Abdul Wahid Qureshi

Raed Adill

Hannon Altamimy

Ali El Habhab

Hanine El

Muhammad Akmal Farooq

Fatiha Zobairi

Hira Hasan

Lamia Amoura

Mohamad Kassem

Cyril Auger

A W Qureshi

R Altamimy

H El Itawi

V Schini-Kerth

Dr F Toti
email: toti@unistra.fr

V Schini- Kerth

Prs V Schini-Kerth

Toti Florence

Ageing enhances the shedding of splenocyte microvesicles with endothelial pro-senescent effect that is

Keywords: Microparticles, endothelial senescence, spleen leukocyte, oxidative stress, angiotensin system, omega-3 polyunsaturated fatty acids

prevented by Losartan and associated with endothelial oxidative stress. M-SMVs induced an up-regulation of senescence markers (p16, p21, p53), proatherothrombotic (VCAM-1, ICAM-1, tissue factor) and pro-inflammatory markers (pNF-κB, COX-2) and proteins of the angiotensin system (ACE, AT1-R). Conversely, endothelial NO synthase was down-regulated. Intake of EPA:DHA 1:1 and 6:1 by middle-aged rats decreased SMV shedding by 14% and 24%, respectively. Only EPA:DHA 6:1 intake abolished the M-SMVs-induced endothelial senescence and reduced the prosenescent action of O-SMVs by 45%. Protection of ECs was not observed in response to SMVs from EPA:DHA 1:1 treated rats.

Conclusion:

Ingestion of EPA:DHA 6:1 by middle-aged or old rats, respectively abolished or limited both the shedding of SMVs and their pro-senescent, prothrombotic and pro-inflammatory effects in ECs, most likely by triggering the local angiotensin system. EPA:DHA 6:1 may help to delay ageing-related endothelial dysfunction.

Introduction

Cardiovascular diseases (CVDs) account for 39.6% of age-related chronic diseases [START_REF] Fajemiroye | Aging-Induced Biological Changes and Cardiovascular Diseases[END_REF]. The age-driven production of reactive oxygen species (ROS) within the vessel wall is a well-known contributor to endothelial dysfunction and vascular tone alteration [START_REF] Tan | Antioxidant and Oxidative Stress: A Mutual Interplay in Age-Related Diseases[END_REF]. Endothelial senescence is an emerging cause of the age-related progressive vascular dysfunction. It is however also early detected in middle-aged individuals at thrombogenic vascular sites, such as in coronary arteries, which are prone to flow disturbance. Senescence is characterized by an irreversible cell cycle arrest, morphological changes and a pro-atherogenic endothelial phenotype, favoring a loss of endothelial-dependent vascular function and progressive cardiovascular damages accompanying hypertension or atherosclerosis [START_REF] Herrera | Endothelial dysfunction and aging: an update[END_REF][START_REF] Khemais-Benkhiat | The Redox-sensitive Induction of the Local Angiotensin System Promotes Both Premature and Replicative Endothelial Senescence: Preventive Effect of a Standardized Crataegus Extract, The journals of gerontology[END_REF]. In senescent endothelial cells (ECs), Reactive oxygen species (ROS) from mitochondrial sources and generated by up-regulated cytoplasmic NADPH oxidase, cyclooxygenases (COXs) initiate redox-sensitive processes that prompt endothelial dysfunction thereby shifting the hemostatic features of the healthy endothelium towards a pro-inflammatory, procoagulant and vaso-constricting phenotype. In addition, P53 and down-stream cyclin-dependent kinase inhibitors, P21 and P16, are up-regulated while the cytoprotective endothelial nitric oxide synthase (eNOS) is down-regulated. Ultimately, senescent ECs promote the release of endothelial microvesicles able to disseminate a pro-senescent signal [START_REF] Burger | Microparticles induce cell cycle arrest through redox-sensitive processes in endothelial cells: implications in vascular senescence[END_REF][START_REF] Abbas | Endothelial Microparticles From Acute Coronary Syndrome Patients Induce Premature Coronary Artery Endothelial Cell Aging and Thrombogenicity[END_REF].

Microvesicles (MVs), are sub-micron plasma membrane vesicles shed by cells in response to stress. MVs convey active cytoplasmic proteins, lipids as well as miRNA that characterize the cell lineage and stress at the origin of their release [START_REF] Benameur | Molecular Mechanisms Underpinning Microparticle-Mediated Cellular Injury in Cardiovascular Complications Associated with Diabetes[END_REF]. In blood, they circulate as surrogate and pathogenic markers of primary and secondary cardiovascular risk [START_REF] Koenen | Editorial: Extracellular Vesicle-Mediated Processes in Cardiovascular Diseases[END_REF]. Indeed, circulating MVs constitute a storage pool of effectors of multiple cell origin orchestrating vascular cell cross-talk. MVs are pro-coagulant because they expose phosphatidylserine (Phtdser), an anionic phospholipid that catalyzes the assembly of blood coagulation complexes, and when stemmed from endothelial cells and monocyte, tissue factor (TF), the cellular initiator of blood coagulation [START_REF] Morel | Microparticles: a critical component in the nexus between inflammation, immunity, and thrombosis[END_REF]. MVs of endothelial origin from patients with acute coronary syndrome (ACS) have been shown to induce coronary endothelial cell dysfunction, premature senescence and thrombogenicity through the activation of the local angiotensin system [START_REF] Abbas | Endothelial Microparticles From Acute Coronary Syndrome Patients Induce Premature Coronary Artery Endothelial Cell Aging and Thrombogenicity[END_REF].

Interestingly, leukocyte-derived MVs (LMVs), were shown crucial to thrombus growth and vascular remodeling [START_REF] Swystun | The role of leukocytes in thrombosis[END_REF]. Their elevated plasma level was correlated with the Framingham score in patients with primary and secondary cardiovascular adverse outcomes [START_REF] Chironi | Cardiovascular risk as assessed by traditional risk factors[END_REF]. LMVs are recruited at the surface of the inflamed endothelium, constitute an additional source of TF and mediate cell interactions within the thrombus [START_REF] Falati | Accumulation of tissue factor into developing thrombi in vivo is dependent upon microparticle P-selectin glycoprotein ligand 1 and platelet P-selectin[END_REF][START_REF] Grover | Tissue Factor: An Essential Mediator of Hemostasis and Trigger of Thrombosis[END_REF]. In human atherosclerotic plaques, 55% of MVs originate from leukocytes [START_REF] Leroyer | Cellular origins and thrombogenic activity of microparticles isolated from human atherosclerotic plaques[END_REF]. Animal models of atherothrombosis or ischemia reperfusion indicate that LMVs contribute to tissue remodeling by limiting the endothelial NO and promoting redox-sensitive pathways, among which the up-regulation of proinflammatory cytokines and of adhesion proteins favoring diapedesis. In mice, the injection of LMVs from diabetic patients alters the vascular tone by reducing the acetylcholine-evoked endothelial relaxation of the aorta [START_REF] Tesse | Upregulation of Proinflammatory Proteins Through NF-{kappa}B Pathway by Shed Membrane Microparticles Results in Vascular Hyporeactivity[END_REF][START_REF] Angelillo-Scherrer | Leukocyte-derived microparticles in vascular homeostasis[END_REF]. Altogether, TF + -MVs prompt coagulation and exacerbate inflammation through multiple amplification loops involving TF-driven cell responses [START_REF] Leroyer | Endothelial-derived microparticles: Biological conveyors at the crossroad of inflammation, thrombosis and angiogenesis[END_REF]. Interestingly, MV shedding seems linked to the lateral lipid organization of the plasma membrane, lipid rafts favoring their TF enrichment when released from activated macrophages [START_REF] Pollet | Plasma Membrane Lipid Domains as Platforms for Vesicle Biogenesis and Shedding?[END_REF].

The lipid composition of the plasma membrane of immune cells influences membrane fluidity and down-stream production of lipid mediators. In humans consuming western diet, polyunsaturated fatty acids (PUFAs) in the plasma membrane of circulating neutrophils, lymphocytes, monocytes are mainly composed of 10%-20% of arachidonic acid, 0.5%-1% eicosapentaenoic acid (EPA) and 2%-4% docosahexaenoic acid (DHA). Intake of omega-3 PUFAs enhances the EPA /DHA membrane proportion at the expense of the n-6 PUFAs, mainly arachidonic acid and derived metabolites, alters the membrane order, and raft-dependent downstream pathways [START_REF] Calder | Omega-3 fatty acids and inflammatory processes[END_REF].

Clinical trials and animal models have shown that EPA and DHA reduce the risk of cardiovascular diseases [START_REF] Dinicolantonio | Omega-3s and cardiovascular health[END_REF][START_REF] Iwamatsu | Which has the stronger impact on coronary artery disease, eicosapentaenoic acid or docosahexaenoic acid?[END_REF]. Daily consumption of omega-3 PUFAs is protective against primary or secondary cardiovascular events [START_REF] Kromhout | Fish oil and omega-3 fatty acids in cardiovascular disease: do they really work?[END_REF]. While EPA and DHA attenuate redox-driven DNA damages in aorta cells [START_REF] Sakai | Fish oil omega-3 polyunsaturated fatty acids attenuate oxidative stress-induced DNA damage in vascular endothelial cells[END_REF], vasoprotection by omega-3

PUFAs was shown dependent on the EPA/DHA ratio. We previously showed that maximal vaso-relaxation of healthy pig coronary artery is only obtained with omega-3 EPA:DHA 6:1 as compared to omega-3 EPA:DHA 1:1 [START_REF] Zgheel | Redox-sensitive induction of Src/PI3-kinase/Akt and MAPKs pathways activate eNOS in response to EPA:DHA 6:1[END_REF]. Intake also improves the endothelial dysfunction and reduces systolic blood pressure by 53% in angiotensin-II-induced hypertensive rats [START_REF] Niazi | EPA:DHA 6:1 prevents angiotensin IIinduced hypertension and endothelial dysfunction in rats: role of NADPH oxidase-and COXderived oxidative stress[END_REF].

