Modelling farm vulnerability to flooding Towards the appraisal of adaptation policies Case study

Our study aims at developing a methodology to appraise this kind of policy using Cost-Benefit Analysis.

Measures to mitigate vulnerability enhance farm capacity to adapt and recover after flooding. The expected benefits of their implementation rest upon the improvement of farm management during and after flooding. Current appraisal methods are not sufficient: they rely on direct damage estimation and do not consider farm level.

We developed a conceptual model using UML (Unified Modelling Language) at the farm scale which takes into account:

1. spatial distribution of farm buildings and land plots, 2. time scale (mid and long term effects), 3. work management to achieve production and recovery tasks.

Contributions of modelling vulnerability at the farm scale 2

Perspectives of application to the downstream area of the Rhône river 3

The Rhône river program, a contract of agreed objectives between French government and local authorities, plans to implement measures to mitigate farm vulnerability in flood prone areas of the Rhône river downstream.

Expected

advantages of farm vulnerability mitigation are: 1.

to adapt farms to maintain agricultural activities in floodplains, 2. to compensate "over-flooding" impacts in case of risk transfer at basin level. Effects of a list of measures to mitigate farm vulnerability will be evaluated using a "farm scale" vulnerability model and will be compared with the costs of implementation (Cost-Benefit Analysis) to help decision makers to prioritize funding. The modelling step highlights a need of data and help us to establish a methodology to collect it.

Organisational dimension Our model takes into account work management after flooding (competition between production and recovery tasks).

Few examples of implementation of vulnerability mitigation measures exist. To help decision making, local authorities asked for an economic method to evaluate the program of farm vulnerability mitigation.

Effects taken into account with current methods and with a farm scale approach

Current methods of damage appraisal appraise direct damage (building and crop). Focusing on the farm scale enables to take into account induced effects on farm management after flooding.

Spatial dimension

Current models (left) only take into account land use, our model (right) also links plots / buildings to farms.


Claire Cuyamendous

Aurélien De

Yiu La Torre

Yiu Lee

Kin Sum Leung

Alexandre Guy

Valérie Bultel-Poncé

Jetty Jean-Marie Galano

Jetty Chung-Yung Lee

Camille Oger

Thierry Durand
email: thierry.durand@umontpellier.fr

Elien De La Torre

Val Erie Bultel-Ponc

The novelty of phytofurans, isofurans, dihomo-isofurans and neurofurans: Discovery, synthesis and potential application

Keywords:

come

L'archive ouverte

Introduction

Reactive oxygen species (ROS) are free radicals generated under physiological conditions, but also under the so-called oxidative stress condition (OS) when natural antioxidant defences are overwhelmed by the amount of ROS generated. ROS target oxidation prone polyunsaturated fatty acids (PUFA) to undergo lipid peroxidation (LPO) [START_REF] Gu Eraud | Chemistry and biochemistry of lipid peroxidation products[END_REF]. The hydroxyl radical ( OH) is believed to be the most reactive radical in vivo, however other radicals can also participate in the processes of LPO [START_REF] Yin | Free radical lipid peroxidation: mechanisms and analysis[END_REF]. However, no contentious exists if ROS can be specific of certain lipid and therefore of certain type of oxygenated metabolites. Furthermore, it is also believed that the non-enzymatic free radical mechanism of LPO that occurs in three phases (initiation, propagation, termination) could be more subtle than anticipated [START_REF] Schneider | Routes to 4-Hydroxynonenal: fundamental issues in the mechanisms of lipid peroxidation[END_REF][START_REF] Morita | The real radical generator other than main-product hydroperoxide in lipid autoxidation[END_REF]. OS and LPO have been clearly associated to several pathologies and diseases, and one particular biomarker of OS was discovered in the nineties and called F 2 -isoprostanes (F 2 -IsoPs). They are generated from autooxidation of arachidonic acid bounded phospholipid (AA, C20:4 n-6) and are circulating in biological fluids as free form mainly. Today they are currently considered as the best marker of OS in biological systems. In the biosynthesis route of IsoPs, an endoperoxide carbon radical (B) undergoes a 5-exo-trig cyclization to form the cyclopentane ring of IsoPs [START_REF] Morrow | A series of prostaglandin F2-like compounds are produced in vivo in humans by a noncyclooxygenase, free radical-catalyzed mechanism[END_REF][START_REF] Jahn | Beyond ProstaglandinsdChemistry and biology of cyclic oxygenated metabolites formed by free-radical pathways from polyunsaturated fatty acids[END_REF] (Scheme 1). However, at high oxygen tension further transformations can occur and oxygen molecule may react with the endoperoxyde carbon radical to generated tetrahydrofuran ring containing compounds. Those new metabolites were termed isofurans (IsoFs). This second route may prevent the IsoPs pathway and limit their formation [START_REF] Fessel | Discovery of lipid peroxidation products formed in vivo with a substituted tetrahydrofuran ring (isofurans) that are favored by increased oxygen tension[END_REF]. It is thus reasonable to measure IsoFs in addition to IsoPs in the case of high oxygen tension injury e.g. hyperoxia, ischemia-reperfusion etc. Depending on the parent PUFA that at least would possess two skipped diene units (i.e; three alkenes separated by a CH 2 ), other furanoid metabolites can be formed and were discovered in human tissues or fluids e.g. neurofurans (NeuroFs) from docosahexaenoic acid (DHA, 22:6 n-3), dihomo-isofurans (dihomo-IsoFs) from adrenic acid (AdA) and in plants like the phytofurans (PhytoFs) from a-linolenic acid (ALA, C18:3 n-3). Those metabolites should be added to the list of metabolites to quantitate OS. The present review aims to provide a concise overview on these furanic PUFA metabolites, from how they are formed in vivo and how they are named. We also would like to highlight that these metabolites are not commercially available and need to be prepared by organic chemists before being evaluated. Accordingly, the syntheses developed until now will be presented. Finally, the last part will be focused on the identification of those metabolites and their implication in biological systems. More importantly, this review will also give keys to the researchers in the preparation of the samples and the measurements of such metabolites.

Biosynthesis

In 1990, Morrow and co-workers revealed the formation of IsoPs from arachidonic acid esterified as phospholipid, and explained their formation via a radical initiated mechanism [START_REF] Morrow | A series of prostaglandin F2-like compounds are produced in vivo in humans by a noncyclooxygenase, free radical-catalyzed mechanism[END_REF]. Hydrogen abstraction of the bis-allylic hydrogen atom of AA ester lead to isomeric pentadienyl radicals which are oxygenated into pentadienylperoxyl radicals (A) (Scheme 1). Peroxyl radicals A can also undergo kinetically controlled 5-exo cyclization with suitably positioned alkene units to generate the endoperoxide intermediate B (dioxolanylcarbinyl radical). Irreversible carbon-centered radical intramolecular attack onto a double bond generates the cyclopentane ring with the appropriate configuration (cis) characteristic of the IsoPs. However, another fate of dioxolanylcarbinyl radical B was revealed by Fessel and coworkers in 2002. It relies on the diradical character of O 2 and its high reactivity toward carbon-centered radicals, and permits to access IsoFs [START_REF] Fessel | Discovery of lipid peroxidation products formed in vivo with a substituted tetrahydrofuran ring (isofurans) that are favored by increased oxygen tension[END_REF]. Interestingly, early in vitro experiment revealed the importance of oxygen concentration on the preferential generation of IsoFs versus IsoPs, and while the IsoPs concentration reached a plateau at 21% oxygen, the concentration of IsoFs increased further as the oxygen tension increased. This observation also proved to be true for organs of known different oxygen tension like liver versus lung.

The mechanism of formation was closely investigated and so far three biosynthetic pathways are proposed for the formation of IsoFs. From those and by competitive pathways, two types of IsoFs are synthesized, namely the alkenyl-IsoFs and the enediol-IsoFs, each of them present as a mixture of five regioisomeris and diastereomers. As shown in Scheme 1, we represented two out of the three biosynthetic pathways which all agree with the in vitro isotopic labelling experiments [START_REF] Jahn | Beyond ProstaglandinsdChemistry and biology of cyclic oxygenated metabolites formed by free-radical pathways from polyunsaturated fatty acids[END_REF].

