instance, in P. monodon, a Type IIa crustin is essentially active against Gram-positive bacteria, while Type IIb crustins are active against both Gram-positive and Gram-negative bacteria [START_REF] Ranganathan | The whey acidic protein family: a new signature motif and three-dimensional structure by comparative modeling[END_REF][START_REF] Supungul | Cloning, expression and antimicrobial activity of crustinPm1, a major isoform of crustin, from the black tiger shrimp Penaeus monodon[END_REF]. Interestingly, Type III and Type IV crustins show antimicrobial and/or antiprotease activity [START_REF] Jiang | A new group of antilipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response[END_REF], whereas Type III crustins lacking the N-terminal proline/arginine-domain and Type IV crustins from shrimp [START_REF] Amparyup | Molecular cloning, genomic organization and recombinant expression of a crustin-like antimicrobial peptide from black tiger shrimp Penaeus monodon[END_REF][START_REF] Du | A double WAP domain (DWD)-containing protein with proteinase inhibitory activity in Chinese white shrimp, Fenneropenaeus chinensis[END_REF] only have antiprotease activity [START_REF] Jiang | A new group of antilipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response[END_REF]. Type IV crustins from crabs exhibit both antimicrobial and antiprotease activities [START_REF] Du | A double WAP domain (DWD)-containing protein with proteinase inhibitory activity in Chinese white shrimp, Fenneropenaeus chinensis[END_REF][START_REF] Li | A Double WAP Domain-Containing Protein Es-DWD1 from Eriocheir sinensis Exhibits Antimicrobial and Proteinase Inhibitory Activities[END_REF].

Stylicins

Stylicins are anionic (pI 5) multi-domain peptides of 8.9 kDa that are composed of a N-terminal proline/arginine-rich domain followed by a C-terminal domain containing 13 cysteine residues (Table 1) [START_REF] Du | A novel single WAP domain-containing protein isoform with antibacterial relevance in Litopenaeus vannamei[END_REF]. The recombinant Ls-Stylicin-1 (from Litopenaeus stylirostris) is active against the filamentous fungus Fusarium oxysporum, but is not antibacterial. As observed for Group B ALFs, Ls-Stylicin-1 shows potent LPS-binding activity and was also able to agglutinate Gramnegative bacteria in vitro [START_REF] Du | A novel single WAP domain-containing protein isoform with antibacterial relevance in Litopenaeus vannamei[END_REF].

Shrimp AMPs encrypted in multifunctional proteins

Hemocyanin-derived fragments

In addition to gene encoded AMPs, diverse AMPs from crustaceans are encrypted in large proteins carrying non-immune functions. Haemocyanins are respiratory proteins found in arthropods. Interestingly, crustacean haemocyanins release histidine-rich antimicrobial peptides in response to microbial challenge [START_REF] Rolland | Stylicins, a new family of antimicrobial peptides from the Pacific blue shrimp Litopenaeus stylirostris[END_REF][START_REF] Destoumieux-Garzon | Crustacean Immunity: Antifungal peptides are generated from the c terminus of shrimp hemocyanin in response to microbial challenge[END_REF]. In penaeid shrimp, strictly antifungal peptides are released from the C-terminus of haemocyanins [START_REF] Rolland | Stylicins, a new family of antimicrobial peptides from the Pacific blue shrimp Litopenaeus stylirostris[END_REF]. Recently, the 3D-structure of the antifungal peptide PvHCt from L. vannamei was determined, and its mechanism of action was investigated. This histidine-rich AMP was shown to selectively bind to the fungal cell wall and permeabilize fungal membranes by adopting an amphipathic alphahelical structure. Insertion of PvHCt into the plasma membrane disrupts its integrity as a permeability barrier, leading to a disruption of internal homeostasis and the death of the fungal pathogen [START_REF] König | The diversity and evolution of anuran skin peptides[END_REF].

Histones and derived fragments

Histones are essential protein components of the chromatin architecture. The antimicrobial activity of histones was first described in deuterostomes (mammalians) [START_REF] Lee | Processing of an Antibacterial Peptide from Hemocyanin of the Freshwater Crayfish Pacifastacus leniusculus[END_REF]. Histones and derived fragments are active against Gram-negative and Gram-positive bacteria, fungi and viruses with various modes of action, including the permeabilization of bacterial cell membrane and binding to bacterial DNA and/or RNA [START_REF] Miller | Antibacterial properties of protamine and histone[END_REF]. The role of histones in shrimp defence was first determined in L. vannamei [START_REF] Kawasaki | Potential roles of histones in host defense as antimicrobial agents[END_REF]. The extracellular release of histones is associated with a defence reaction named ETosis, in which phagocytes release histones associated with extracellular traps (ETs) of DNA that entangle and eventually kill microbes [START_REF] Patat | Antimicrobial activity of histones from hemocytes of the Pacific white shrimp[END_REF]. ETs have now been described in deuterostomes [START_REF] Guimarães-Costa | ETosis: A Microbicidal Mechanism beyond Cell Death[END_REF] and protostomes, including species of Ecdysozoa (insects [START_REF] Brinkmann | Neutrophil extracellular traps kill bacteria[END_REF], crustaceans [START_REF] Altincicek | Host-Derived Extracellular Nucleic Acids Enhance Innate Immune Responses, Induce Coagulation, and Prolong Survival upon Infection in Insects[END_REF]) and Lophotrochozoa (molluscs [START_REF] Ng | The DNA fibers of shrimp hemocyte extracellular traps Page 11 of 13[END_REF]). This process is triggered by infection and/or tissue damage. ROS production is a signal that triggers ET formation in mammals [89] and lophotrochozoans [START_REF] Ng | The DNA fibers of shrimp hemocyte extracellular traps Page 11 of 13[END_REF]. In shrimp, haemocytes also release ETs in response to ROS inducers [START_REF] Altincicek | Host-Derived Extracellular Nucleic Acids Enhance Innate Immune Responses, Induce Coagulation, and Prolong Survival upon Infection in Insects[END_REF]. It will be of great interest to identify the haemocyte types that are involved in ETosis and determine whether the AMPs that are stored in haemocytes, like penaeidins, contribute to the antimicrobial activity of shrimp ETs.

AMPs in marine invertebrate immune-microbiota interactions

Marine invertebrates host a broad diversity of microorganisms in their tissues and haemolymph, including vibrios [START_REF] Schmitt | The Antimicrobial Defense of the Pacific Oyster, Crassostrea gigas. How Diversity may Compensate for Scarcity in the Regulation of Resident/Pathogenic Microflora[END_REF], which have the potential to become pathogenic and cause severe disease outbreaks (see section 2). Some adopt intracellular stages and are able to survive inside phagocytes [START_REF] Keshari | Reactive oxygen speciesinduced activation of ERK and p38 MAPK mediates PMAinduced NETs release from human neutrophils[END_REF][START_REF] Ma | Translocation of a Vibrio cholerae Type VI Secretion Effector Requires Bacterial Endocytosis by Host Cells[END_REF]. As illustrated in section 3, haemolymph, phagocytes and epithelial tissues are rich in AMPs. We are therefore facing a puzzling paradox, that is, microorganisms have evolved the ability to colonize immune cells/tissues that produce high local concentrations of AMPs. Although still incompletely understood, the recent literature sheds some light on the role of AMPs in the control of microbiota (including pathogens) and the mechanisms by which microorganisms avoid the complex chemical defences of their hosts.