Still, the beneficial effects of omega-3 PUFAs in cardiovascular health are debated. Clinical trials such as DART, GISSI-Prevenzione, GISSI-HF, JELIS have shown beneficial effects of omega-3 PUFAs in reducing the risk of cardiovascular diseases, whereas, others such as ALPHA OMEGA, SU.FU.OM3, ASCEND, VITAL did not observed promising proofs of reduction [START_REF] Bowen | Omega-3 Fatty Acids and Cardiovascular Disease: Are There Benefits?[END_REF][START_REF] Abdelhamid | Omega-3 fatty acids for the primary and secondary prevention of cardiovascular disease[END_REF]. Most recently, the REDUCE-IT trial which included hyperlipidemic patients with previously established cardiovascular diseases and receiving statin therapy, showed that 4 g/day ingestion of icosapent ethyl EPA led to a 19% reduction in plasma triglycerides and a 25% drop in major cardiovascular events after 4.9 years follow-up [START_REF] Bhatt | Cardiovascular Risk Reduction with Icosapent Ethyl for Hypertriglyceridemia[END_REF][START_REF] Grzegorz | Omega-3 polyunsaturated fatty acids: is their future VITALized or REDUCEd?[END_REF].

Strikingly, mechanisms of endothelial protection by omega-3 PUFAs against

LMVs remain yet to be characterized. In post-myocardial 60 years-old patients, elevated levels of circulating MVs of leukocyte and platelet origin were slightly but significantly lowered by a 12-week supplementation with 5.2 g/day omega-3 PUFA (33% EPA 60%DHA w:w) given 6 month later [START_REF] Del Turco | Effect of the administration of n-3 polyunsaturated fatty acids on circulating levels of microparticles in patients with a previous myocardial infarction[END_REF], while MV procoagulant potential was reduced in vitro and involved both TF-dependent and -independent coagulation [START_REF] Del Turco | Effect of the administration of n-3 polyunsaturated fatty acids on circulating levels of microparticles in patients with a previous myocardial infarction[END_REF]. However, it still remains unclear whether omega 3 PUFA prevent the release of LMVs or rather shift noxious LMVs into cytoprotective actors and if so, which leukocyte population becomes beneficial to the ageing endothelium.

In vitro, omega-3 PUFAs directly modulate ion channels endocytosis, trigger PPAR-α or HNF-4α nuclear receptor dependent-responses, G-protein mediated-activation and ultimately ERK ½ kinases and NF-ƙB signaling. They also may alter cell responses by incorporating into the plasma membrane, where they are converted by COXs and LOXs into key anti-inflammatory specialized pro-resolving lipid mediators, including D-and E-series resolvins and protectins, maresin, [START_REF] Mozaffarian | Omega-3 fatty acids and cardiovascular disease: effects on risk factors, molecular pathways, and clinical events[END_REF][START_REF] Molfino | The role for dietary omega-3 fatty acids supplementation in older adults[END_REF][START_REF] Sokoła-Wysoczańska | Polyunsaturated Fatty Acids and Their Potential Therapeutic Role in Cardiovascular System Disorders-A Review[END_REF].

This study was aimed at investigating whether the intake of the omega-3 EPA:DHA 6:1 new formulation modifies the ability of ageing immune cells to release MV, and whether it prevents their pro-senescent action towards the endothelium. MVs were isolated from the spleen of middle-aged (48 weeks), young (12 weeks) and old (72 weeks) rats. The ability of the spleen-derived leukocytes MVs (SMVs) to promote premature endothelial senescence was examined in young primary coronary endothelial cells (ECs).

Materials and Methods

Chemicals

All the chemicals and solvants, unless indicated, were from Sigma-Aldrich (Sigma-Aldrich, Saint Quentin Fallavier, France). Losartan was obtained from Merck

Research Laboratories (Boston, MA, USA).

Ethics statement

Wistar rats (Janvier-labs

Preparation of Omega-3 PUFAs

Purified formulations of omega-3 EPA and DHA were obtained from Pivotal Therapeutics, Inc. (Woodbridge, ON, Canada). EPA:DHA ratios of 6:1 and 1:1 (w/w) were prepared by mixing each EPA:DHA according to their relative purity under nitrogen flux to avoid oxidation, and stored in amber colored glass vials at 4°C. In several experiments (impact of Losartan on SMV-mediated endothelial senescence, determination of SMV phenotypes), the EPA:DHA 6:1 solution was replaced by the content of Vascazen capsules (Pivotal Therapeutics Inc) and compared to the content of Omacor capsules (EPA:DHA 1.2:1).

Rat treatment

Middle-age male wistar rats (M, 48-week old) were weighed and assigned to four groups (9 rats/group). Rats were given 500 mg/kg/day oral dose of either EPA:DHA 6:1, EPA:DHA 1:1, corn oil as an isocaloric control without omega-3 or tap water for a week. Young (Y, 12-weeks old) and old (O, 72-weeks-old) male wistar rats, respectively treated for 7 and 14 days, were used for comparative purpose. After treatment, rats were weighed and euthanized by I.P injection of pentobarbital (150 mg/kg).

Spleen-derived leukocytes culture (splenocytes)

Freshly isolated rat spleens were washed with sterile PBS (phosphate buffered saline, Lonza, USA), cleaned of any fat debris and weighed. Under sterile conditions, spleens were cut using razor blades into five or six large pieces further homogenized in PBS using the plunger of 2 mL syringes and filtered through a strainer (100-µm Nylon, Falcon, USA) over a 50-mL tube. Filtrate was then centrifuged at 450g for 5min. at room temperature (RT). The pellet was gently re-suspended in 3 mL ammonium chloride potassium (ACK) erythrocyte lysis buffer (0.15M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA, pH 7.2-7.4), mixed under gentle shaking for 3 min, recentrifuged (450g, 5min, RT) and re-suspended with RPMI medium-1640 (Gibco, life technologies limited, UK) supplemented with L-glutamate (2 mM), penicillin (100 U/mL), streptomycin (100 U/mL), fungizone (250 mg/mL) and 15% fetal bovine serum (FBS) (Gibco, Saint Aubin, France). Cells were counted, seeded at 3 x 10 6 cells/mL in T75 flasks and incubated in humidified incubator at 37 °C, with 5% CO2 for 24 hours.

Isolation, quantification and characterization of splenocyte microvesicles

After 24 hours culture, supernatants were collected under sterile conditions and splenocytes were counted using Trypan blue to determine the number of surviving cells. Splenocytes and cell debris were discarded by centrifugation at 450g,15min, RT. Supernatants were centrifuged twice at 14,000g, 60min, 4 °C, and washed SMVs pelleted and concentrated in Hanks Balanced Salt Solution (HBSS, without phenol red, without Ca +2 and Mg +2 , Lonza, Belgium) and stored at 4°C for a maximum of 1 month. Prothrombinase assay was performed to quantify SMVs after their capture on Annexin-A5 coated micro-wells using a microplate spectrophotomer in kinetic mode. Annexin-5 has high affinity for phosphatidylserine (PhtdSer) exposed at the surface of MVs. In this assay, PhtdSer is the rate-limiting factor of the generation of thrombin from prothrombin detected at 405 nm using a chromogenic substrate (PNAPEP-0216, cryopep, Montpellier, France). SMV concentration was referred to as nanomolar PhtdSer equivalent (nM phtdser), by reference to a standard curve constructed with synthetic vesicles with known concentration of PhtdSer [START_REF] Hugel | Measuring circulating cell-derived microparticles[END_REF]. The cell origin of SMVs was characterized by capturing SMVs onto biotinylated antibodies against leukocytes CDs before quantification by prothrombinase assay as described [START_REF] Delabranche | Microparticles are new biomarkers of septic shock-induced disseminated intravascular coagulopathy[END_REF] using the following IgGs : anti-CD45 for leukocyte common antigen, anti-CD3 for T lymphocyte population, anti-CD4 for T helper cells, anti-CD8b for T cytotoxic cells, anti-CD161a for natural killer cells, anti-CD25 for IL-2 receptor T cells and splenic dendritic cells, anti-CD31, mainly for endothelial cells, anti-CD11b/c for monocyte/macrophage and granulocyte, anti-CD11b for neutrophil. Biotinylated monoclonal antibodies were insolubilized onto the streptavidin-coated microtitration plates and incubated with SMVs. All antibodies were purchased from BD Pharmingen, San Jose, USA. The MV concentration was obtained by subtracting the OD values measured using a control istotype biotinylated IgG.

Primary coronary artery endothelial cell culture

Primary coronary artery endothelial cells (ECs) were prepared from left circumflex coronary arteries of pig hearts, collected from local slaughterhouse (COPVIAL, Holtzheim, France) as described by [START_REF] Abbas | Endothelial Microparticles From Acute Coronary Syndrome Patients Induce Premature Coronary Artery Endothelial Cell Aging and Thrombogenicity[END_REF]. Briefly, left circumflex coronary arteries were dissected out of freshly slaughtered pig hearts, cleaned of any adhesive conjunctive tissues and flushed with PBS without calcium to remove all the remaining blood.

Cleaned coronary arteries were then treated with Collagenase type I (Gibco, life technologies corporation, USA) solution at 1 mg/ml prepared in MCDB-131 medium (Gibco, life technologies limited, UK), supplemented with streptomycin (100 U/ml), penicillin (100 U/ml), fungizone (250 mg/ml), and L-glutamine (1 mM, all from Lonza, St Quentin en Yvelines, France) for 15 min at 37°C. ECs were then extracted into 50-mL falcon tubes by circular massage of arteries with frequent flushing with medium. The collected medium containing ECs was then centrifuged (450g, 5min, RT), supernatant was discarded and cells were re-suspended with complete MCDB-131 medium supplemented with 15% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 U/ml), fungizone (250 mg/ml), and L-glutamine (1 mM). ECs of three different coronary arteries were cultured in adherent T25 flask in humidified incubator at 37 °C, with 5% CO2. After 6 hours, cells were washed with PBS to remove any non-adherent cells and fresh complete medium was added (P0 ECs).

Thereafter, first passage ECs (P1ECs) were grown for 72 hours with medium changed every 48 hours.