It is believed that these pathways diverge from dioxolanylcarbinyl radical B. In the first one, oxygen trapping can lead to hydroperoxy-1,2-dioxolane C (Scheme 1, bottom left), which undergoes after single electron reduction of the peroxide bridge, 3exo cyclization and oxygen trapping, the dihydroperoxy epoxide D. Further regioselective intramolecular nucleophilic ring opening of the epoxide by the hydroxyl group and final reduction of the hydroperoxide gives rise to the four regioisomeric alkenyl isofuran classes as mixture of diastereomers.

Another route (Scheme 1, bottom right) is also possible directly from dioxolanylcarbinyl radical B after a facile 1,3-S H i reaction, 3exo cyclization and oxygenation to lead diepoxyhydroperoxide E. After hydrolysis to form the regioisomeric epoxy diols F1 and F2, intramolecular nucleophilic ring opening of the epoxide by a suitably positioned hydroxy group leads to alkenyl-IsoFs according to path 1 and to enediol-IsoFs by path 2.

After the initially discovered isofuranoid derived AA, analogous compounds from other PUFAs were discovered like the NeuroFs in 2008 by FitzGerald [START_REF] Song | Neurofurans, novel indices of oxidant stress derived from docosahexaenoic acid[END_REF] from DHA, as depicted in Table 1. Later, the combined groups of Galano/Durand and Lee discovered the dihomo-IsoFs derived from AdA in pig brain [START_REF] De La Torre | Synthesis, discovery, and quantitation of dihomo-isofurans: biomarkers for in vivo adrenic acid peroxidation[END_REF]. Recently, this join collaborative effort also identified the PhytoFs from ALA identified in seed and nuts [START_REF] Cuyamendous | Synthesis and discovery of phytofurans: metabolites of a-linolenic acid peroxidation[END_REF]. It is obvious that other isofuranoids are probably generated from other PUFA and are still to be discovered.

Nomenclature

Biosynthetically related to IsoPs and containing a furan ring, those metabolites were naturally labeled as IsoFs. As depicted in the biosynthesis part, IsoFs are produced by three different pathways, and lead to the formation of two classes of IsoFs, called alkenyl and enediol. There are thus eight different isofuran regioisomers (four alkenyl and four enediol), which can exist as 16 diastereomers, for a total of 256 enantiomerically-pure metabolites. A systematic nomenclature allows the differentiation of each isomeric structures and established by Taber, Fessel and Roberts [START_REF] Taber | A nomenclature system for isofurans[END_REF]. This nomenclature is based on the relative orientation of the side chains and substituents. Furthermore, this nomenclature system was validated by IUPAC. Several rules must be followed for the generation of a name from a structure (Fig. 1). First, it is noted that: the carbon number one is the carbon atom of the acid function (COOH) and the first ring carbon atom as well as the carbon bearing the hydroxyl groups (OH) on the side chain have, by default, (S) configuration. Secondly, the relative orientation of the alkyl chains must be defined: syn (S) or anti (A), prior to the relative orientation of the ring hydroxyl group and the adjacent alkyl chain: cis (C) or trans (T). Then, the carbon number of the E double bond should be addressed as a superscript after the D sign, followed by the carbon number of the first ring carbon atom. The abbreviation IsoF, for isofuran, is appended thereafter and reveals the parent PUFA: IsoF (AA and EPA), PhytoF (ALA), dihomo-IsoF (AdA) and NeuroF (DHA).

Great care should be paid to the configuration of the first ring carbon atom and the carbon atoms bearing hydroxyl groups on the side chain ((S) by default). If the configuration of the hydroxyl is inverted, the carbon number followed by "epi" is used as a prefix. If the first ring carbon atom configuration is reversed, the prefix "ent" is used and all the hydroxyl groups on the side chains are considered (R) as default.