Essential role of AMPs in the control of the microbiota

The application of gene silencing to non-model organisms has opened the way for in vivo functional studies that provide a more exhaustive view on the role of AMPs at the interface between hosts and microorganisms (Table 1). RNA interference (RNAi) confirmed the essential role of AMPs in controlling infections, as demonstrated for Type I crustins from Marsupenaeus japonicus, which participate in bacterial clearance in shrimp haemolymph [START_REF] Duperthuy | Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus[END_REF][START_REF] Yang | Catalase eliminates reactive oxygen species and influences the intestinal microbiota of shrimp[END_REF]. It also confirmed the functional divergence of AMP variants. Indeed, in vivo, LvALF1 (Group A ALF) protected L. vannamei against V. penaeicida and F. oxysporum, but not WSSV [START_REF] Liu | A novel crustin from Marsupenaeus japonicus promotes hemocyte phagocytosis[END_REF], whereas ALFPm6 (Group C ALF) in P. monodon protected against both V. harveyi and WSSV [START_REF] De La Vega | Antilipopolysaccharide factor in Litopenaeus vannamei (Lv ALF): A broad spectrum antimicrobial peptide essential for shrimp immunity against bacterial and fungal infection[END_REF]. However gene silencing also showed a more complex role of AMPs in the immune response of shrimp. Indeed, Type II crustins, which are not active against Gram-negative bacteria in vitro [START_REF] Smith | Antimicrobial proteins: From old proteins, new tricks[END_REF], participate in the resistance of L. vannamei and M. japonicus to Gram-negative V. penaeicida in vivo [START_REF] Ponprateep | Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections[END_REF][START_REF] Shockey | The role of crustins in Litopenaeus vannamei in response to infection with shrimp pathogens: An in vivo approach[END_REF]. In addition, through the use of gene silencing, AMPs were shown to orchestrate a key interface with the resident microbiota in marine invertebrates. In arthropods (shrimp), ALFs controlled shrimp-associated microbial communities [START_REF] De La Vega | Antilipopolysaccharide factor in Litopenaeus vannamei (Lv ALF): A broad spectrum antimicrobial peptide essential for shrimp immunity against bacterial and fungal infection[END_REF][START_REF] Hipolito | Role of Marsupenaeus japonicus crustin-like peptide against Vibrio penaeicida and white spot syndrome virus infection[END_REF]. Indeed, silencing of ALFPm3 (Group B ALF) in P. monodon caused a rapid propagation of bacteria, resulting in the death of the animals [START_REF] De La Vega | Antilipopolysaccharide factor in Litopenaeus vannamei (Lv ALF): A broad spectrum antimicrobial peptide essential for shrimp immunity against bacterial and fungal infection[END_REF]. In freshwater cnidarians (Hydra), species-specific AMPs called arminins were found to define host-species-specific bacterial associations [START_REF] Wang | A Shrimp C-type Lectin Inhibits Proliferation of the Hemolymph Microbiota by Maintaining the Expression of Antimicrobial Peptides[END_REF]. With those findings, host-specific AMPs were proposed to have evolved in early branching metazoans because of the need to control the resident beneficial microbes and not because of invasive pathogens [100]. Novel functions (non-antimicrobial) of AMPs were also discovered through gene silencing. Thus, silencing of PEN3 from the shrimp P. monodon resulted in a decrease in the β-integrin-dependent adhesion properties of shrimp haemocytes, revealing a previously unknown immunomodulatory function for penaeidins [101]. Gene silencing techniques applied to anionic AMPs, such as Group D ALFs [START_REF] Yang | NMR structure of rALF-Pm3, an antilipopolysaccharide factor from shrimp: model of the possible lipid A-binding site[END_REF] and stylicins [START_REF] Du | A novel single WAP domain-containing protein isoform with antibacterial relevance in Litopenaeus vannamei[END_REF], which show poor antimicrobial activity in vitro, should enable the characterization of their role in shrimp defence.

Responses of pathogens to AMPs

Immune suppression of AMPs by pathogens

Among strategies to escape the immune response, the suppression of AMP expression was shown in different phyla of marine invertebrates, including arthropods and cnidarians. In the shrimp L. vannamei, an infection by the fungal pathogen Fusarium solani dramatically repressed the expression of penaeidins, stylicins and Type II crustin [102]. Similarly, immune suppression of AMPs was reported in the coral P. damicornis, in which V. coralliilyticus exhibits temperaturedependent virulence, resulting in coral bleaching above 24 °C and tissue lysis above 25 °C [START_REF] Ben-Haim | Vibrio coralliilyticus sp. nov., a temperature-dependent pathogen of the coral Pocillopora damicornis[END_REF]. In its non-virulent state (below 24 °C), V. coralliilyticus induces the transcription of several coral immune genes [103] and the release of damicornin in coral mucus [START_REF] Saito | A novel big defensin identified in horseshoe crab hemocytes: isolation, amino acid sequence, and antibacterial activity[END_REF]. In contrast, in its virulent state, vibrios penetrating into coral tissues [104] induce a strong transient expression followed by a dramatic repression of damicornin transcription [START_REF] Saito | A novel big defensin identified in horseshoe crab hemocytes: isolation, amino acid sequence, and antibacterial activity[END_REF]. To date, the molecular mechanisms underlying the immune suppression of AMPs remains to be discovered.

Resistance to AMPs in vibrios

The membranes of microorganisms are an important interface with host AMPs (see section 3). Consequently, in vibrios, many of the most important mechanisms of AMP-resistance rely on outer membrane remodelling. Another important mechanism of resistance is the active efflux of AMPs from the bacterial cell. Those mechanisms have been recently reviewed in detail [105]. The most significant are those in which the minimum inhibitory concentrations (MICs) increase by several fold and are summarized below.

Membrane charge alteration.

As electrostatic interactions often initiate the binding of cationic AMPs to bacterial membranes, bacteria colonizing metazoan hosts have evolved strategies to lower the net negative charge of cell surface molecules; in Gram-negative bacteria, such modifications are often observed on LPS, the major constituent of their outer membrane [106,107]. Vibrios, including human pathogens, live in close association with marine invertebrates [108], which in turn may have selected AMP-resistant phenotypes among vibrios. A structural modification of LPS was recently found to be responsible for the different AMP-resistant phenotypes observed in V. cholerae. Indeed, the Lipid A structure (the anionic membrane anchor of LPS) of V. cholerae O1 and O139 [109] contains a hydroxylated secondary acyl chain that is also found in the squid symbiont V. fischeri [100]. This structure plays an important role in the resistance to AMPs [109,110], as it can be substituted by di-Glycine residues that lower the negative charge of the whole molecule [111].

Release of outer membrane vesicles. Upon outer membrane stress, such as that created by AMPs, bacteria can use the alternate σE factor to promote the expression of factors that help preserve and/or restore cell envelope integrity. The release of outer membrane vesicles (OMVs) is a σE-dependent mechanism [112], whose role in AMP resistance has been recently shown in vibrios. Vibrios, including the oyster pathogen V. tasmaniensis LGP32, were shown to release OMV protection against membrane-active AMPs [113,114]. Interestingly, OMV release was triggered by oyster plasma, suggesting, as shown in E. coli [115], that the membrane-active agents that are present in oyster plasma can trigger OMV release in vibrios. While the major protective effect of OMVs against AMPs has now been shown in two vibrio species and in E. coli [116], it is still unknown whether this effect results from a membranous shield-like effect in which OMVs surround vibrios and trap membrane-active AMPs, thereby preventing their interaction with the membranes of the bacterial cell, or a membrane renewal mechanism eliminating AMP-damaged membranes to maintain membrane integrity. The σE-dependent induction of OMV release tends to support the second hypothesis. Active efflux of AMPs. Once AMPs have breached the membrane barriers of bacteria and reached the cytoplasmic space, they can be expelled into the extracellular milieu by diverse efflux pumps. Pumps of the resistance-nodulation-cell division superfamily (RND) contribute to AMP-resistance in vibrios. However, among the 6 RND efflux pumps of V. cholerae [117], only VexAB-TolC is required for AMP resistance in vitro [118,119]. The VexAB-TolC pump is structurally and functionally similar to the E. coli and Salmonella enterica AcrAB-TolC pump [120,121] and the Pseudomonas aeruginosa MexAB-OprM systems [122]. VexAB is also the main efflux pump that is involved in the resistance to bile acids, detergents, antibiotics, and PmB [119,123]. To date, among vibrios, V. cholerae remains the only species in which AMP-resistance was mediated by an RND efflux system.

Marine invertebrate AMPs in an applied context

AMPs as therapeutic drugs

Since their first discovery in the 1980s, AMPs have been considered to be promising candidates for therapeutic uses in humans, animals and plant health. However, only a few AMPs have reached phases of clinical and preclinical pipelines [124,125]. AMPs with potential interest for biopharmaceutical companies have been isolated from marine invertebrates. Some AMPs, such as mollusc defensins, have very low MICs in the nanomolar range against Gram-positive bacteria [START_REF] Gonzalez | Evidence of a bactericidal permeability increasing protein in an invertebrate , the Crassostrea gigas Cg-BPI[END_REF]. Their fungal homologue, plectasin, is considered to be a major candidate for therapeutic use [126]. Importantly, AMPs are currently being used for drug development due to their activity as immune modulators, which gives them clinical potential beyond the treatment of antibiotic-resistant strains [127]. Among AMPs from marine invertebrates, ALFderived peptides have been shown to modulate the inflammatory response in murine macrophage cell lines [128] and display antitumor activity against HeLa cells through the alteration of the cell membrane [129]. Those novel activities may open the way to future drug developments.