Treatment of ECs by splenocyte MVs

Following trypsinization (Trypsin, Gibco, Life Technologies SAS, St Aubin, France)

Measurement of Senescence-Associated β-galactosidase activity

The fluorogenic cell permeable substrate C12FDG (5-dodecanoylaminofluorescein Di-β-D-galactopyranoside, Invitrogen, ThermoFisher, Illkirch, France) was used to determine the senescence-associated β-galactosidase activity (SA-β-gal) in treated

ECs by flow cytometry, as described previously [START_REF] Abbas | Endothelial Microparticles From Acute Coronary Syndrome Patients Induce Premature Coronary Artery Endothelial Cell Aging and Thrombogenicity[END_REF]. Briefly, ECs were alkalinized (pH raised to 6) with chloroquine (300 μM) for 1 h, followed by 1 h incubation with C12FDG (33 μM), a fluorogenic substrate of SA-β-gal [START_REF] Debacq-Chainiaux | Protocols to detect senescence-associated beta-galactosidase (SA-βgal) activity, a biomarker of senescent cells in culture and in vivo[END_REF]. Cells were thereafter washed with ice-cold PBS, harvested with trypsin and freshly analyzed using the CellQuest software (FACScan, Becton Dickinson, San Jose, CA, USA). Light scattering parameters were set to eliminate dead cells and subcellular debris. The green C12-fluorescein signal was measured and SA-β-gal activity estimated using the mean fluorescence intensity (MFI) of the cell population. Auto-fluorescence gains were determined in unlabeled cells and set at the first logarithmic decade.

Western blot analysis

After

Characterization of oxidative stress in spleen tissue and ECs

Oxidative stress in spleen tissue or ECs was determined using DiHyroEthidium To identify the sources of oxidative stress, ECs were pre-treated for 30 min, with an NADPH oxidase inhibitor (5 μM VAS-2870), a cyclooxygenase inhibitor (30 μM indomethacin), antioxidant (1mM N-acetylcysteine), or a mixture of mitochondrial inhibitors of the respiratory chain (myxothiazol, potassium cyanide and rotenone, 1

μM each) prior addition of DHE alone (tissue) or combined with SMVs (ECs).

Measurement of endothelial apoptosis

Apoptosis was measured by flow cytometry using Annexin-5 and propidium iodide double labeling. ECs treated by 10 nM or 30 nM SMVs for 48 h were incubated with fluorescent Annexin-5 (5 μg/mL ImmunoTools, Friesoythe, Germany) and 2.5 μg/ml propidium iodide (Milteny Biotec SAS, Paris, France) in dark for 15 min, at room temperature. Early apoptotic cells were defined as Annexin-5 stained and PI unlabeled. Fluorescence acquisition was performed by Guava EasyCyte Plus FlowCytometry System (Millipore). Analysis was constructed from a minimum of 2000 events.

Statistical Analysis

Data are expressed as mean ± standard error mean (S.E.M) for n different experiments and analyzed by Graphpad Prism 5. Statistical variance between two groups was determined by applying unpaired-T test. Differences were considered statistically significant at P < 0.05.

Results

Microvesicle shedding from primary splenocytes increases with age and is associated with enhanced pro-senescent potential

Using freshly harvested rat spleens from young (Y), middle-aged (M) and old (O) rats as a convenient source of primary spleen-derived leukocytes, thereafter termed splenocytes, we measured the microvesicle (SMV) shedding in cell supernatants after 24h culture. The proportion of surviving splenocytes was reduced by half in old compared to young splenocyte cultures (figure 1c), while SMV shedding significantly increased with age (3.3 folds in O, 1.7 folds in M vs. Y, p < 0.01). After 48h incubation with 10 nM SMVs, premature endothelial senescence of young primary coronary endothelial cells at passage 1 (P1ECs) was indicated by significantly enhanced SA-β-gal activity only in response to SMVs from old and middle-aged rats (O: 2 folds, M: 1.5 folds vs. untreated P1 ECs, p < 0.01). Conversely, SMVs from young rats had no significant effect (figure 1b). On line with other reports, [START_REF] Luceri | Aging related changes in circulating reactive oxygen species (ROS) and protein carbonyls are indicative of liver oxidative injury[END_REF] we also identified an age-related ROS formation in the corresponding spleen tissues with a significant amount of ROS in middle-aged rats by DHE staining (M: 1959.91

A.U. vs. Y: 1492.75 A.U., figure 2). Altogether, data suggest a progressive alteration of splenocytes with ageing that is associated with redox-sensitive mechanisms and leads to an increased shedding of pro-senescent SMVs.

Since omega-3 PUFA were reported to incorporate in rafts and modulate the plasma membrane lateral organization of lymphocytes [START_REF] Fan | Remodelling of primary human CD4+ T cell plasma membrane order by n-3 PUFA[END_REF], we investigated the impact of an EPA:DHA short-term intake on SMVs and first focused on splenocytes freshly isolated from middle-aged rats.

EPA:DHA 6:1 intake reduces oxidative stress in middle-aged rats with no incidence on spleen or total bodyweight

No significant changes were observed in spleen or total body weight of middle-aged rats after 7 days of treatment with either corn oil or omega-3 (figure 3a-b 5a). SMV CTL also induced significant up-regulation of senescence markers p53 (1.6 fold) and down-stream p21 and p16 (1. A.U., p<0.05, SMV 6:1 vs. SMV CTL ), an observation that was also confirmed by assessment of the expression of p53, p21 and p16 senescence protein markers.

In addition, the fact that splenocyte supernatants depleted of SMV CTL by high-speed centrifugation, did not induce significant SA-β-gal activity, confirmed that SMV CTL were the true inducers of premature senescence in the cell medium. Of note, none of the SMV subsets affected the degree of apoptosis in P1ECs, confirming the specific pro-senescent nature of the signal delivered by SMVs (figures 5a, 5c).

EPA:DHA 6:1 intake by middle-aged rats prevents the endothelial proinflammatory and pro-thrombotic responses induced by SMVs

We previously had reported that endothelial senescence is characterized by a proinflammatory and procoagulant phenotype [START_REF] Abbas | Endothelial Microparticles From Acute Coronary Syndrome Patients Induce Premature Coronary Artery Endothelial Cell Aging and Thrombogenicity[END_REF]. We therefore examined the eventual protection by omega-3 PUFA intake using the above SMV-mediated endothelial senescence model. Endothelial pro-inflammatory and pro-coagulant protein markers were assessed by western blot after treatment for 6h or 48h with 10 nM washed SMV CTL , SMV CO , SMV 1:1 , or SMV 6:1 . After 48 h incubation, SMV CTL induced the upregulation of COX-2, but not of COX-1, by 3 folds, ICAM-1 and TF by 1.5 fold, VCAM-1 by 1.6 fold (figure 6). Interestingly, a significant 1.9 fold rise in phosphorylated NF-kB was detectable as early as 6 h after the addition of SMVs, pointing at a pivotal role of the inflammatory signaling in the progression of senescence. Conversely, the expression of pro-inflammatory and procoagulant makers was abolished in the presence of SMV 6:1 but not corn oil or SMV 1:1 , indicating a specific effect of EPA:DHA 6:1 (p<0.05 SMV 6:1 vs. SMV CTL , figure 6).

EPA:DHA 6:1 intake by middle-aged rats limits oxidative stress in

SMVs-treated ECs

Since the presence of ROS was early detected in middle-aged spleen and because oxidative stress contributes to endothelial senescence via reduced NO formation [START_REF] Khemais-Benkhiat | The Redox-sensitive Induction of the Local Angiotensin System Promotes Both Premature and Replicative Endothelial Senescence: Preventive Effect of a Standardized Crataegus Extract, The journals of gerontology[END_REF],

we further examined whether 10 nM SMVs could act as early contributors to oxidative stress in ECs and whether omega 3 intake would be beneficial. After 6h incubation, SMV CTL significantly increased the amount of ROS by 2 folds in young P1ECs (SMV CTL : 2673.65 A.U. vs. untreated P1ECs: 1315.37 A.U, p< 0.01).

Consistent with the whole spleen tissue measurements (figure 3), P1ECs showed significantly lowered amounts of ROS in response to SMV 6:1 , but not to SMV CO or SMV 1:1 , suggesting that omega-3 EPA:DHA 6:1 intake prevents the SMVs-induced oxidative stress (figure 7). Pharmacological inhibition by Indomethacin, VAS-2870 and the mitochondrial respiration complex inhibitors significantly blunted the SMVsinduced formation of ROS, indicating that COXs, NADPH oxidase and mitochondria contribute to the SMVs-induced oxidative stress. Consistent with the observation, western blot analysis indicated that SMV CTL down-regulated eNOS by 30% in P1ECs after 48 h (SMV CTL vs. untreated ECs, p<0.05, figure 7). Altogether, intake omega-3 EPA:DHA 6:1, but not of corn oil or omega-3 EPA:DHA 1:1, was a cytoprotective treatment against SMVs-induced endothelial dysfunction.

EPA:DHA 6:1 intake by middle-aged rats reduces the SMVs-mediated activation of local angiotensin system in ECs

Because the local angiotensin system contributes to the induction of endothelial senescence and is associated with increased endothelial expression of the angiotensin converting enzyme (ACE) and its angiotensin type-1 receptor (AT1R) [START_REF] Khemais-Benkhiat | The Redox-sensitive Induction of the Local Angiotensin System Promotes Both Premature and Replicative Endothelial Senescence: Preventive Effect of a Standardized Crataegus Extract, The journals of gerontology[END_REF], we investigated the impact of omega-3 intake. The expression of ACE and AT1-R was significantly up-regulated in P1ECs treated with SMV CTL for 48h (SMV CTL vs.

untreated P1EC, p<0.05, figure 8a). Only SMV 6:1 showed reduced ACE and AT1-R expression by 44% and 45%, respectively, suggesting that EPA:DHA 6:1, but not corn oil or EPA:DHA 1:1, prevent the SMV-mediated activation of the local angiotensin system. Furthermore, the SMV CTL -induced SA-β gal activity was reduced to baseline in the presence of Losartan, an AT1R antagonist, thereby indicating the pivotal role of the angiotensin system in the SMV-induced premature senescence (figure 8b).

Altogether, our data indicate that SMVs from middle-aged rats are pro-senescent endothelial effectors acting via redox-sensitive pathways, and that their noxious effects can be abolished by a 7-day omega-3 EPA:DHA 6:1 intake.

EPA:DHA 6:1 intake is beneficial to old rats

Because the shedding of SMVs was significantly higher in old rats, we also verified that a 14 days intake of only EPA:DHA 6:1, but not of corn oil, significantly reduced the ability of old splenocytes to shed SMVs. EPA:DHA 6:1 significantly reduced the shedding of SMVs by 39% while a symmetrical 22% splenocyte survival was observed as compared to treated rats (p<0.05 O 6:1 vs. O CTL , figure 9a-b).

Conversely, a 14-day corn oil ingestion had an inverse effect with a 25% fold increase in SMV release (figure 9a) and a reduced splenocyte survival (figure 9b).

Using the SMV-induced senescence model, we observed a significant 35% reduction of the P1CEs SA-β-gal activity in the presence of SMV 6:1 (figure 9c).