Total synthesis of furanoids in the literature

The total synthesis of furanoid PUFA metabolites is of great interest because little is known about their biochemistry. Thus, four synthetic strategies were developed by organic chemists. The first one was described by Taber et al. in 2004 through a diol epoxide benzenesulfonate cyclization and permits the synthesis of AA derived IsoFs both alkenyl and enediol type [12e14]. More recently, Zanoni's group used Trost asymmetric alkylation for the first synthesis of a neurofuran [START_REF] Valli | Stereodivergent strategy for neurofuran synthesis via palladium-catalyzed asymmetric allylic cyclization: total synthesis of 7-epi-ST-D8-10-Neurofuran[END_REF]. Finally, the Galano and Oger group developed in parallel two strategies toward furanoid derivatives based on Borhan cyclization or a one-pot Payne rearrangement/5exo-tet cylisation, giving access to enediol and alkenyl furanoids derived from ALA, AdA and DHA [START_REF] De La Torre | Synthesis, discovery, and quantitation of dihomo-isofurans: biomarkers for in vivo adrenic acid peroxidation[END_REF][START_REF] Cuyamendous | Synthesis and discovery of phytofurans: metabolites of a-linolenic acid peroxidation[END_REF][START_REF] De La Torre | Total syntheses and in vivo quantitation of novel neurofuran and dihomo-isofuran derived from docosahexaenoic acid and adrenic acid[END_REF]. So far, none of the isofuranoids synthesized are commercially available but can be available from the Zanoni and Galano/Oger/Durand teams (isofuranoids from Taber were kindly given to the Montpellier group).

Total syntheses of IsoFs developed by the group of Taber

The first strategy developed by Taber in 2004 gave access to the alkenyl furans and relied on an epoxide intermediate 1 where the lateral chains are introduced later on the synthesis (Scheme 2A) [START_REF] Taber | A flexible enantioselective synthesis of the isofurans[END_REF].

The epoxide moiety 1 comes from an epoxide opening cyclization where the benzenesulfonate unit is substituted (compound 2). Thanks to Sharpless epoxidation and dihydroxylation reactions, all stereogenic centers are controlled and may be changed by a simple modification of the chiral ligand. Moreover, even if the SC structure is obtained herein, a Mitsunobu reaction on epoxide 2 may allow the change of configuration of the hydroxyl group on the furan and thus the synthesis of ST structures. This strategy permitted the synthesis of the 8-epi-SC-D 13 -9-IsoF and its epimers on the 15th carbon atom, in 16 steps and 0.8% global yield. The same cyclization key step was used for the synthesis of enediol structures. However, the cyclization substrate had to be slightly modified to get the ent-SC-D 13 -8-IsoF and it epimer on the 15th carbon (Scheme 2B) in 21 steps and in 1.7% global yield [START_REF] Taber | Synthesis of the enediol isofurans, endogenous oxidation products of arachidonic acid[END_REF].

Total synthesis of NeuroF by the group of Zanoni

The discovery of the NeuroFs in 2008 led by Zanoni group to develop a new strategy for the synthesis of furanoids by making the first total synthesis of one NeuroF, named 7-epi-ST-D 8 -10neurofuran (Scheme 3) [START_REF] Valli | Stereodivergent strategy for neurofuran synthesis via palladium-catalyzed asymmetric allylic cyclization: total synthesis of 7-epi-ST-D8-10-Neurofuran[END_REF].

The key step of the strategy is a diastereo-and enantioselective cyclization using Tsuji-Trost alkylation, starting from diene-diol 6, to obtain the furan intermediate 5 in good yield (86%) and good enantiomeric excess (96%). This intermediate 5 allowed the insertion of the lateral chains by Julia-Kocienski coupling and Wittig reaction, to access to the 7-epi-ST-D 8 -10-neurofuran in 18 steps and 1.6% global yield.