AMPs in aquaculture

Disease prevention in marine invertebrate aquaculture has been traditionally based on the control of pathogens or the selection of animals that are resistant to diseases. AMPs of marine invertebrates have been considered for both applications.

AMPs in the control of pathogens in aquaculture. In the context of intensifying aquaculture, antibiotics have been used for disease prevention and management, which has resulted in increased bacterial resistance in the environment, and favoured the emergence of resistant strains of major human pathogens [130]. If AMPs are proposed as an alternative to the use of antibiotics, which should be reduced or avoided in aquaculture (FAO/OIE/WHO), there is a risk of promoting the emergence of AMP-resistant strains (see section 4), and this resistance can readily be achieved experimentally [131]. However, AMPs present some important advantages over antibiotics, including the following: (i) they are much less stable in the environment; (ii) they often combine multiple mechanisms of action, e.g., they can both disrupt bacterial membranes and inhibit metabolic pathways; and (iii) they do not increase bacterial mutation rates [132]. However, most of all, it is now recognized that the antimicrobial activity of AMPs largely relies on their immune regulatory properties, including the recruitment and differentiation of immune cells [133], against which microorganisms cannot evolve simple mechanisms of resistance. The finding that AMPs are not simple antimicrobials but are complex orchestrators of host defences has opened new perspectives to combat bacterial infections. AMPs from marine invertebrates have appeared to be attractive candidates to reduce the impact of diseases in closed aquaculture systems that have a low impact on the environment [START_REF] Bachère | Antiinfectious immune effectors in marine invertebrates: potential tools for disease control in larviculture[END_REF]. However, only rare applications have been reported. AMPs have been recently applied in the Polynesian pearl industry [134]. For example, tachyplesin was combined with exopolysaccharides as filming agents instead of the antibiotics that are traditionally used in the grafting process to reduce oyster post-operative mortality and increase pearl quality. The result of this alternative process was similar to that of commercial nuclei that were treated with antibiotics [135]. To date, the use of AMPs as immune modulators has not been reported in aquaculture.

AMPs as markers for survival capacity in shrimp. AMPs control homeostasis in marine invertebrates and maintain individuals in a healthy state (see section 4). Inspired by studies in humans, which have investigated the associations between the copy number of AMP encoding genes and susceptibility to diseases [136,137], signatures of AMP expression have been studied in marine invertebrates that are susceptible or resistant to infectious diseases. Thus, in the shrimp L. stylirostris, the basal expression of AMP-encoding genes (PEN2 and PEN3, ALF-D, Type II crustin and lysozyme) has been correlated with the capacity of the shrimp to circumvent V. penaeicida infections. As a consequence, signatures of AMP expression have been proposed as original molecular markers for selection programs that are dedicated to shrimp resistance to bacterial infections [138].

Conclusion

Based on our current knowledge, there is probably not a general scheme but a diversity of roles for AMPs in the homeostasis of "marine invertebrates". Some AMP families are highly abundant in host cells and tissues, whereas others are expressed at levels below inhibitory concentrations. Although the antimicrobial activity of AMPs has been the main focus of research, the immune regulatory properties of AMPs in the defence of marine protostomes and cnidarians now deserve a much greater research effort. In addition, some AMPs appear to have evolved within specific phyla or species, whereas others evolved from a common ancestor and are widespread in the tree of life. The inspiring example of freshwater cnidarians prompts us to investigate how specific AMP repertoires may have shaped the host-specific microbiota formation of stable holobionts and compare those examples to other metazoan species with less specific AMP repertoires and more versatile microbiota. This research effort is now needed to obtain a more integrated view of the role of AMPs in symbiotic interactions that range from mutualistic to pathogenic to develop a better understanding of the role of AMPs in protostome and cnidarian health and disease.

  the clearance of Escherichia coli. Dev. Comp. Immunol. 48, 229-233.

  mc.manuscriptcentral.com/issue-ptrsb Submitted to Phil. Trans. R. Soc. B -Issue

Table 1 :

 1 Principal families of AMPs in penaeid shrimp.

http://mc.manuscriptcentral.com/issue-ptrsb Submitted to Phil. Trans. R. Soc. B -Issue

Funding

The authors received financial support from the ANR (Vibriogen project ANR-11-BSV7-0023 and Adacni project ANR-12-ADAP-0016), CAPES (CIMAR 1974(CIMAR /2014)), CNPq (MEC/MCTI/CAPES/CNPq/FAPs PVE 401191/2014-1) and FONDECYT 11150009-2015. Cairé Barreto was supported by a M.Sc. scholarship provided by CNPq-Brazil.

Authors' Contributions

Authors have written and approved the final version of this manuscript.

Competing Interests

We have no competing interests.


V Murugesh

Apurba Ranjan Sahoo

Mathieu Achard

V M Gangavaram

Christian Sharma

Surisetti Bruneau
email: surisetti@iict.res.in

Suresh

Gangavaram V M Sharma

Christian Bruneau

Surisetti Suresh
email: suresh.surisetti@yahoo.in.

Ruthenium Catalyzed Regioselective beta-C(sp(3))-H Functionalization of N-Alkyl-N '-p-nitrophenyl Substituted Piperazines using Aldehydes as Alkylating Agents

Keywords: Regioselective, C(sp 3 )-H functionalization, piperazine, ruthenium catalysis, alkylation

come

Identical reactivity of C(sp [START_REF]For selected reviews on α-C(sp 3 )-H functionalization of saturated nitrogen heterocycles, see: a)[END_REF] )-H bonds in saturated heterocyclic molecules makes selective C(sp [START_REF]For selected reviews on α-C(sp 3 )-H functionalization of saturated nitrogen heterocycles, see: a)[END_REF] )-H bond functionalization as an attractive challenge for the researchers. Literature witnessed the directing group guided or catalyst controlled site selective C(sp [START_REF]For selected reviews on α-C(sp 3 )-H functionalization of saturated nitrogen heterocycles, see: a)[END_REF] )-H bond functionalizations. [1] However, this requires installation of specific directing groups and specially designed catalysts/ligands. Site-selective C(sp [START_REF]For selected reviews on α-C(sp 3 )-H functionalization of saturated nitrogen heterocycles, see: a)[END_REF] )-H functionalization based on natural reactivity of substrate attracts the chemists attention. Innate C(sp [START_REF]For selected reviews on α-C(sp 3 )-H functionalization of saturated nitrogen heterocycles, see: a)[END_REF] )-H functionalization [2] is demanding compared to the guided one [1] because of its merits such as obviating the use of the directing group installation and its removal, designed ligands and catalysts. Although α-C(sp [START_REF]For selected reviews on α-C(sp 3 )-H functionalization of saturated nitrogen heterocycles, see: a)[END_REF] )-H functionalization of saturated nitrogen heterocycles has been well-reported, [START_REF]For selected reviews on α-C(sp 3 )-H functionalization of saturated nitrogen heterocycles, see: a)[END_REF] the β-C(sp [START_REF]For selected reviews on α-C(sp 3 )-H functionalization of saturated nitrogen heterocycles, see: a)[END_REF] )-H functionalization has received significantly less attention. In this context, the research groups of Bruneau and Seidel, independently, made seminal contributions to the development of β-C(sp [START_REF]For selected reviews on α-C(sp 3 )-H functionalization of saturated nitrogen heterocycles, see: a)[END_REF] )-H functionalization of saturated nitrogen heterocycles without using directing groups. [4,5] Regioselective C(sp 3 )-H bond functionalization of challenging saturated N-heterocyclic compounds [6] like piperazine remains a critical issue and a welcoming challenge in organic synthesis. This is due to the reason that piperazine is the 3 rd most appeared 'privileged' Nheterocyclic core structure in the marketed drugs. [7] Among the top 100 best selling drugs, piperazine containing drugs occupy a significant number. In particular, C-substituted piperazines have received considerable attention from the scientific community owing to their potential therapeutic values being useful for the treatment of infections, viral diseases, malaria and CNS related disorders (Figure 1). [8] Figure 1. Drugs and biologically active compounds containing C-substituted piperazine moiety.