Discussion

The present findings indicate that age is a strong and progressive trigger for the shedding of pro-senescent spleen-derived leukocytes microvesicles (SMVs) that prompt premature senescence in primary coronary endothelial cells, on line with our previous observation [START_REF] Habhab | PB 671 Immune Response Favors the Release of Leukocyte-derived Microparticles Mediating Endothelial Senescence, and Procoagulant and Pro-inflammatory Phenotype[END_REF]. Senescence was characterized by SA-β-gal activity, prooxidative, pro-inflammatory and procoagulant and pro-atherogenic responses.

Interestingly, the release of pros-senescent MVs was a characteristic feature of both old and middle-aged rat splenocytes, thereby suggesting the benefit of an early pharmacological control. We therefore investigated the possibility that a short-term omega-3 intake prevents the age-related SMV effects in middle-aged rats. The intake dose was equivalent to 5.67 g/day/70 kg body weight in humans [START_REF] Reagan-Shaw | Dose translation from animal to human studies revisited[END_REF], on line with the 0.18 to 10 g/day ranges given in clinical studies [START_REF] Calder | Immunomodulation by omega-3 fatty acids[END_REF][START_REF] Delgado-Lista | Long chain omega-3 fatty acids and cardiovascular disease: a systematic review[END_REF][START_REF] Enns | The impact of omega-3 polyunsaturated fatty acid supplementation on the incidence of cardiovascular events and complications in peripheral arterial disease: a systematic review and meta-analysis[END_REF][START_REF] Barden | Short-term n-3 fatty acid supplementation but not aspirin increases plasma proresolving mediators of inflammation[END_REF];

4.1

The ratio of EPA:DHA Omega 3 is critical in the prevention of the SMVinduced premature senescence.

Using an original SMV-endothelial cross-talk model, we compared two different formulations of omega 3 PUFA and clearly demonstrate that only the EPA:DHA 6:1 formulation has strong anti-senescent effects. Omega-3 EPA:DHA 6:1 limited the release of splenocyte SMVs and significantly blunted their pro-senescent properties whereas EPA:DHA 1:1 had no effect. This observation is consistent with a previous ex-vivo study in healthy pig coronary arteries showing that the EPA:DHA 6:1 is an optimal ratio to induce endothelium-dependent vaso-relaxation, predominantly involving eNOS and hence restoring NO availability [START_REF] Zgheel | Redox-sensitive induction of Src/PI3-kinase/Akt and MAPKs pathways activate eNOS in response to EPA:DHA 6:1[END_REF]. Interestingly, in old (see companion article by Farooq A.M. et al.) or hypertensive rats EPA:DHA 6:1. Chronic intake was also associated to an improved endothelial-dependent vaso-relaxation measured in mesenteric arteries while limiting hypertension in the latter model [START_REF] Niazi | EPA:DHA 6:1 prevents angiotensin IIinduced hypertension and endothelial dysfunction in rats: role of NADPH oxidase-and COXderived oxidative stress[END_REF].

Similarly, EPA:DHA 6:1 appeared also beneficial in old SMV-induced premature endothelial senescence. Our data also indicate that the oxidative stress was specifically reduced by EPA:DHA 6:1 in spleen tissues from which SMVs were harvested, thereby confirming organ targeting.

EPA:DHA 6:1 protection against age-related SMV-induced endothelial senescence and inflammaging.

As previously reported, inflammaging, characterized by the impairment of the immune system and chronic low-grade inflammation, contributes to persistent tissue damages altering cells of both naïve and adaptive systems. Accumulation of senescent cells also impacts tissue repair and amplifies the senescence associated secretory syndrome (SASP) favoring pro-inflammatory and pro-oxidative loops and the secretion of cytokines and mediators acting as paracrine inflammatory effectors [START_REF] Figueira | Interventions for age-related diseases: Shifting the paradigm[END_REF].

Endothelial-derived and splenocyte-derived MVs also act as cellular mediators of autocrine [START_REF] Burger | Microparticles induce cell cycle arrest through redox-sensitive processes in endothelial cells: implications in vascular senescence[END_REF][START_REF] Abbas | Endothelial Microparticles From Acute Coronary Syndrome Patients Induce Premature Coronary Artery Endothelial Cell Aging and Thrombogenicity[END_REF] and paracrine induction of premature endothelial senescence [START_REF] Habhab | PB 671 Immune Response Favors the Release of Leukocyte-derived Microparticles Mediating Endothelial Senescence, and Procoagulant and Pro-inflammatory Phenotype[END_REF]. It is therefore likely that pro-senescent MVs contribute to age-related senescence in clinical settings. While the proportion of pro-senescent MVs remains to be characterized, endothelial and leukocyte-derived MVs circulate at high levels in patients with cardiovascular diseases and chronic inflammatory disorders like type 2 diabetes [START_REF] Pollet | Plasma Membrane Lipid Domains as Platforms for Vesicle Biogenesis and Shedding?[END_REF][START_REF] Ridger | Microvesicles in vascular homeostasis and diseases[END_REF].

In the present study, EPA:DHA 6:1 omega 3 intake modifies the pattern of the cellular origin of spleen leukocyte-derived MVs and their spontaneous release from the innate and adaptive immune cells. Furthermore, in middle-aged rats, the prosenescent features of SMVs leading to coronary endothelial senescence are abolished by a short-term EPA:DHA 6:1 intake, that completely blunts the endothelial dysfunction and SMV-borne pro-oxidative, pro-coagulant and proinflammatory potentials, most likely by reducing oxidative stress, enhancing NO bioavailability and preventing the activation of the local angiotensin system. In addition, the rapid blunting of the inflammatory stress via an early targeting of the NF-K B activation would also contribute to prevent the up-regulation of TF, an early responsive gene with high thrombogenic potential [START_REF] Grover | Tissue Factor: An Essential Mediator of Hemostasis and Trigger of Thrombosis[END_REF].

Since endothelial senescence is promoted by flow disturbance favoring MVendothelial interactions and early detected at coronary branches, it is tempting to speculate that EPA:DHA 6:1 also blunts the pro-senescent signaling of LMVs at such endothelial sites prone to athero-thrombosis, thereby limiting the low grade but sustained endothelial inflammation in middle-aged and old rats and deferring the progression of cardiovascular events with age.

4.3

Ageing-related endothelial dysfunction and targets of EPA:DHA 6:1

We could identify NADPH oxidase cyclooxygenases (COXs) and the mitochondrial respiratory chain as the endothelial main sources of ROS accumulation during agerelated SMV induced endothelial senescence. In a previous report from the team, EPA:DHA 6:1 was shown a potent inducer of NO formation targeting endothelial Src/PI3-kinase/Akt and MAPkinase pathways and eNOS activation [START_REF] Zgheel | Redox-sensitive induction of Src/PI3-kinase/Akt and MAPKs pathways activate eNOS in response to EPA:DHA 6:1[END_REF].

Nevertheless, the EPA:DHA 6:1 direct targets remain poorly characterized, despite clear anti-inflammatory effects [START_REF] Calder | Omega-3 fatty acids and inflammatory processes[END_REF]. In the splenocyte membrane, non-exclusive mechanisms would be (i) the modification of the cavaeole lipid microenvironment [START_REF] Li | Eicosapentaenoic acid modifies lipid composition in caveolae and induces translocation of endothelial nitric oxide synthase[END_REF] and/or membrane composition possibly leading to the accelerated endocytosis of TF, ACE or AT1, (ii) the modification of the lateral membrane proteo-lipidic domains (rafts) involved in pro-inflammatory signaling favoring cytokine release [START_REF] Li | Topological localization of monomeric C-reactive protein determines proinflammatory endothelial cell responses[END_REF],

thereby blunting the down-stream up-regulation of TF, ICAM, VCAM, or (iii) the sorting of noxious active proteins exported in the MV membrane. To the best of our knowledge, the impact of EPA:DHA on lipid transporters across the membrane bilayer remains unknown.

Of note, the EPA:DHA 6:1 intake was not associated with any sign of bleeding nor had any effect on SMV-induced endothelial senescence in healthy young rats, confirming the absence of safety issues after short-term intake and indicating that ageing cells are specific targets of treatment. An eventual explanation is the reported enhanced degree of EPA incorporation in cells from old individuals [START_REF] Calder | Immunomodulation by omega-3 fatty acids[END_REF].

Relevance to cardiovascular diseases

Angiotensin II is an inducer of premature endothelial senescence [START_REF] Shan | From autophagy to senescence and apoptosis in Angiotensin II-treated vascular endothelial cells, APMIS : acta pathologica, microbiologica[END_REF]. Although we demonstrated that EPA:DHA 6:1 protection against SMV-induced senescence was blunted after ECs pre-treatment with Losartan, an inhibitor of the AT1 receptor, the beneficial effect of EPA:DHA 6:1 and the ECs cytoprotection against SMV-induced premature senescence remains to be established in young and older hypertensive rats as already reported for endothelial-dependent vascular dysfunction. Indeed, a spleen-cardiac axis was recently proposed, pointing at the role of immune cells and chronic low-grade inflammatory state in cardiovascular diseases. In addition, in aged patients with chronic heart failure, splenomegaly and a higher rate of peripheral monocytes were indicative of non-responders to resynchronization therapy and higher prevalence of new hospitalization due to disease progression [START_REF] Fujinami | Association between the baseline peripheral blood monocyte counts, the size of spleen, and the response to cardiac resynchronization therapy[END_REF].

Furthermore, in a mouse model of prolonged cardiac ischemic insult, a cardio HMGB1-splenic RAGE signaling axis was evidenced during the infarct reperfusion,

showing that cardiac tissues release mediators contributing to infarct exacerbation through the activation of splenic neutrophil in a FPR-1-dependent manner and their migration to the heart [START_REF] Tian | The spleen contributes importantly to myocardial infarct exacerbation during post-ischemic reperfusion in mice via signaling between cardiac HMGB1 and splenic RAGE[END_REF].

Conclusion

Ageing is associated with increased leukocyte ability to shed MVs that are strong promoters of age-associated endothelial dysfunction due to their pro-senescent, proinflammatory and pro-atherothrombotic properties. Short-term ingestion of the omega-3 optimized formulation EPA:DHA 6:1 not only reduces such shedding but also protects the endothelium against their prosenescent potential, most probably by reducing oxidative stress, preserving eNOS and preventing the activation of the local angiotensin system. Moreover, spleen appears to be a convenient and rich source of leukocyte-MVs reflecting the immune competence of the individual and enabling the study of pharmacologigal monitoring. The data further suggest that omega-3 EPA:DHA 6:1 may help to prevent or delay the age-related endothelial dysfunction mediated by prosenecent leukocyte-derived MVs.