Total syntheses of dihomo-IsoFs, NeuroF and PhytoFs by Galano and Oger

The recent advances in the field of non-enzymatic lipid metabolites prompted our group to develop synthetic strategies to access all PUFA furan metabolites, even those not yet discovered at that time, such as PhytoFs derived from ALA or dihomo-IsoFs derived from AdA. Thus, two strategies were developed in parallel to access those metabolites. In the first strategy the key step is an intramolecular cyclization of a hydroxyl onto an orthoester (Scheme 4) [START_REF] De La Torre | Synthesis, discovery, and quantitation of dihomo-isofurans: biomarkers for in vivo adrenic acid peroxidation[END_REF] whereas a Payne rearrangement followed by a basic mediated intramolecular 5-exo-tet cyclization is used in the second one (Scheme 5) [START_REF] Cuyamendous | Synthesis and discovery of phytofurans: metabolites of a-linolenic acid peroxidation[END_REF].

The strategy goes through intermediate 9, common to the alkenyl and enediol classes. The regioselective monoprotection of the hydroxyl group in a (compound 10) or b (compound 8) of the ester 9 allows either a 5-exo-tet cyclization (compound 7) or a 5endo-tet cyclization (compound 11). Then, the lateral chains are introduced by Wittig or Horner-Wadsworth-Emmons reactions, This strategy permitted the first syntheses of dihomo-IsoFs derived from AdA: the 10-epi-17(RS)-SC-D 15 -11-dihomo-IsoF and the 7(RS)-ST-D 8 -11-dihomo-IsoF, as well as the first enediol-NeuroF, the 4(RS)-ST-D 5 -8-NeuroF, in 21e23 steps and 1.5e2% global yield [START_REF] De La Torre | Synthesis, discovery, and quantitation of dihomo-isofurans: biomarkers for in vivo adrenic acid peroxidation[END_REF][START_REF] De La Torre | Total syntheses and in vivo quantitation of novel neurofuran and dihomo-isofuran derived from docosahexaenoic acid and adrenic acid[END_REF].

The second strategy allows the acquisition of both alkenyl and enediol furanoids through common intermediate 14. The bisepoxide 15 is easily obtained from diyne-diol 16 by a trans-reduction of the diyne followed by a Sharpless bis-epoxidation. Thus, subjected to basic conditions (KOH, 80 C), the bis-epoxide leads to a Payne rearrangement followed by an epoxide opening and a 5exo-tet cyclization (compound 14).

The orthogonal protections of the hydroxyl moieties and regioselective deprotection lead to two intermediates (compounds 12 and 13), needed for the lateral chains insertion by Wittig or Horner-Wadsworth-Emmons reactions.

This strategy permitted the first syntheses of PhytoFs derived from ALA: the ent-16(RS)-9-epi-ST-D 14 -10-PhytoF, the ent-16(RS)-13-epi-ST-D 14 -9-PhytoF and the ent-9(RS)-12-epi-ST-D 10 -13-PhytoF, in 20 steps and 1% global yield [START_REF] Cuyamendous | Synthesis and discovery of phytofurans: metabolites of a-linolenic acid peroxidation[END_REF].

Identification and implication of isofuranoids in biological systems

The synthesis of these novel compounds allows us to expand their measurement in biological samples. To date, the most robust measurement of the isofuranoids is by gas-chromatography-mass spectrometry (GC-MS) or liquid chromatography tandem mass spectrometry (LC-MS/MS). These instruments rely on the mass-tocharge ratio (m/z) and additional transition ion in the case for LC-MS/MS. The disadvantage of GC-MS is the requirement of a derivatizing process and inability to separate isomeric compounds with same molecular weight. Nevertheless, it should be noted the sensitivity of isofuranoids determination also depends on the preparation of the samples. In the literature, not all methods of isofuranoids and other oxidized lipid product measurement are in alignment between researchers; this depend on how lipid portion of the samples are extracted prior to purification by thin layer chromatography (TLC) or solid phase extraction (SPE) for example which are well reported [17e19].