The synthesis of C-substituted piperazines, from the parent piperazine core, mainly relies on the reduction of pyrazinium salts [9] and C(sp 3 )-H bond functionalization reactions. [10] Literature survey revealed that most of the methods for activating C(sp 3 )-H bonds on piperazine depend on α-C(sp 3 )-H functionalization [11,6a] in the presence of stoichiometric amounts of strong bases (Scheme 1a), while very few methods were reported under photoredox catalysis conditions (Scheme 1b). [10b,c] Catalytic approaches to construct regioselective C(sp 3 )-C bonds from piperazine's inert-C(sp 3 )-H bonds is a challenging transformation due to the Accepted Manuscript presence of two nitrogen atoms at 1,4-positions in its ring. Our previous ruthenium-catalyzed protocol [12] on the intramolecular C(sp 3 )-H functionalization for the synthesis of piperazine fused indoles (Scheme 1c) paved a way and inspired us to develop a regioselective intermolecular β-C(sp 3 )-H bond functionalization method on piperazines. We envisaged the regioselective functionalization of piperazine C(sp 3 )-H bonds by varying the substitution pattern on the nitrogen atoms of the saturated ring. The N-substitution pattern on the two nitrogen atoms plays a crucial role. Piperazine having an electron withdrawing group on the nitrogen N(4), which is adjacent to the targeted C(sp 3 )-H bond and the other nitrogen N(1) bearing an alkyl substituent, would serve as a model substrate to test the β-C(sp 3 )-H bond functionalization. Under ruthenium catalysis conditions, we anticipated the regioselective formation of C-C bond through the events of the dehydrogenation of piperazine, formation of iminium species to propagate enamine that could undergo nucleophilic addition to the aldehyde followed by hydrogenation to eventually result in the regioselective β-C(sp 3 )-H alkylation (Scheme 1d and vide infra).

Scheme 1. Literature reports on C(sp 3 )-H bond functionalization on piperazine derivatives and This work.

To prove our hypothesis, we designed a substrate such as 1a having an electron-withdrawing pnitrophenyl group on nitrogen (N4) which is adjacent to the targeted C(sp 3 )-H bond and an electron-rich methyl substituent on the other nitrogen (N1). 1-Methyl-4-(4-nitrophenyl)piperazine 1a was treated with benzaldehyde (2a) in the presence of [Ru(pcymene)Cl2]2/ camphorsulfonic acid (CSA) catalytic system followed by the addition of formic acid as the external hydrogen source. [4] Delightfully, the expected alkylated product 3a was obtained in 62% yield (Table 1, entry 1). Encouraged by this finding, we conducted an optimization assay (Table 1) for this transformation in order to improve the yield of 3a. We screened different Ru(II), Ir(III)-catalysts for this regioselective transformation. The Ru-catalyst B and Ir-catalyst C, bearing phosphino-benzene sulfonate ligands, gave only moderate yields of 3a (Table 1, entries 2 and3). Other ruthenium catalysts D or E bearing triphenylphosphine or bipyridine ligands were not efficient for this regioselective alkylation (Table 1, entries 4 and5). The ruthenium(0) complex Ru3(CO)12 F did not provide the desired product 3a (Table 1,entry 6). Additives such as HCOOH, trifluoroacetic acid (TFA) or methanesulfonic acid (MSA) did not give better results (

Accepted Manuscript

Note that the optimized conditions (Table 1, entry 10) enabled gram scale synthesis of 3a in 60% yield (Scheme 2). After selecting the optimized reaction conditions, we turned our attention to check the substrate scope of the ruthenium-catalyzed β-C(sp 3 )-H bond functionalization of piperazine (Scheme 2). Firstly, we examined the scope of the ruthenium-catalyzed C-alkylation of 1a with various aldehydes. The regioselective alkylation tolerated substituted benzaldehydes containing different functional groups like halogens, electron-donating and electron-withdrawing groups. Halogen substituted benzaldehydes produced the corresponding Calkylated piperazine derivatives 3b-g in moderate to good yields, while the ortho-substituted ones gave lower yields. The benzaldehydes containing electrondonating groups such as o-OH, o-OMe, p-OMe, p-Me and p-NMe2 furnished the corresponding C-alkylated piperazine derivatives 3h-l in moderate to good yields. The benzaldehyde bearing a p-CF3 electronwithdrawing group tolerated for this transformation as the reaction of 1a and 4-(trifluoromethyl)benzaldehyde afforded the corresponding C-alkylated piperazine 3m in 48% yield. The structure of 3m was further confirmed with the aid of X-ray crystal structure analysis [14] besides the standard spectroscopic characterization methods (see the Supporting Information, for X-ray data details). The benzaldehydes comprising multiple substituents also worked well as 2,5-dibromobenzaldehyde, 2bromo-5-fluorobenzaldehyde, and 6-bromopiperonal proved to be successful alkylating agents to deliver the corresponding C-alkylated products 3n-p in 61%, 62% and 52% yields, respectively. Polyaromatic aldehydes such as 2-naphthaldehyde and biphenyl-4carboxaldehyde gave their respective C-alkylated piperazines 3q and 3r in 66% and 58% yields, respectively.

Aliphatic aldehydes including cyclohexanecarboxaldehyde, cyclopropanecarboxaldehyde, and 2,2diphenylacetaldehyde delivered the corresponding Calkylated products 3s-u in reasonable yields. Heteroaromatic aldehydes such as furan-2carbaldehyde and thiophene-2-carbaldehyde were suitable reagents to alkylate 1a to produce the corresponding C-alkylated products 3v and 3w in 61% and 52% yields, respectively. However, our efforts towards the regioselective alkylation with ketones were not successful. Later we checked the scope with different substituents on N(1) of piperazine. Piperazine derivatives comprising N(1)ethyl, -propyl, -phenethyl, -benzyl substitution furnished their corresponding C-alkylated products 3x-aa in low to moderate yields upon reaction with benzaldehyde under the ruthenium catalysis settings. Some unidentifiable byproducts were observed in the cases of the products with low to moderate yields. Based on the substitution pattern of the piperazine substrate, there are two possibilities for the present regioselective C(sp 3 )-H functionalization: (i) β-C(sp 3 )-H functionalization to the N(1) atom and (ii) α-C(sp 3 )-H functionalization to the N(4) atom. To investigate the possibilities, we conducted control experiments. By using the optimized reaction conditions, we treated benzaldehyde with 1-(4nitrophenyl)piperidine 4a or 4-(4nitrophenyl)morpholine 4b, which lack the second

Accepted Manuscript

nitrogen atom in the saturated rings of 4a or 4b compared to that of the piperazine 1. However, we did not observe the corresponding alkylation products 5a (or) 5b, which implies that the β-C(sp 3 )-H functionalization on piperazines 1 is operating from N(1). It may be noted that under ruthenium catalysis conditions N-methylpiperidine underwent β-C(sp 3 )-H functionalization by using benzaldehyde as alkylation agent. [4c,d] These results may also suggest the crucial role of the methylene group [4] on the N(1) nitrogen of the piperazine 1, which may be acting as an innate directing group for the β-C(sp 3 )-H functionalization of 1 (Scheme 3). A plausible mechanism was proposed for the present regioselective β-C(sp 3 )-H functionalization on piperazine based on the control experiments and literature reports (Scheme 4) [4a-b,12,13] . The iminium cation intermediate I would form from the piperazine substrate 1 by the action of ruthenium catalyst via hydrogen atom transfer process. [4a-b] Then an azomethine intermediate II would result from the intermediate I via proton abstraction of the ruthenium hydride species. [4a-b] In the presence of acid, an enamine intermediate III would form from intermediate II. [4a-b,12,13] The enamine III would add to aldehyde 2 to give intermediate IV. Dehydration of the intermediate IV would provide intermediate V. In the presence of ruthenium species and acidic source, the imine functionality and unsaturation in the intermediate V might undergo reduction [4a-b] to produce the C-alkylated piperazine derivative 3. shown the proposed mechanism might operate in two successive catalytic cycles. The presence of C-H bond at N(1) allows the azo-methine formation this nitrogen position only. On the other hand, the attracting p-nitrophenyl group at N(4) polarizes the enamine double bond to favour the regioselective formation of C-C bond at C(3). The presence of the electron deficient -NO2 moiety on the resulting products can be judiciously used for further post-functionalization. Accordingly, we synthesized 4-(2-benzyl-4-methylpiperazin-1yl)aniline 6 by reducing the nitro group on 3a (Scheme 5), which will increase its synthetic utility for further transformations including cross coupling reactions, arylations, alkylations, amidations, Schiff's base formation reactions etc. [15] Scheme 4. Plausible mechanism. Scheme 5. Reduction of nitro group on 3a.