Figures legends

(

  DHE), a redox-sensitive fluorescent probe. Spleen tissues, embedded in histomolds containing Tissue-Tek optimum cutting temperature (OCT) compound (Sakura 4583, Leiden, Netherlands) and snap-frozen in liquid nitrogen, were cryosectionned (25 μm) and mounted on slides. For ECs, P1ECs were seeded in Labtek chambers (Millicell EZ slide, Ireland) and incubated with 10nM PhtdSer eq. SMVs in humidified incubator at 37 °C, with 5% CO2 for 6 hours. Slides (spleen tissue or ECs) were then incubated with DHE (2.5 μM) for 30min, at 37ºC in a light protected humidified chamber. Slides were washed three time with PBS and mounted under coverslip using fluorescence mounting medium (DAKO, USA), dried in dark and analyzed by confocal microscopy (Leica SP2 UV DM IRBE; Leica, Heidelberg, Germany) with a 20X magnification lens. Level of oxidative stress was quantified using the Image J software.

  7 and 2 folds, respectively), thereby confirming the pro-senescent potential of SMVs detected by SA-β-gal activity (figures 5b, and above). Intake of EPA:DHA 6:1, but not of corn oil or EPA:DHA 1:1, abolished the pro-senescent effect of SMVs by SA-β-gal activity (17.7 A.U. vs 10.8

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 : 1 (Figure 6 :Figure 7 :Figure 8 :Figure 9 : 1 (O 6 : 1 )

 1234516789161 Figure 1: Ageing enhances the release of pro-senescent MVs from

  SMV treatment, ECs were washed with cold PBS, and proteins were extracted

	Membranes were washed three times with TBS-T (TBS-Tween, Euromedex) and
	incubated with peroxidase-labelled secondary antibodies; anti-rabbit (7074s) and
	anti-mouse (7076s), (3:10000, Cell signaling technology, USA) for 60 min, at room
	temperature. Pre-stained markers (protein ladder, Euromedex) were used for
	molecular mass determination. Immunostaining was revealed by chemiluminescence
	by RIPA lysis buffer (20mMTris/HCl, 150mM NaCl, 1mM Na3VO4, 10mM sodium solution (ECL, Bio-Rad laboratories, USA). The chemiluminescence signal was
	pyrophosphate, 0.01mM okadaic acid, 20mM, a tablet of protease inhibitor (Roche, recorded with ImageQuant LAS4000 system (GE Healthcare Europe GmbH, Velizy-
	Basel, Switzerland), and 1% Triton X-100 (Euromedex, Souffelweyershem, France).
	Abcam), rabbit monoclonal anti-COX-1 (ab109023, 1:1000, Abcam), rabbit
	polyclonal anti-COX-2 (ab15191, 1:1000, Abcam), mouse monoclonal anti-tissue
	factor (4509, 1:1000, Sekisui Diagnostics, Germany), mouse monoclonal anti-eNOS
	(610297, 1:1000, BD Biosciences, France), mouse monoclonal anti-p21 (sc-817,
	1:1000; Santa Cruz Biotechnology, USA) and rabbit polyclonal anti-p53 (sc-6243,

Proteins (15 μg or 20 μg) were separated by 10 % or 12 % SDS-PAGE at 100 V for 2 hours and further electrophoretically transferred onto polyvinylidene difluoride (PVDF) membrane (GE Healthcare, VWR, Fontenay-sous-Bois, France) at 100 V for 2 hours. Membrane non-specific binding sites were blocked in Tris-buffered saline (TBS) solution containing 5% BSA (Bovine Serum Albumin) and 0.1% Tween-20 (Euromedex) for 1 h at room temperature. Proteins of interest were probed with specific primary antibodies in blocking solution, rabbit monoclonal anti-VCAM-1 (ab215380, 1:1000, Abcam, UK), mouse monoclonal anti-ICAM-1 (ab171123, 1:1000, Abcam), rabbit polyclonal anti-phosphorylated NF-kB (ab86299, 1:1000, 1:1000; Santa Cruz Biotechnology), rabbit polyclonal anti-p16 (250804, 1:500, Abbiotec, USA) and rabbit polyclonal anti-ACE (250450, 1:500, Abbiotec), rabbit polyclonal anti-AT1R (sc-1173, 1:500, Santa Cruz Biotechnology), at 4°C overnight.

Villacoublay, France) and analyzed using ImageQuant TL software (version 8.1, GE Healthcare). Quantitative normalization with respect to housekeeping protein (mouse monoclonal anti-GAPDH, ab125247, 1:1000, Abcam) was done for each protein of interest.

1 intake by middle-aged rats prevents the splenocyte shedding

	and prime targets of omega-3 PUFA. No statistical difference was observed with
	respect to splenocyte survival (figure 4c).
	3.4	). Treatment EPA:DHA 6:1 intake blunts the endothelial pro-senescent impact of
	with EPA:DHA 6:1, but not corn oil or EPA:DHA 1:1, significantly lowered the age-splenocyte-derived MVs
	related amount of ROS by 19.5% in spleen tissues (EPA:DHA 6:1 : 1575.9 vs. Young primary coronary endothelial cells (P1ECs) were incubated for 48 h with 10
	control : 1959.91 A.U., p<0.05, figure 3c). Pharmacological inhibition by nM PhtSer eq. of washed SMVs isolated from either one of the 4 different M-rat
	Indomethacin (IND), VAS-2870, and mitochondrial respiration complex inhibitors subsets (SMV CTL , SMV CO , SMV 1:1 , SMV 6:1 ). SMV CTL significantly increased the SA-β-
	(MIT) reduced the amount of ROS in spleen tissue, indicating multiple sources of gal activity by 55% in P1ECs (p<0.05, SMV CTL vs. P1ECs), reaching values similar to
	mitochondrial and cytoplasmic origins (figure 3d). those induced by H 2 O 2 , a known inducer of premature endothelial senescence
	(figure
	3.3	EPA:DHA 6:The
	shedding of neutrophils, T lymphocytes, natural killer cells, monocytes/macrophages
	and granulocytes, was approximately reduced by half, reaching a  60% drastic
	decrease for natural killers and T lymphocytes (figure 4b and table 1), thereby
	suggesting that EPA:DHA 6:1 strongly targets cells of the innate immune system. By
	comparison, the low CD31 + -SMVs shedding remained statistically unchanged,
	confirming that, in this model, immune cells are the main contributors to MV release

Intake of omega-3 EPA:DHA 6:1 and EPA:DHA 1:1, but not of corn oil, significantly reduced the shedding of procoagulant SMVs measured by prothrombinase assay in the splenocyte supernatants, by 24% and 14%, respectively, suggesting that omega-3 PUFA significantly limit plasma membrane remodeling in situ (figure

4a

).

Acknowledgments

Authors are indebted to Pivotal therapeutics Inc. for the gift of EPA:DHA 6:1 and EPA:DHA 1:1 solutions and Vascazen capsules. This work was partly support by the grant ANR ENDOPAROMP (ANR-17-CE17-0024 01) A.W. Qureshi was supported by a doctorate fellowship from the Higher Education Commission (HEC) Of Pakistan, R. Altamimy was supported by a PhD fellowship from the Ministry of Higher Education and Scientific Research of Iraq.

Conflicts of interest:

Authors have no conflict of interest to declare.


Bernard Boitrel
email: bernard.boitrel@univ-rennes1.fr

Morgane Bouget

Pradip K Das

Stéphane Le Gac

Thierry Roisnel

Manel Hanana

Hélène Arcostanzo

Renaud Cornut

Bruno Jousselme

Stéphane Campidelli
email: stephane.campidelli@cea.fr

Oxygen reduction reaction catalyzed by overhanging carboxylic acid strapped iron porphyrins adsorbed on carbon nanotubes

Keywords: Porphyrins, dioxygen reduction, Synthesis, X-ray diffraction, Catalysis

teaching and research institutions in France or abroad, or from public or private research centers.

INTRODUCTION

Oxygen Reduction Reaction (ORR) is the paramount reaction taking place at the cathode of a fuel cell. In aerobic life on Earth, Cytochrome c Oxidase, the oxygen electrode of Nature's fuel cell is a famous example of protein performing the reduction of oxygen through the four-electron four-proton mechanism [1]. Thus, a plethora of catalysts based on iron "porphyrinoid" derivatives, mimicking the structure of the active center of the protein have been developed and extensively investigated for ORR [START_REF] Lemon | Handbook of Porphyrin Science[END_REF][START_REF] Bezerra | [END_REF]4]. However, where in nature a bimetallic center is required particularly under rate-limiting electron flux [5], it has been shown that "iron-only" tris(2-aminoethyl)amine (tren)-capped porphyrins, that is without any copper in the cap, could be efficient catalysts of the 4-electron reduction of dioxygen as long as electron supply is not a limiting factor as at the surface of an electrode [6]. This surprising result was then shown true for strapped or quinolinoyl picket porphyrins [7,8,9]. Finally, by grafting a carboxylic acid group around the binding site of the macrocycle, the hangman catalyst family was also found to be effective monometallic catalysts for the ORR [10,11].

This effectiveness was rationalized by the possible role of the carboxylic acid group as a proton-relay. The same concept has been extended to the study of meso-tetra-arylporphyrins with four carboxylic acid groups either in ortho or para position of the meso aromatic cycles. In the case of the ortho substitution, the selectivity was high for the four-electron process with a production of hydrogen peroxide quasi inexistent [12].

One of us, in previous work [13], has reported that the ORR activity of several non functionalized Co and Fe phthalocyanines and porphyrins immobilized by π-stacking interactions on various types of carbon nanotubes such as SWNTs, DWNTs, oxidized and non-oxidized MWNTs, was improved by the presence of the carbon nanotubes.