Preparation for measurements

Mammalian samples

The detection and quantification of isofuranoids from tissues and/or cells mandatorily require the initial step of sample extraction. To avoid and minimize potential ex vivo generation of lipid metabolites via either enzymatic or non-enzymatic pathways, a small amount of BHT and indomethacin are commonly added into the sample before any homogenization procedures [START_REF] Leung | Current development in nonenzymatic lipid peroxidation products, isoprostanoids and isofuranoids, in novel biological samples[END_REF][START_REF] Lee | Prenatal exposure to the contaminant perfluorooctane sulfonate elevates lipid peroxidation during mouse fetal development but not in the pregnant dam[END_REF]. Accurate quantities of specimens are often weighed in a highly precise balanced weighing machine, usually based on wet weight. Alternatively, in the case of sampling from cell culture system, the estimated number of cells is determined using various cell counting methods before adding in cell lysis buffer and lipid extracting solvent. It is important to note that having a fully homogenized sample is one of the critical steps in quantifying isofuranoids accurately, as the received shearing force are often closely related to the amount of cellular components being released during the process.

Homogenization of samples is usually performed under physical disruption of tissues with the use of sharp blades or small silica beads and even ultrasound [START_REF] Araujo | Extraction of lipids from microalgae by ultrasound application: prospection of the optimal extraction method[END_REF]. While these homogenization methods are well recognized, blade homogenizer is widely adopted possibly due to its lower cost and maintenance. The heat generated during homogenization is one of the main concerns by general users. Although the rotor-stator homogenizer is efficient in producing high shearing force upon the sample, large amount of heat is also generated from the fast turning rotor and transferred to the homogenizing sample through the attaching blade. Exposing samples to high levels of heat during preparation might contribute to the formation of artefacts, or ex vivo generation of molecules, which ultimately could lead to false positive-negative results. To reduce the amount of heat retained in the sample, one generally uses ice-cold solvents during homogenization as well as cooling the outer surface of the sample tube with ice-cold water or ice itself. In addition, it is encouraged to wash or blend-pass through homogenizing blade with methanol and MilliQ water in between samples, even though they might be from the same treatment group. This additional step prevents cross contamination and maximizes the confidence of gaining highly accurate quantitation in the later stage.

Isofuranoids extraction from different types of tissues using this rotor-stator homogenizer however, is not always reproducible. The extraction of total lipids from tissues is generally based on two established methods, i.e. Folch and Bligh/Dyer. These two methods commonly utilized organic solvents (Folch solution: chloroform:methanol, 2:1; Bligh/Dyer: chloroform:methanol, 1:2) for their lipid extraction [START_REF] Folch | A simple method for the isolation and purification of total lipides from animal tissues[END_REF][START_REF] Bligh | A rapid method of total lipid extraction and purification, can[END_REF]. Mammalian tissues, e.g. kidney and liver, tend to be hardened when directly exposed to organic solvents, increasing the difficulty in successive homogenization. Other tissue types, like the brain, are comparatively softer even in the presence of organic solvents, but brain homogenates tend to stick to the homogenizing blade, inducing great loss if they were not recovered. Hence, multiple washes of the homogenizing blade with extracting solution are needed and should then be pooled together to maximize the tissue recovery. To overcome the structural changes of tissues in organic solvents, some co-workers [START_REF] Newberry | Decreased hepatic triglyceride accumulation and altered fatty acid uptake in mice with deletion of the liver fatty acid-binding protein gene[END_REF][START_REF] Guan | Non-targeted profiling of lipids during kainate-induced neuronal injury[END_REF] have successively extracted total lipids by homogenizing the tissues first in PBS, and followed by adding appropriate organic solvents. Alternatively, some other researchers have reported to extract lipids by first grinding freshly harvested tissues into powder form in the presence of liquid nitrogen, and subsequently weigh accurate portion for lipid extraction using organic solvents [START_REF] Kang | A simplified method for analysis of polyunsaturated fatty acids[END_REF][START_REF] Viant | An NMR metabolomic investigation of early metabolic disturbances following traumatic brain injury in a mammalian model[END_REF]. These two alternative methods provided a detour to total lipid extraction by organic solvents from tissues after they were fully disrupted.