In summary, we designed and developed a regioselective, intermolecular β-C(sp 3 )-H functionalization method to access C-substituted piperazines by using aldehydes as alkylating agents in the presence of ruthenium catalysis. A wide range of aldehydes including aromatic, aliphatic, heteroaromatic aldehydes have been employed as alkylating agents for the C-alkylation of piperazines to access the corresponding C-alkylated piperazine derivatives in moderate to good yields. Therefore, the presented strategy offers a useful protocol to alkylate the "privileged" piperazine moieties with high levels of predictable regioselectivity.

Experimental Section

Synthesis of 3a to 3aa

In a dry schlenk tube 5 mol% of [RuCl2(p-cymene)]2 (0.025 mmol, 0.015 g), 20 mol% of CSA (0.1 mmol, 0.023 g) and 0.6 mmol of piperazine 1 were taken, vacuum was applied (2-5 sec) to the reaction tube and degassed with N2. Then added 0.5 mmol of aldehyde 2 under nitrogen atmosphere followed by the addition of 2 mL of degassed toluene. The reaction tube was placed on a preheated metal-heating block and stirred at 150 oC for 18 h. The reaction was cooled to room Accepted Manuscript temperature, then added 2 equiv of formic acid (1 mmol, 0.037 mL) and stirred at 150 oC for 12 h. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (2 x 20 mL), washed with water (2 x 20 mL) followed by brine. The organic layer was dried over anhydrous Na2SO4, filtered and the solvent was removed under the reduced pressure to afford a crude residue. The crude was subjected to silica gel column chromatography by using EtOAc/hexane as eluent to obtain pure C-alkylated piperazine derivatives 3a-3aa.

Scheme 2 .

 2 Scheme 2. Substrate scope of the regioselective β-C(sp 3 )-H functionalization of piperazines.

Scheme 3 .

 3 Scheme 3. Control experiments to confirm β-C(sp 3 )-H functionalization.

Table 1 .

 1 Table 1, entries 7-9). We observed better yields of 3a with 20 mol% of CSA (Table 1, entry 10). Further increase in the amount of CSA did not improve the yield of 3a (Table 1, entry 11). No reaction took place in the absence of catalyst (Table 1, entry 12), which highlights the crucial role of the catalyst in the C(sp 3 )-H bond functionalization (see supporting information, for an extensive optimization study). Optimization study a . Reaction conditions: 1a (0.6 mmol), 2a (0.5 mmol), catalyst (5 mol%), additive (x mol%), toluene (2 mL), at 150 o C for 18 h, then HCOOH (1 mmol), at 150 o C for 12 h; b) Yields are of isolated products.

	Entry a Catalyst Additive (mol%) %Yield of 3a b
	1	A	CSA (10)	62
	2	B	CSA (10)	46
	3	C	CSA (10)	15
	4	D	CSA (10)	27
	5	E	CSA (10)	18
	6	F	CSA (10)	-
	7	A	HCOOH (10)	29
	8	A	MSA (10)	22
	9	A	TFA(10)	32
	10	A	CSA (20)	73
	11	A	CSA (30)	62
	12	-	CSA (20)	-
	a)

Acknowledgements

We thank the Indo-French Centre for the Promotion of Advanced Research for financial support. SS thanks the Science & Engineering Research Board (SERB), Department of Science and Technology (DST), India for an Extramural Research grant (EMR/2017/002601). VM thanks UGC, New Delhi, for a fellowship. ARS thanks CEFIPRA for financial support. We thank Dr. N. JagadeeshBabu, CSIR-IICT for single crystal X-ray structure analysis. We thank the Director, CSIR-IICT for the support (Communication No. IICT/Pubs./2020/085)

COMMUNICATION


Daiwang Xu

Angèle Chiaroni

Maurice-Bernard Fleury

Martine Largeron

Electrochemically Induced Cascade Reaction for the Assembly of Libraries of Biologically Relevant 1,4-Benzoxazine Derivatives

Abstract. The scope and mechanism of an electrochemically induced cascade reaction, which leads to highly substituted 1,4-benzoxazine derivatives, have been explored through the variation of the structure of the o-azaquinone mediator. This reaction sequence, wherein both cycloaddition partners are generated in situ, at room temperature, under metal-free conditions, allows the regiospecific inverseelectron-demand Diels-Alder (IEDDA) reaction of an o-azaquinone heterodiene and a secondary alkylenamine dienophile, two chemically nonaccessible unstable entities. The cascade reaction was Introduction 1,4-Benzoxazine derivatives have received great attention in chemical and medicinal research due to their natural occurrence and important biological activities. Some of them are central nervous system depressants, antipsychotic agents, calcium antagonists and antibacterial agents, while others are potential drugs for treating neurodegenerative, inflammatory, autoimmune, cardiovascular and diabetic disorders. 1 Past syntheses of 1,4-benzoxazine derivatives have relied predominantly upon multistep procedures, using 2-nitrophenols or 2-aminophenols as the starting materials. However, these methods suffered some limitations in the generation of molecular diversity. 2 The recent development of new synthetic strategies such as solid state synthesis, use of palladium catalysis or microwave irradiation, which allow one-pot multiple functionalization, has therefore reinforced the interest in the 1,4-benzoxazine core. 1a,2a Due to its efficiency for introducing high degree of diversity through the variation of the structure of both cycloaddition partners, the Diels-Alder reaction may also constitute an attractive strategy for the synthesis of the 1,4-benzoxazine scaffold. 3 Despite this potential, the synthetic scope of this reaction is limited by the requirement of o-azaquinone heterodienes, which are not readily accessible stable compounds. Most of the work has been mainly restricted to o-quinone monooximes 4 and o-quinonemonoimides. [START_REF]Å 3 , dcalc = 1.259[END_REF] To the best of our knowledge, only a few reports described the synthesis of 1,4benzoxazine derivatives from in situ chemically generated o-azaquinone heterodienes. 5j,l A few years ago, we showed that electrogenerated 3,4-azaquinone 1ox (Scheme 1) behaved as an effective organic catalyst for the autorecycling oxidation of aliphatic primary amines. The catalytic cycle produced the reduced catalyst 1red and an imine as the product of amine oxidation. 6 However, in the case of R 1 R 2 CHCH2NH2 amines, the catalytic process ceased after a few turnovers, as the catalyst 1ox was trapped through inverse-electron-demand Diels-Alder (IEDDA) reaction with the simultaneously electrogenerated tautomeric enamine form of the alkylimine extruded during the catalytic process. This cascade sequence, wherein both cycloaddition partners were generated in situ, at room temperature, under metal-free conditions, allowed the rapid synthesis of polyfunctionalized 1,4benzoxazine derivatives. 7a Although similar reactions with in situ chemically generated tertiary enamine are known, 8 the electrochemically induced enamine cycloaddition reaction represented uncharted terrain. Consequently, in a preceding work, we examined the scope of this cascade reaction, first with respect to the secondary enamine dienophile. 7b Especially, to increase the molecular diversity, we generated enamines in which the substituents on the amino group were different from those linked to the double bond. For this purpose, the amine R 1 R 2 CHCH2NH2 was catalytically oxidized by o-azaquinone 1ox in the presence of a second primary aliphatic amine R 3 NH2 (Scheme 1). Then, the 1ox-mediated cascade reaction led to the construction of complex 1,4-benzoxazine derivatives in a regiospecific manner and allowing diastereospecific annellation. 7b Interestingly, some of these compounds were found to be significant targets as neuroprotective agents both in vitro and in vivo. 7c, d

HN O OH O H 2 N HO OH O R 1 NH 2 R 2 R 1 R 2 N R 3 R 1 R 2 N R 3 H H N O O O H R 1 R 2 N H R 3 + R 3 -NH 2
1 ox 1 red Scheme 1. 1 ox -mediated cascade reaction leading to highly functionalized 1,4-benzoxazine derivatives

At this stage, a sole heterodiene (1ox) was engaged in the cascade reaction with diverse enamine dienophiles. For a more comprehensive investigation of the scope of the cascade reaction, the variation of the heterodiene-part proved to be essential. In this prospect, there were several challenges inherent to this reaction sequence which needed to be addressed. As a continuation of our preceding work, 7b we present hereafter the results of new experiments aimed at exploring further the synthetic potential of this cascade reaction using a variety of electrogenerated o-azaquinone entities, with the ultimate goal of developing libraries of biologically relevant 1,4-benzoxazine derivatives.