However, as discussed by Rigsby et al. the nature and structure of the adsorbed catalyst are crucial parameters as well [14]. Thus, the formulation itself of a catalyst ink containing the same iron porphyrin, that is either deposited directly on glassy carbon (GC) or mixed with Vulcan and then deposited on GC electrode, has an effect at least as important as changing the structure of the molecular catalyst itself. In the same line, and more recently, we have reported on the synergistic effect on ORR of strapped porphyrins polymerized around carbon nanotubes [15]. It was shown that the presence of a potential proton relay in the hybrids materials in comparison with those lacking such a group did not generate any significant improvement. However, it is worth to mention that the spatial arrangement of the molecular catalyst, namely the iron porphyrin, properly functionalized with peripheral groups for the polymerization was expected to be somehow controlled by the covalent linkages between the porphyrin units. Thus, in the present work, we now report on the ORR activity of MWNTs functionalized only by physisorption with specific iron (III) strapped porphyrins in a pH range from 13 to 6. These porphyrins contain a bridge bearing one or two carboxylic functions between the phenyl groups in 5 and 15 meso positions (Scheme 1). The overhanging bridge should prevent the aggregation of the porphyrins compared to the previous studies while preserving one side available for the interaction with the nanotubes by π-stacking. The goal of this study is first to measure the ORR properties of strapped porphyrins bearing either one or two polar overhanging carboxylic acid(s) and second to evaluate the influence of the communication between the nanotube and the catalytic centers as well as the effects of the non-aggregation of the porphyrins adsorbed on the nanotube as depicted in Figure 1.

RESULTS AND DISCUSSION

Strapped-porphyrin derivatives 1Fe and 2Fe bearing one and two carboxylic acid functions respectively were prepared according to a synthetic pathway previously reported in the case of a single strap porphyrin bearing 3,5dihydroxyphenyl in the 10,20 meso positions [16]. Chiefly, the synthesis of 2, summarized in Scheme 1, began by condensation of 5-(4-methoxyaryl)dipyrrylmethane 3 with 2-nitrobenzaldehyde in acidic conditions to obtain porphyrin 4 whose nitro groups were reduced by tin chloride in acidic medium leading to a mixture of the two atropisomers of the resulting aminophenyl porphyrin. Then, the atropisomer αα 5 was separated by silica gel flash chromatography and acylated with 3-chloromethyl benzoyl chloride in the presence of triethylamine to obtain porphyrin 6. Condensation of diethyl malonate in basic conditions allowed the formation of the strap in the 5,15 meso positions of the resulting porphyrin 7 and finally treatment by BBr 3 of the latter supplied free base porphyrin 2. Actually, both porphyrins 1Fe and 2Fe were obtained by refluxing free-base porphyrin 2 in THF in the presence of iron(II) bromide and 2,6-lutidine in a glove box. During this process, the decarboxylation reaction leading partially to 1Fe was observed and the two complexes were obtained in roughly equal proportions as indicated by TLC analysis and were separated by silica gel chromatography out of the glovebox after oxidation and identified by MALDI-TOF mass spectrometry (see experimental part).

Among the two final iron porphyrins, we were able to obtain single crystals of 1Fe and to solve its X-ray structure (Figure 2 and Table 1). The latter indicates that the iron cation is square-pyramidal five-coordinate with the intramolecular carboxylate bound on it with a Fe-O1 bond lenght of 1.997 Å. This intramolecular binding implies a significant distortion of the strap wich retains a "W-shape" as observed on the apical view (Figure 2, right). Although a similar coordination polyhedron has already been reported for the bis-carboxylato analogous complex of 1Fe [15], in the present case, the strap is more distorted with the carbon atom C2 diving toward the macrocycle plane (3.935 Å from iron against 4.301 Å for the non-decarboxylated complex) and with a distance of the iron atom to the mean porphyrin plane of 0.449 Å, against 0.512 Å for the non-decarboxylated complex. The very same distorted conformation was also found in an analogous zinc complex in which the distance of the metal to the mean porphyrin plane was only 0.386 Å [17], clearly indicating that this type of strap remains quite flexible. The macrocycle is strongly saddle-shaped with almost no ruffling, as indicated by the angle with the mean plane of the two pairs of diametrically opposed pyrrole rings (N1 and N3: -13.02° and -12.68°; N2 and N4: 11.14° and 14.06).

-.Å -3 a R int = ∑ |F o 2 -< F o 2 >| / ∑[F o 2 ] b S = {∑ [w(F o 2 -F c 2 ) 2 ] / (n -p)} 1/2 c R1 = ∑ | |F o | -|F c | | / ∑ |F o | d wR2 = {∑ [w(F o 2 -F c 2 ) 2 ] / ∑ [w(F o 2 ) 2 ]} 1/2 w = 1 / [σ(F o 2 ) + aP 2 + bP] where P = [2F c 2 + MAX(F o 2 , 0)] /3

A c c e p t e d M a n u s c r i p t

In order to prepare the hybrid materials, MWNT (Nanocyl NC3100 TM ) were purified by treatment with nitric acid (35%) at 100°C for 5h (see experimental section). After treatment the nanotubes were diluted with iced-water, filtered through 0.45µm PTFE membrane and extensively washed with water and then with methanol. The catalysts were prepared by mixing MWNT with porphyrins 1Fe or 2Fe (in a 1:1 ratio in weight) in THF under bath sonication. After mixing, THF was evaporated and the catalyst inks were prepared by dispersing, using bath sonication, the different MWNT/porphyrin hybrids (3 mg) in 750 µL of ethanol and 75µL of Nafion solution (5% in alcohol). Similarly, the inks of the reference compounds MWNT, 1Fe and 2Fe were prepared by mixing the nanotubes or the porphyrins in 750 µL of ethanol and 75µL of Nafion solution (5% in alcohol). The catalysts were drop-casted on the Glassy Carbon disk and tested in a series of Rotating Ring Disk Electrode experiments at pH 13 (NaOH, 0.1M), pH 10, 8 and 6 (phosphate buffers).

We first compared the electrocatalytic properties of the different components: MWNT, iron porphyrins 1Fe and 2Fe

and the 1:1 mixture MWNT-1Fe and MWNT-2Fe at pH 13 (Figure 3) and pH 10 (Figure 4). All the curves correspond to the average (reduction and reoxidation) of the cyclic voltammetry curves. porphyrins deposited on different carbon supports [14]. In addition, it is interesting to notice that the voltammetry curves of MWNT-1Fe and MWNT-2Fe exhibit two waves at around -0.4 and -0.7 V, explained by the own activity of the nanotubes (see below). Figure 3c shows the activity of MWNT and Figure 3d compares the voltammetry curves (0 and 2000 rpm) of MWNT, 1Fe and MWNT-1Fe. First, we observe that, at pH 13, MWNT reduced oxygen with lower overpotential and higher current density than 1Fe. Second, the reduction starts roughly at the same potential (-0.2V vs Ag/AgCl) for MWNT and MWNT-1Fe. Multi-walled carbon nanotubes are active materials for oxygen reduction in alkaline media [18,19] and it is likely that the nanotubes are more competitive than the nanotube/porphyrin hybrids at pH 13. It can explain the two waves observed on the reduction curves of the nanotube/porphyrin hybrids. We believe that the reduction initiated on the nanotubes is then performed by the porphyrin at lower potential. Nevertheless, when the plateau is reached at -0.8V vs Ag/AgCl the current density is larger of ca. 2 mA/cm2 for MWNT-1Fe compared to MWNT alone. (and of the reference compounds) exhibit the same trend with respect to those observed at pH 13 and 10. We also performed tests at pH 6 and pH 8 with porphyrin that does not exhibit proton relay and similar results to those obtained for MWNT-1Fe were obtained [15]. The presence of the proton in close proximity to the iron center does not seem to be mandatory to improve the reaction in our case. Indeed the presence of Nafion in the mixture probably ensures the availability of proton close to the reaction center. It is worth mentioning that at pH 6 and lower, the ORR activity of the porphyrins decreases rapidly and almost completely vanishes after the 3 cycles requiring the deposition of a new catalyst ink for each rotation cycle. This loss of ORR activity can be due to demetalation of the porphyrin during the electrochemical processes [20,21], this effect being well documented in the case of iron phthalocyanine [22,23,24] but still subject to debate in the case of porphyrins.

Figure 5 shows the RRDE curves registered at a rotation rate of 400 rpm for MWNT, MWNT-1Fe and 1Fe at pH 13 and 10. The numbers of electrons n involved in the reduction, summarized in Table 2 were determined following the equation n = 4I d /( I d + I r /N c ) as recommended by Qiao et al. [25], from the disk and ring currents and with a collection coefficient N c = 0.2 determined using the one-electron Fe(CN) 6 3-/Fe(CN) 6 4-redox couple. From the curves, it is observed that the reduction of O 2 is accompanied by the production of hydrogen peroxide both for MWNT and 1Fe.

Conversely, for MWNT-1Fe, almost no production of H 2 O 2 is detected at the plateau. At pH 13 a bump in the ring current between -0.20 and -0.70V reflecting the production of H 2 O 2 is observed; however, this phenomenon is attributed to the initial reduction of oxygen by the nanotubes and appears before the plateau at disk is reached. However, at both pH, the evaluated value of n remains close to the ideal value of 4. A c c e p t e d M a n u s c r i p t

CONCLUSION

Herein we formulated a series of catalyst inks for oxygen reduction reaction containing strapped porphyrins and MWNTs. The combination of the nanotubes with the iron porphyrins systematically gives better catalytic properties than those of two components taken separately. While carbon nanotubes are known to be slightly active in oxygen reduction, they produce significant amounts of hydrogen peroxide. When porphyrins are simply mixed in Nafion and deposited on the glassy carbon electrode, a low efficiency is generally observed. We attribute this behavior to the lack of electrons available for the reduction. Indeed in the catalyst ink, the porphyrins are embedded in Nafion and only the porphyrins close to the glassy carbon disk can benefit from efficient electron transfers from the electrodes. Conversely, carbon nanotubes create a percolating pathway for the charges in the catalyst film that ensure electron availabilities for the reduction of oxygen. In this case almost no hydrogen peroxide is produced and the reduction of oxygen is mostly performed via a 4 electrons pathway. However, under acidic conditions, at pH 6 and lower, the catalytic activity of these inks decreased rapidly.

EXPERIMENTAL Materials and methods

Mass

Steric decompression of two atropisomers.

In a 1 L two necks round bottom flask equipped with a stir bar and a condenser, 200 g of silica (60 µm) were added to toluene (400 mL). The reaction mixture was heated to 80°C and degassed with argon during 45 min. Then 2.9 g of the αβ and αα atropisomers of the bis-2-aminophenylporphyrin obtained previously were dissolved in toluene and added to the silica gel mixture in toluene. After evaporation of the solvent, the compound was dissolved in minimum amount of CH 2 Cl 2 and purified by column chromatography. The two atropisomers were separated on a silica gel chromatography eluted with CH 2 Cl 2 /MeOH (αβ 0.2%, αα 0.5%). Yield: 5 αα (1.9 g, 66%), αβ (0.9 g, 34%). It is worth to note that the atropisomer αα 5 remains contaminated with by-products resulting from scrambling reactions not separable by silica gel flash chromatography and has not been fully characterized.