Results and Discussion

The scope of the cascade reaction was investigated from newly synthesized o-aminophenol derivatives 2 (Table 1), under the optimum conditions previously reported 7b which required a mercury anode, methanol as the solvent, tetraethylammonium hexafluorophosphate (TEAHFP) as the supporting electrolyte and 20.0 equiv of amine substrate per molecule of o-aminophenol derivative 2. Under these conditions, the cyclic voltammogram of compound 2ared (2 mM), in deaerated MeOH containing isobutylamine (40 mM) chosen as an example of amine substrate, showed, at a dropping mercury electrode, an oxidation peak Pa at -50 mV, due to a two-electron process, the sweep rate being 0.5V s -1 .

As can be seen in Figure 1, a cathodic peak Pc appeared on the reverse sweep at -160 mV vs SCE, illustrating the quasireversibility of the two-electron transfer that could be assigned to the oaminophenol 2ared/o-iminoquinone 2aox redox couple. When the controlled potential of the mercury pool was fixed at +50 mV vs SCE, which is at a potential for which 2ared could be oxidized to the iminoquinone form 2aox, the system of peaks (Pa, Pc) remained unchanged for a certain time, consistent with steady-state catalytic behavior (Figure 1).

Accordingly, the anodic current remained constant over a certain time, while a value of 12 was found for the total number of electrons (n) transferred per molecule of 2ared in the catalytic process (6 turnovers). These results indicated that the 2ared/2aox system behaved as a redox mediator for the electrochemical oxidation of isobutylamine to the corresponding alkylimine (Scheme 1), according to the transamination mechanism previously reported. 6b Further, the catalytic process ceased and close inspection of the exhaustively oxidized solution revealed that electrogenerated o-azaquinone 2aox was trapped with the tautomeric enamine form of the alkylimine, to give the substituted 2-alkylamino-1,4benzoxazine 3a in 71% yield (Table 1, entry 1).

The progress of the electrolysis was simultaneously followed by monitoring the UV-vis absorption spectrum. After application of the anodic potential (E = +50 mV vs SCE), no spectral changes were observed for a time, consistent with steady-state catalytic behavior (Figure 2). After the consumption of 7 F mol -1 , a decrease in the UV-vis absorption band shown by the monoanionic form of 2red at 334 nm (/mol -1 L cm -1 = 16500) was observed, while new bands at 302 nm and 254 nm developed. Spectral changes showed two isosbestic points at 313 and 268 nm, indicating that a simple equilibrium between two species was shifted. The new bands at 302 and 254 nm could be assigned to the 2-alkylamino-1,4benzoxazine 3a, as corroborated after recording the UV-vis absorption spectrum of the isolated product. With the reliable set of conditions in hand, the electrochemically induced cascade reaction was investigated using different o-aminophenol substrates 2a-i. Table 1 shows some examples of the molecular diversity that is accessible through this reaction sequence, which encompasses an IEDDA reaction between the electron-poor o-azaquinone heterodiene and the electron-rich secondary alkylenamine dienophile. This cycloaddition reaction occurred at room temperature, within 5-8 h, with complete regioselectivity. The more electron-rich carbon atom of the enamine dienophile added to the nitrogen atom of the o-azaquinone heterodiene. In all cases (Table 1, entries 1-9), the methodology was successful, producing the 1,4-benzoxazine derivatives in good yields. Interestingly, bromosubstituted 3,4-aminophenol derivative 2d reacted effectively to furnish the desired cycloadduct 3d in 80% yield (Table 1, entry 4). [START_REF]Bromosubstituted aromatic rings are particularly attractive in diversity-oriented synthesis because they can be easily transformed into differently substituted aromatics by cross-coupling reactions. (10) As shown in Figure 3, in both compounds, a strong intramolecular hydrogen bond is established between the hydroxyl groups O9-H and the oxygen atoms O10 (for 3f: distances[END_REF] In the specific case of 3,4-aminophenol derivatives bearing a benzophenone framework, the scope of the cascade reaction could be also extended by the attachment of substituents on the benzoyl moiety (Table 1, entries 5-8). Surprisingly, the yield of the cycloadduct was good regardless of the electronic and/or steric effects of the substituents. This outcome could be rationalized from X-ray crystallographic analyses of benzoxazines 3f and 3h, which showed the presence of an hydrogen bond between the 2-hydroxyl substituent and the carbonyl group of the benzophenone skeleton (Figure 3). As a consequence, the substituted phenyl group twisted out of the plane, 10 then affecting the transmission of the substituent effects. Similarly, an hydrogen bond could be expected on the 3,4-azaquinone heterodiene which would produce the same effects. A few other attempts to accomplish the cascade reaction which are not listed in Table 1 deserve special note. The electrochemical oxidation of 3,4-aminophenol derivatives 4a and 4b (Table 2), which lack the 2-hydroxyl substituent, was performed under the experimental conditions reported above. However, as these substrates were oxidizable at a higher potential, the mercury anode, which has a low anodic decomposition potential, was replaced by a platinum anode. Then, the cyclic voltammogram of compound 4ared (2 mM) showed an oxidation peak Pa due to a two-electron process at + 200 mV vs SCE, the sweep rate being 0.1 Vs -1 (Figure 4). On the reverse sweep, the lack of peak Pc illustrated the irreversibility of the overall two-electron oxidation process, indicating that the produced 3,4-azaquinone 4aox was reactive enough to be engaged in a fast follow-up chemical reaction. When the controlled potential of the Pt anode was fixed at + 350 mV vs SCE, which is at a potential for which 4ared could be oxidized to the 3,4-azaquinone form 4aox, a decrease in both anodic electrolysis current and Pa height was observed (Figure 4). At the same time, the solution became dark, while a coulometric value of 4.0 was found for n. These results indicated that the produced 3,4-azaquinone 4aox was probably converted into a Michael adduct which, after a subsequent two-electron oxidation reaction, spontaneously decomposed to melanin-like polymers. Accordingly, no stable product could be detected at the end of the electrolysis. To evaluate the ability of the 2-substituent to prevent the competing formation of Michael adduct, 3,4-aminophenol 4b (Table 2), bearing a 2-methyl group, was used as the substrate and treated under the same experimental conditions. Similarly, no stable product could be isolated at the end of the electrolysis.

H 2 N HO O OH H 2 N HO O OH SO 2 Me O OH H N O N H F O OH H N O N H OMe O OH H N O N H O OH H N O N H
At this point, it could be concluded that the presence of the 2-hydroxyl substituent was of overriding importance for the success of the cascade reaction. On the basis of the low value found for n using 4a or 4b as the substrate (4 instead of 10-12 in the case of examples collected in Table 1), we suspected that the requirement for a 2-hydroxyl group was mainly inherent to the catalytic process. So, to circumvent the limitation related to the catalytic process, we envisioned a variant of our initial procedure in which the o-azaquinone entity was electrogenerated in the presence of a preformed enamine. In other words, we sought to design an alternative procedure which could be generally applied to construct polyfunctionalized 1,4-benzoxazine derivatives, without the need to employ a 2-aminoresorcinol derivative as the starting material (Table 1).