α-5,15-bis-(2-[{3-chloromethyl}benzoylamido]-phenyl)-10,20-bis-(4-methoxyphenyl)-porphyrin 6.

A 500 mL two neck round bottom flask equipped with a stirrer and cooled in an ice bath was charged with compound 5 (0.99 mmol, 700 mg), dry CH 2 Cl 2 (300 mL) and NEt 3 (350 µL, 2.5 equiv.). 3-(chloromethyl)benzoyl chloride (420 µL, 3 equiv.) was then added dropwise under argon atmosphere. The reaction mixture was allowed to stir for three hours. Then the reaction was quenched by water and the organic layer was separated. The solvent was removed under vacuum. The resulting solid was dissolved in CH 2 Cl 2 and directly loaded on a silica gel chromatography column. The expected compound eluted with 0.2% CH 2 Cl 2 /MeOH, was obtained in 90% yield (902 mg). 1 H NMR (CDCl was dissolved in DCM (100 mL). After 12 h of stirring at RT, the reaction was completed. The mixture was quenched by water. The precipitated compound was filtered and the green solid was washed with water at pH =7. The product was purified by silica gel chromatography column and eluted with CHCl 3 /MeOH/AcOH (90/9/1). Yield: 80% (440 mg). MWNT. MWNTs (60 mg) were sonicated in nitric acid (35 vol %) (150 mL) with a sonic bath (Fisherbrand, 37kHz, power 100% for 10 min and then 40% for 30 min) and then heated at 100°C for 5 h. The suspension was then cooled and vacuum filtered through a 0.2 µm PTFE membrane and washed with water. The nanotubes were redispersed in NaOH 2 M (100 mL) using the sonic bath (100% for 10 min) and then filtered through a PTFE membrane and washed with deionized water, and then HCl 1 M followed by deionized water until the filtrate was neutral.

Electrochemical experiments. Sample preparation. For the preparation of the sample containing nanotubes (MWNT-1Fe, MWNT-2Fe), a mixture of purified MWNT (9 mg) and porphyrin 1Fe (9 mg) or porphyrin 2Fe (9 mg) in dry THF (10 ml) was homogenized using a sonic bath (Fisherbrand, 37kHz, power 100%) for 15 min. The THF was gently evaporated with a steam of N 2 and the mixtures were dried under vacuum. 3mg of mixture were dispersed in 750 µl of ethanol and 75 µl of Nafion solution (5% in alcohol). The mixtures were homogenized using a sonic bath until they formed homogenous inks. For the MWNT ink, the same procedure was followed but without adding porphyrins. °, V = 3124.0(8) Å 3 . Z = 2, d = 1.086 g.cm -3 , µ = 0.291 mm -1 . The structure was solved by dual-space algorithm using the SHELXT program [26], and then refined with full-matrix least-squares methods based on F 2 (SHELXL) [27]. The contribution of the disordered solvents to the calculated structure factors was estimated following the BYPASS algorithm [28], implemented as the SQUEEZE option in PLATON [29]. A new data set, free of solvent contribution, was then used in the final refinement. All non-hydrogen atoms were refined with anisotropic atomic displacement parameters. H atoms were finally included in their calculated positions and treated as riding on their parent atom with constrained thermal

Figure 1 .

 1 Figure 1. Schematic representation of the nanotube/porphyrins hybrids.

Scheme 1 .

 1 Scheme 1. Synthesis of iron porphyrins 1Fe and 2Fe bearing one and two overhanging carboxylic acid groups respectively, for MWNT coating. (i) 2-nitrobenzaldehyde, CH 2 Cl 2 , BF 3 -Et 2 O, 2 h, then DDQ, 20%; (ii) SnCl 2 , HCl, 80%; (iii) silica gel

Figure 3a -

 3a Figure 3a-b present the comparison between the ORR activity of MWNT-1Fe, MWNT-2Fe and those of the iron porphyrins deposited directly on the Glassy Carbon (GC) electrode. The curves show that the catalyst inks made by mixing the nanotubes with the porphyrins exhibit higher current density and lower overpotential (of about 0.1 V) than porphyrins alone. This result is not surprising since similar observations were made by Rigsby et al. in the case of iron

Figure 4

 4 Figure 4 shows the same comparison between the ORR activity of MWNT, MWNT-1Fe, MWNT-2Fe and those of 1Fe and 2Fe deposited on the GC electrode, but at pH = 10. Once again, the curves clearly demonstrate that the catalyst inks made by mixing the nanotubes with the porphyrins exhibit higher current density than porphyrins alone. The reduction of oxygen starts with an overpotential of almost 0.4 V for 1Fe and 2Fe compared to the same porphyrin deposited on the nanotubes. MWNT-1Fe and MWNT-2Fe exhibit very similar properties, it appears that the presence of one or two carboxylate functions does not influence the properties under alkaline conditions. At pH 10, MWNT become less competitive for oxygen reduction than MWNT-1Fe or MWNT-2Fe and only one reduction wave is observed. The comparison of the ORR activity (RDE and RRDE) of MWNT, 1Fe, 2Fe, MWNT-1Fe and MWNT-2Fe for pH 8 and pH 6 are given in Figure S1 and Figure S2, respectively. The catalytic activities of MWNT-1Fe and MWNT-2Fe

Figure 3 .

 3 Figure 3. Polarization curves at different rotation rates (0, 400, 800, 1200, 1600 and 2000 rpm) recorded for ORR in O 2 -saturated 0.1 M NaOH solution (pH 13) (scan rate = 5 mV/s, room temperature) on GC with predeposited (a) 1Fe (pink) and MWNT-1Fe (blue); (b) 2Fe (green) and (c) MWNT-2Fe (orange); MWNT (black). (d) Comparison between MWNT, MWNT-1Fe and 1Fe at 0 and 2000 rpm.

Figure 4 .

 4 Figure 4. Polarization curves at different rotation rates (0, 400, 800, 1200, 1600 and 2000 rpm) recorded for ORR in O 2 -saturated at pH 10 (phosphate buffer solution) (scan rate = 5 mV/s, room temperature) on GC with predeposited (a)1Fe (pink) and MWNT-1Fe (blue); (b) 2Fe (green) and MWNT-2Fe (orange); (c) MWNT (black). (d) Comparison between MWNT, MWNT-1Fe and 1Fe at 0 and 2000 rpm.

A c c e p t e d M a n u s c r i p t

Figure 5 .

 5 Figure 5. RRDE measurements of oxygen reduction (negative current) and hydrogen peroxide oxidation (positive current) for MWNT (black), 1Fe (pink) and MWNT-1Fe (blue) at (a) pH 13 and (b) pH 10 in O 2 -saturated solutions. The ring electrode was polarized at 0.260 V vs Ag/AgCl. Rotation rate: 400 rpm; scan rate: 5 mV s -1 .

α- 5 ,

 5 15-bis-(2-aminophenyl)-10,20-bis-(4-methoxyphenyl)-porphyrin 5. αβ and αα atropisomers of the dinitroporphyrin 4 (5.2 mmol, 4 g) were dissolved in the mixture of CH 2 Cl 2 -MeOH (100 mL), taken in a 2 L conical flask along with a reducing agent SnCl 2 .2H 2 O (11.8 g, 10 equiv.) and concentrated HCl (200 mL) was added slowly to the mixture. The resulting green solution was stirred for 2 days at RT. After completion of the reaction (monitored by MALDI), it was quenched by aqueous KOH solution at 0 °C under ice. The resulting violet solution was washed several times with water and CHCl 3 . The organic layers were collected and dried over MgSO 4 . Yield: 2.95 g (80 %).

2 .

 2 Boron tribromide (2.5 mL, 50 equiv.) was added to compound 7 (0.54 mmol, 600 mg)

  PHOTON 100 detector founded by FEDER, Mo-Kα radiation (λ = 0.71073 Å, multilayer monochromator), T = 150(2) K; triclinic P -1 (I.T.#2), a = 14.499(2), b = 14.729(2), c = 17.848(3) Å, α = 90.838(5), β = 109.567(6), γ = 117.362(5)

A c c e p t e d M a n u s c r i p t

, BF 3 -Et 2 O, 2 h, then DDQ, 20%; (ii) SnCl 2 , HCl, 80%; (iii) silica gel chromatography, CH 2 Cl 2 , 66%; (iv) 3-(chloromethyl)benzoyl chloride (3 eq), NEt 3 , THF, 85%; (v) CH 2 (CO 2 Et) 2 (10 equiv.), THF, EtONa, room temp., 12 h, 80%; (vi) BBr 3 , CH 2 Cl 2 , room temp., 12 h, 80%; (vii) FeBr 2 , 2,6-lutidine, THF, reflux overnight, silica gel column chromatography after air oxidation and HCl (1M) washing, 1Fe (41%), 2Fe (37%).

	Figure 2. X-ray structure of 1Fe. Top: ORTEP lateral view (30% thermal ellipsoids) and bottom: apical rods view with the carbon
	atoms of the strap colored in black. Selected distances (Å): N1-Fe 2.066, N2-Fe 2.049, N3-Fe 2.061, N4-Fe 2.056, O1-Fe 1.997, C1-
	O1 1.232, C1-O2 1.230, (Fe, 24MP) 0.449
	Empirical formula	C 62 H 41 Fe N 6 O 6
	Formula weight	1021.86 g/mol
	Temperature	150(2) K
	Wavelength Crystal system, space group Unit cell dimensions Volume Z, Calculated density Absorption coefficient F(000) Crystal size Crystal color Theta range for data collection 3.026 to 27.484 ° 0.71073 Å triclinic, P -1 a = 14.499(2) Å, α = 90.838(5) ° b = 14.729(2) Å, β = 109.567(6) ° c = 17.848(3) Å, γ = 117.362(5) 3124.0(8) Å 3 2, 1.086 g.cm -3 0.291 mm -1 1058 0.320 x 0.260 x 0.110 mm black h_min, h_max -18, 18 a n u s c r i p t k_min, k_max -19, 19 l_min, l_max -23, 23 M Reflections collected / unique 57073 / 13966 [R(int) a = 0.1159]
	Reflections [I>2σ]	9825
	Completeness to theta_max Absorption correction type Max. and min. transmission Refinement method Data / restraints / parameters b S (Goodness-of-fit) Final R indices [I>2σ] A c c e p t e d 0.975 multi-scan 0.968, 0.776 Full-matrix least-squares on F 2 13966 / 0 / 672 1.036 R1 c = 0.0888, wR2 d = 0.2340 R indices (all data) R1

c = 0.1213, wR2 d = 0.2579 Largest diff. peak and hole 1.852 and -1.601 e

Table 1

 1

. X-ray structural data of 1Fe.