We first performed the anodic controlled-potential electrolysis of 3,4-aminophenol 4a (Table 2 andScheme 2), in the presence of 4-cyclohexylidenemethylmorpholine. The optimized conditions required 5 equiv of enamine per molecule of 4a. However, as the basicity of the alkylenamine was not sufficient to produce the phenolate anion of 4ared, which is the sole form that can be oxidized to 3,4-azaquinone 4aox, 1 equiv of morpholine was added to the bulk solution as the ancillary base. Then, the reaction could go to completion by acid-base equilibrium displacement. When E was fixed at + 450 mV vs SCE, that is at a potential for which 4ared could be oxidized to the 3,4-azaquinone form 4aox (Figure 4), a coulometric value of 2.2 was found for n, consistent with a twoelectron transfer. After applying the potential, a decrease in the UV-vis absorption bands shown by the major neutral form of 4ared at 343 and 244 nm was observed, while two new bands at 360 and 254 nm developed (Figure 5). Spectral changes showed two isosbestic points at 355 and 310 nm, indicating that a simple equilibrium between two species was shifted. Reagents and conditions: (4a-h) = 2 mM, (enamine) = 10 mM, MeOH, rt, Pt anode (E = + 450 mV vs SCE), 4h; 1equiv of morpholine was added to the bulk solution for producing the phenolate anion of 4ah. b Yields refer to chromatographically pure isolated products.

After 4h, treatment of the exhaustively electrolyzed solution afforded the desired 2-alkylamino-1,4benzoxazine derivative 5a, in 56% yield, as a single regioisomer (Table 2, entry 1). Conclusive evidence of the structure of the regioisomer was provided by X-Ray crystallographic analyses of the benzoxazine derivatives 5e and 5f (Figure 6). The scope of the tandem oxidation-IEDDA reaction was then investigated using a series of oaminophenol derivatives 4a-h. As illustrated in Table 2, the method was sufficiently general to accommodate a variety of substituents.

As predicted for a IEDDA reaction, electrogenerated o-azaquinone heterodienes, with a pronounced electron-poor character, produced the desired cycloadduct in good yield (Table 2, entries 1-6), whereas introduction of an amide substituent resulted in lower yield (Table 2, entry 7). Anodic oxidation of 2aminophenol generated a poor reactive o-iminoquinone heterodiene, which mainly decomposed to melanin-like polymers, giving only 10% of the desired cycloadduct (Table 2, entry 8).

In general, the tandem oxidation-IEDDA reaction produced slightly lower yields of cycloadducts than those yielded by the aforementioned cascade reaction, presumably due to the replacement of the Hg anode by a Pt anode rather than due to the lack of the 2-hydroxyl substituent. To ensure that, we carried out the anodic controlled-potential electrolysis of the 2-aminoresorcinol derivative 2i, in the presence of 4-cyclohexylidenemethylmorpholine using the two anodes, alternatively (Scheme 3). Accordingly, we found that replacing the Hg anode by a Pt anode lowered the yield of cycloadduct 6 from 70 to 58%. Note that o-azaquinone heterodienes displayed an interesting reactivity in the sense that the cycloaddition reactions were regiospecific, irrespective of the nature of the 2-substituent, thus ensuring their synthetic utility. These results contrast with our preceding observations concerning related oquinone heterodienes, generated from pyrogallol derivatives, for which the 2-hydroxyl group is required for inducing the regiospecificity. [START_REF] Xu | [END_REF] Although the o-quinone entities have been the most widely utilized for IEDDA reaction, [START_REF]For a review on cycloaddition reactions of o-quinone heterodienes see[END_REF] the o-iminoquinone species proved to be more versatile heterodienes which would merit further developments.

All these results indicated that, in the course of the cascade reaction, the presence of the 2-hydroxyl substituent constituted a prerequisite only to the development of the catalytic process. Then, we questioned about the exact role of the 2-hydroxyl substituent to convert a catalytic inert o-azaquinone species into a highly effective organic catalyst. For this purpose, we thoroughly reexamined the ionic transamination mechanism we have previously reported for the autorecycling oxidation of aliphatic amines. 6b This process involves -proton abstraction and the subsequent electron flow from the carbon to the o-azaquinone moiety, which aromatizes to the Schiff base 2'ox (Scheme 4). At this stage, the activation of the imine function for further nucleophilic attack of amine would be provided by an intramolecular hydrogen bond between the 2-hydroxyl group and the imine-nitrogen, generating the highly reactive cyclic transition state 2'ox. This activated nucleophilic attack of amine would constitute the driving force of the overall transamination mechanism, then preventing any competitive Michael addition reaction. Similar effects of 2-phenolic hydroxyl group on the reactivity of ketimine derivatives have been recently reported in the literature. 14

Experimental Section

All apparatus, cells and electrodes were identical with those described previously. 15 Chemicals were commercial products of the highest available purity and were used as supplied. 2-amino-4-nitrophenol 4f and 2-aminophenol 4h were commercially available compounds. Substituted o-aminophenol starting materials 2i, 4a, 4c and 4e were synthesized through previously reported procedures. 16 The syntheses of new 2-aminoresorcinol derivatives 2a-h together with o-aminophenol substrates 4b, 4d and 4g, are reported in the supporting information.

2ox-mediated cascade reaction leading to benzoxazine derivatives 3a-i. General procedure A: oaminophenol substrate 2 (0.5 mmol) and excess isobutylamine (10 mmol) were dissolved in methanol (250 mL), which contained tetraethylammonium hexafluorophosphate (TEAHFP) as the supporting electrolyte (5 mmol). The resulting solution was then oxidized under nitrogen, at room temperature, at a mercury pool whose potential was fixed at +50 mV vs SCE (initial current 50 mA). After exhaustive electrolysis (5-8h, n=10-12), that is, when a negligible current was recorded (1 mA), the solution was neutralized with dry ice and the solvent was removed under reduced pressure. The brown oil residue was then poured into diethyl ether (20 mL). Insoluble TEAHFP was filtered off and the filtrate was evaporated under reduced pressure, at 30°C. Flash chromatography of the residue on silica gel afforded the expected 1,4-benzoxazine derivatives.

[(R,S)-3,3-dimethyl-5-hydroxy-2-isobutylamino- 3, 132.4, 123.7, 119.8, 117.9, 115.8, 103.5, 95.6, 67.1, 52.5, 48.9, 35.1, 34.0, 25.2, 21.4, 21.2 3.80-3.60 (m, 4 H), 3.40-3.20 (m, 2 H), 2.70-2.40 (m, 2 H), 1.80-1.00 (m, 10 H); [START_REF]For a review on cycloaddition reactions of o-quinone heterodienes see[END_REF] C NMR (75 MHz, CDCl3)150. 3, 141.5, 131.8, 115.6, 114.9, 110.1, 96.0, 67.1, 52.5, 48.9, 35.1, 33.9, 25.2, 21.4, 21.

  Angèle Chiaroni ‡ , Maurice-Bernard Fleury † , and Martine Largeron* , † † UMR 8638 CNRS-Université Paris 5, Synthèse et Structure de Molécules d'Intérêt Pharmacologique, Faculté des Sciences Pharmaceutiques et Biologiques, 4 avenue de l'observatoire, 75270 Paris cedex 06, France and ‡ Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette cedex, France E-mail : martine.largeron@univ-paris5.fr

Figure 1 .

 1 Figure 1. Voltammetric changes accompanying the electrochemical oxidation of 2a (2 mM) at a mercury anode (E = +50 mV vs SCE), in deaerated MeOH containing tetraethylammonium hexafluorophosphate (20 mM) and isobutylamine (40 mM): continuous line (before electrolysis); dashed line (a) 2-6; (b) 8; (c) 10; (d) 12 mol of electrons. Arrowheads indicate the direction of the potential sweep; v = 0.5 V s -1 . The vertical arrow indicates the initial potential point.

Figure 2 .

 2 Figure 2. Spectrophotometric changes accompanying the electrochemical oxidation of 2a (2 mM) at a

3ea

  Reagents and conditions: (2a-i) = 2 mM, (isobutylamine) = 40 mM, MeOH, rt, Hg anode (E = +50 mV vs SCE), 5-8h. b Total number of electrons transferred per molecule of mediator. c Yields refer to chromatographically pure isolated products.

Figure 3 .

 3 Figure 3. Ortep view of 3f and 3h. Displacement ellipsoids are drawn at the 30% probability level.

Figure 4 .