Table 2 .

 2 Number of electrons involved in the reduction of O 2 at -0.75V a vs Ag/AgCl

		1Fe	MWNT	MWNT-1Fe
	pH 10	3.22	3.54	3.98
	pH 13	2.81	2.82	3.97

a potential chosen on the plateau for MWNT-1Fe.

  BrukerAvance500 or BrukerAvance400 spectrometers equipped with a BBFO probe. Spectra were referenced with residual solvent protons. UV/ Vis spectra were recorded on an Uvikon XL spectrometer. Chemicals were purchased from Aldrich and were used as received. Solvents were purchased form Aldrich or VWR and were used as received.resulting solid was dissolved in CH 2 Cl 2 and directly loaded on a silica gel chromatography column. The desired compound eluted with 70% CH 2 Cl 2 -cyclohexane was obtained in 50% yield (11.65 g, 46.17 mmol).1 H NMR (CDCl 3, 298 K, 500.13 MHz): δ 7.90 (2H, pyr NH ), 7.18 (2H, d, J = 8.61 Hz, aro 2 ), 6.91 (2H, d, J = 8.61 Hz, aro 3 ), 6.71 (2H, m, pyr 4 ), 6.22 (2H, m, pyr 3 ), 5.97 (2H, m, pyr 2 ), 5.44 (1H, CH α ), 3.84 (3H, s, OMe). 13 C NMR (CDCl 3, 298 K, 500.13 MHz): δ 158.5, 134.3, 132.9, 129.4, 117.2, 114, 108.3, 107.2, 55.3, 43.2. ESI-HRMS: calcd m/z = 275.1154 [M-H+Na] + for C 16 H 16 N 2 NaO, found 275.1159. L round-bottomed flask containing molecular sieves, degassed with a stream of Ar for 15 min. Then BF 3 -Et 2 O(110 µL, 0.1 equiv.) was added slowly over 30 s. The reaction was stirred at room temperature and monitored by TLC and MALDI. After 2 hours, DDQ (2.7 g, 1.5 equiv.) was added, and the reaction mixture was stirred at room temperature for a further 1 h. The complete reaction mixture was quenched by Et 3 N and evaporated under reduced pressure to give a black solid which was dissolved in CH 2 Cl 2 . The mixture of two atropisomers i.e. αβ and αα-bis-2nitrophenylporphyrin were purified by silica gel column chromatography using CH 2 Cl 2 as eluent. The atropisomers could not be separated by the usual method of column chromatography on silica gel due to the same polarity. Overall

	5,15-bis-(2-nitrophenyl)-10,20-bis-(4-methoxyphenyl)-porphyrin 4. Samples of 4-methoxyphenyldipyrromethane 3
	M (7.9 mmol, 2 g) and 2-nitrobenzaldhyde (1.19 g, 1 equiv.) were dissolved in freshly prepared distilled CH 2 Cl 2 (600 mL) a n u s c r i p t in a 1
	THF (K/benzophenone, N 2 ) was distilled before use. MWNT commercial grade NC3100 (>95%) were purchased from A c c e p t e d
	Nanocyl.

spectra: ESI: Micromass MS/ MS ZABSpec TOFF spectrometer MALDI-TOF: Microflex-LT Bruker Daltonics were performed at the C.R.M.P.O. (University of Rennes 1). 1 H-and 13 C-NMR spectra were recorded either on Synthesis. Porphyrin ligands. (4-methoxyphenyl)-dipyrromethane 3. In a two neck round bottom flask equipped with a stir bar and a gas inlet, 4-methoxy benzaldehyde (92.8 mmol, 11.3 mL) and pyrrole (161.2 mL, 25 equiv.) were mixed. The reaction mixture was degassed for 15 minutes in argon under dark at room temperature, and then TFA (707 µL, 0.1 equiv.) was added. The solution was stirred for further half an hour. The reaction was monitored by TLC, after that the reaction mixture was quenched by Et 3 N. The excess pyrrole was recovered under reduced pressure. The yield: 600 mg (20 %).

  NHCO), 7.59 (2H, t, J = 7.49 Hz, aro 4' ), 7.28 (4H, bs, aro 8 , aro 8' ),6.73 (2H, d, J = 7.68 Hz, aro 4 ),6.55 (2H, d, J = 7.90 Hz, aro 6 ), 6.49 (2H, t, J = 7.68, aro 5 ), 6.33 (2H, s, aro 2 ), 4.09 (6H, s, OMe), 3.30 (4H, s, CH 2 bz ), -2.61 (2H, s, NH int ).13 C NMR (CDCl 3, 298 K, 500.13 MHz): δ 164.7, 159.7, 138.7, 137.1, 135.7, 135.6, 134.9, 134.7, 133.6, 131.7, 130.8, 129.9, 128.4, 126.2, 126.1, 123.2, 120.9, 120.7, 113.7, 112.5, 112.4, 55.6, 44.3. ESI-HRMS: calcd m/z = 1009.3030 [M+H] + for C 62 H 47 N 6 O 4 35 Cl 2 , found 1009.3031, calcd m/z = 973.3263 [M-HCl+H] + for C 62 H 46 N 6 O 4 Sodium metal (182 mg, 10 equiv.) was added to the absolute alcohol (20 mL) in a small round bottom flask and stirred for few minutes until the complete consumption of Na. Diethyl malonate (1.2 mL, 10 equiv.) was added to this solution at room temperature and stirred for half an hour. The resulting mixture was added to a solution of porphyrin 6 (0.79 mol, 800 mg, 1 equiv.) in CH 2 Cl 2 (600 mL) and the solution was turned immediately from violet to green. After 2h of stirring the reaction was quenched by H 2 O, the organic layer was separated and removed under vacuum. The desired product was purified on a silica gel chromatography column eluted with 0.3% CH 2 Cl 2 /MeOH. The expected compound was obtained in 80% yield (702 mg). 1 H NMR (CDCl 3 , 298 K, 500 MHz): δ

	35 Cl, found
	973.3268. UV-vis (DMF): λ/nm (10 -3 ε, dm 3 mol -1 cm -1 ): 426 (364), 521 (14), 558 (8), 599 (4), 652 (3.4).
	α-5,15-bis-({2,2-(3,3-[2,2-(diethoxycarbonyl)propane-1,3-diyl]-dibenzoyl-amido]-diphenyl)-10,20-bis-(4-
	methoxyphenyl)-porphyrin 7. 9.15 (2H, d, J = 8.39 Hz, aro 2' ), 8.93 (4H, d, J = 4.58 Hz, βpyr), 8.86 (4H, d, J = 4.58 Hz, βpyr), 8.22 (2H, s, NHCO), 8.19 (2H, bs, aro 9 ), 8.01 (2H, d, 2H l , J = 7.54 Hz, aro 5' ), 7.98 (2H, bs, aro 9' ), 7.91 (2H, t, 3 J = 7.70 Hz, aro 3' ), 7.70 (2H, d, 2H h , J = 7.98 Hz, aro 6 ), 7.55 (2H, t, 3 J = 7.70 Hz, aro 4' ), 7.31 (4H, bs, aro 8 , aro 8' ), 6.98 (2H, t, 3 J = 7.90 Hz, aro 5 ), 6.74 (2H, d, J = 7.90 Hz, aro 4 ), 4.99 (2H, s, aro 2 ), 4.08 (6H, s, OCH 3 ), 1.70 (4H, s, CH 2 bz ), 1.23 (4H, bs, CH 2 ester ), -0.55 (6H, t, J = 6.58 Hz, CH 3 ester ), -2.40 (2H, s, NH int ). 13 C NMR (CDCl 3 , 298 K, 500 MHz): δ 167.5, 164.4, 159.6, 138.8, 135.9, 135.2, 134.9, 133.8, 133.7, 132.4, 131.2, 129.9, 128.2, 127.4, 125.9, 122.8, 120.7, 119.7, 113.7, 112.5, a n u s c r i p t 59.9, 58.8, 55.5, 42.5, 11.9. ESI-HRMS: calcd m/z = 1097.4232 [M+H] + for C 69 H 57 N 6 O 8 , found 1097.4234, calcd m/z = 1119.4051 [M+Na] + for C 69 H 56 N 6 O 8 Na, found 1119.4047. UV-vis (DMF): λ/nm (10 -3 ε, dm 3 mol -1 cm -1 ): 425 (380), 520 M (15.8), 558 (8.2), 596 (4.
	A c c e p t e d

3, 298 K, 500.13 MHz): δ 8.93 (4H, d, J = 4.81 Hz, βpyr), 8.87 (4H, d, βpyr), 8.93 (2H, d, J = 8.06 Hz, aro 2' ), 8.13 (2H, d, J =7.49 Hz, aro 5' ), 8.11 (2H, d, J = 7.60 Hz, aro 9 ), 8.02 (2H, d, J = 7.60 Hz, aro 9' ), 7.91 (2H, t, 3 J = 8.06 Hz, aro 3' ), 7.63 (2H, s,

Acknowledgements

Région Bretagne as well as well as CNRS are deeply acknowledged for their significant support to our research team.

This work was also supported by the JST-ANR program TMOL "Molecular Technology" project MECANO (ANR-14-JTIC-0002-01) and project MAGMA (ANR-16-CE29-0027-01).

A c c e p t e d M a n u s c r i p t

(R(F) = 0.0888) for 9825 observed reflections with I > 2σ(I).

Supporting Information composed of the CIF file, detailed spectroscopic data and detailed electrochemical measurements.

Crystallographic data (excluding structure factors) for the structures reported in this paper have been deposited at the Cambridge Crystallographic Data Center under deposition number 1916858. Copies can be obtained on request, free-ofcharge, at www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 1223-336-033 or email: deposit@ccdc.cam.ac.uk).

Oxygen Reduction Reaction Catalyzed by Overhanging Carboxylic Acid Strapped Iron Porphyrins Adsorbed on Carbon

Nanotubes