 4 Figure 4. Voltammetric changes accompanying the electrochemical oxidation of 4a (2 mM) at a platinum anode (E = +450 mV vs SCE), in deaerated MeOH containing tetraethylammonium hexafluorophosphate (20 mM) and isobutylamine (40 mM): (a) 0 (before electrolysis); (b) 1; (c) 2; (d) 3; (e) 4; (dashed line) 4.1 mol of electrons. Arrowheads indicate the direction of the potential sweep; v = 0.1 V s -1 . The vertical arrow indicates the initial potential point.

  Scheme 2. Tandem Oxidation-IEDDA Reaction leading to 2-alkylamino-1,4-benzoxazine derivatives 5a-h

Figure 5 .

 5 Figure 5. Spectrophotometric changes accompanying the electrochemical oxidation of 4a (2 mM) at a platinum anode (E = +450 mV vs SCE), in deaerated MeOH containing tetraethylammonium hexafluorophosphate (20 mM), 4-cyclohexylidenemethylmorpholine (10 mM) and morpholine (2 mM): (a) 0 (before electrolysis); (b) 1; (c) 2.2 mol of electrons. Cell thickness, 0.1 cm.

11

 11

Figure 6 .

 6 Figure 6. ORTEP view of 5e and 5f. Displacement ellipsoids are drawn at the 30% probability level.

  Scheme 3. Tandem Oxidation-IEDDA Reaction using 2i as the starting material

  Scheme 4. Effect of 2-hydroxyl group on the reaction pathway

(

  ; MS (ES+) m/z 314 [M+H] + , m/z 336 [M+Na] + . X-Ray analysis: A little colorless plate of 0.70  0.40  0.10 mm, crystallized from diethyl ether, was used. Empirical formula C18H23N3O2, M = 313.39, T = 293 K. Monoclinic system, space group P 21/c, Z = 4, a = 10.734(4), b = 10.465(4), c = 15.663(6) Å,  = 107.34 (2)° , V = 1679 Å 3 , dcalc = 1.239 g cm -3 , F(000) = 672,  = 0.082 mm -1 , (MoK) = 0.71073 Å. 18442 intensity data were measured with a Nonius Kappa-CCD diffractometer giving 8335 monoclinic reflections, of which 4489 unique. Refinement of 208 parameters on F 2 led to R1(F) = 0.0483 calculated with 2923 observed reflections as I ≥ 2 sigma (I) and wR2(F 2 ) = 0.1286 considering all the 4489 data. Goodness of fit = 1.031. CCDC deposition number : 298623 : 210-212 o C (from diethyl ether and chloroform). 1 H NMR (300 MHz, CDCl3) 7.62 (dd, J = 9.0 and 3.0 Hz, 1 H), 7.50 (d, J = 3.0 Hz, 1 H), 6.82 (d, J = 9.0 Hz, 1 H), 4.63 (s, 1 H), 4.18 (s, 1 H),

2 ;

 2 MS (ES+) m/z 334 [M+H] + , m/z 356 [M+Na] + . X-Ray analysis: A yellow little prismatic crystal of 0.40  0.25  0.125 mm, crystallized from a mixture chloroform/diethyl ether was used. Empirical formula C17H23N3O4, M = 333.38, T = 293 K. Triclinic system, space group P-1, Z = 2, a = 9.373(6), b = 9.802(5), c = 10.391(8) Å  = 71.79(4),  = 70.18(4),  = 72.29(4) °, V = 832 Å 3 , dcalc= 1.331 g cm -3 , F(000) = 356,  = 0.096 mm -1 , (MoK) = 0.71073 Å. 4474 reflections measured with a Nonius Kappa-CCD diffractometer of which 3327 unique. Refinement of 220 parameters on F 2 led to R1(F) = 0.0504 calculated with the 2629 observed reflections having I ≥ 2 sigma (I) and wR2(F 2 ) = 0.1377 considering all the 3327 data. Goodness of fit = 1.022. CCDC deposition number : 298624.

Table 1 .

 1 2ox-Mediated Cascade Reaction Affording 2-Alkylamino-1,4-benzoxazines 3a-i a

	Entry	Substrate	n b		Product		Yield% c
		H 2 N	OH	O			N H	OH	O
	1	HO		2a	12	N H	O		3a	71
	2				11					78
				2b					3b
		H 2 N	OH	O			H N	OH	O
	3	HO H 2 N	OH	O 2c	12	N H	O H N	OH	3c O	76
	4	HO	Br	2d	11	H N	O	Br	3d	80
	5	H 2 N	OH	O	11		H N	OH	O	74
		HO	OH	O 2e	SO 2 Me	N H	O
		H 2 N
	6				12					75
		HO		2f	F				3f
			OH	O	OMe
		H 2 N
	7	HO		2g	11				3g	84
	8				10					72
		H 2 N	OH	NO 2 2h			H N	OH	3h NO 2
	9	HO		2i	12	H N	O		3i	75

Table 2 .

 2 Anodic Oxidation of Various o-Aminophenol Derivatives 4a-h and Trapping of the Electrogenerated o-Azaquinone Heterodiene by 4-Cyclohexylidenemethylmorpholine a

	Entry	Substrate	Product	Yield% b

  colorless crystal. mp: 174-176 o C (from diethyl ether). 1 H NMR (300 MHz, CDCl3)  6.98(d, J = 9.0 Hz, 1 H), 6.83 (s, 1 H), 6.81 (d, J = 9.0 Hz, 1 H), 4.59 (s, 1 H), 4.05 (s, 1 H),3.75-3.55 (m, 4 H), 3.40- 3.25 (m, 2 H), 2.65-2.50 (m, 2 H), 1.80-1.00 (m, 10 H);[START_REF]For a review on cycloaddition reactions of o-quinone heterodienes see[END_REF] C NMR (75 MHz, CDCl3) 148.

	Tandem oxidation-IEDDA reaction leading to benzoxazine derivatives 5a-h. General procedure
	B: Freshly distilled 4-cyclohexylidenemethylmorpholine (2.5 mmol) and morpholine (0.5 mmol) were
	added to a 0.02 M solution of TEAHFP in MeOH. Then, o-aminophenol 4 (0.5 mmol) was added to the
	solution which was oxidized at a platinum anode whose potential was fixed at +450 mV vs SCE, under
	nitrogen, at room temperature. After exhaustive electrolysis (4h, n=2), the solution was treated as above
	(general procedure A) to give the 1,4-benzoxazine derivatives 5a-h.
	[(R,S)-2-morpholino-3-spiro-1'-cyclohexyl-3,4-dihydro-2H-1,4-benzoxazin-6-yl]	nitrile	5e:
	3,4-dihydro-2H-1,4-benzoxazin-6-yl](4-
	fluorophenyl) methanone 3f: orange crystal. mp: 134-136 o C (from diethyl ether and petroleum ether).
	1 H NMR (300 MHz, CDCl3)  12.63 (s, 1 H), 7.80-7.60 (m, 2 H), 7.30-7.10 (m, 2 H), 6.96 (d, J = 9.0

Hz, 1 H), 6.41

(d, J = 9

.0 Hz, 1 H), 4.65 (s, 1 H), 4.02 (s, 1 H), 2.90-2.70 (m, 1 H), 2.60-2.40 (m, 1 H), 1.91 (bs, 1H), 1.80-1.60 (m, 1 H), 1.31 (s, 3 H), 1.23 (s, 3 H), 0.91 (d, J = 6.0 Hz, 3 H), 0.88

(d, J = 9.0

Acknowledgments. This work is dedicated to the memory of Professor Pierre Potier. D.X. thanks Paris 5 University and the MRT for a postdoctoral grant.

Supporting Information Available. Typical experimental procedures and spectroscopic data for new starting materials 2a-h, 4b, 4d and 4g. Analytical data for compounds 3a-e, 3g, 3i, 5a-d, 5g, 5h and 6, including 1 H and [START_REF]For a review on cycloaddition reactions of o-quinone heterodienes see[END_REF] C NMR spectra of all 1,4-benzoxazine derivatives 3a-i, 5a-h and 6 together with crystallographic files (CIF) for compounds 3f, 3h, 5e and 5f. This material is available free of charge via the Internet at http://pubs.acs.org.