Depending on the target species (e.g. isofuranoids) that are of interest in the study, extracted total lipids from specimens are further processed before quantitative analysis. For example, when determining the levels of isofuranoids, which are mostly esterified as phospholipids, those are usually liberated by adding high molar alkaline in organic solvent, e.g. 1 M KOH in methanol. These are then purified from polar lipid species via TLC or SPE columns. Finally, the extracts are subjected to qualitative and quantitative analysis commonly via GC-MS or LC-MS/MS. An additional step of derivatization is necessary when using GC-MS [START_REF] Lawson | Isoprostanes: formation, analysis and use As indices of lipid peroxidation in vivo[END_REF][START_REF] Morrow | Noncyclooxygenase oxidative formation of a series of novel prostaglandins: analytical ramifications for measurement of eicosanoids[END_REF]. Isofuranoids have not been investigated in urine samples, but like for IsoPs they are probably excreted as free forms and conjugated forms (glucoronidates). A validated method for IsoPs was described by Medina et al. [START_REF] Medina | A ultra-pressure liquid chromatography/ triple quadrupole tandem mass spectrometry method for the analysis of 13 eicosanoids in human urine and quantitative 24 hour values in healthy volunteers in a controlled constant diet[END_REF].

In plant samples

Phytofurans (PhytoFs) are newly found oxygenated metabolites produced by non-enzymatic peroxidation of a-linolenic acid (ALA) in plants. It Soxhlet extraction method extracts the total oil from nuts and seeds with chemical solvent such as petroleum ether [START_REF] Su Curovi C | Physicalchemical characteristics and oxidative stability of oil obtained from lyophilized raspberry seed[END_REF] or hexane [START_REF] Caprioli | Lipid nutritional value of legumes: evaluation of different extraction methods and determination of fatty acid composition[END_REF][START_REF] Jiao | Microwave-assisted aqueous enzymatic extraction of oil from pumpkin seeds and evaluation of its physicochemical properties, fatty acid compositions and antioxidant activities[END_REF] in a Soxhlet apparatus for 5e6 h. Afterwards, the solvent is evaporated either under nitrogen gas or in a vacuum evaporator [START_REF] Su Curovi C | Physicalchemical characteristics and oxidative stability of oil obtained from lyophilized raspberry seed[END_REF][START_REF] Jiao | Microwave-assisted aqueous enzymatic extraction of oil from pumpkin seeds and evaluation of its physicochemical properties, fatty acid compositions and antioxidant activities[END_REF]. Oil is extracted from the samples through repeating washing with chemical solvent by reflux in Soxhlet apparatus so the solvent can be used repeatedly and the whole extraction process is automatic. Also, depending on the size of the Soxhlet apparatus, the amount of samples used for the extraction can vary from less than 10 g to 100 g [START_REF] Su Curovi C | Physicalchemical characteristics and oxidative stability of oil obtained from lyophilized raspberry seed[END_REF] allowing the extraction of lipids from samples with low lipid content. However, this method takes a long period of time to extract the lipids from the samples and some of the researchers argued that the heating process during reflux in Soxhlet extraction may cause degradation of lipids that are prone to peroxidation such as PUFA, especially DHA [START_REF] Mansour | Characterization of oilseed lipids from "DHA-Producing camelina sativa": a new transformed land plant containing long-chain Omega-3 oils[END_REF].

For solvent extraction, the most common method used is Folch extraction method [START_REF] Folch | A simple method for the isolation and purification of total lipides from animal tissues[END_REF]. Briefly, the ground samples are homogenized with Folch solution, followed by agitation using orbital shaker at room temperature. The major advantage of Folch extraction is that less solvent is needed for small amount of samples (1 g in 20 ml of solvent mixture) while for Soxhlet extraction, the amount of solvent used depends on the Soxhlet extractor; the latter requires 100 ml of solvent for 1 g sample extraction. Folch extraction method also require less preparation time than Soxhlet extraction, in which the whole process only takes approximately 1e2 h while Soxhlet extraction takes at least 5 h to complete the extraction. Although the time used in Folch extraction is shorter than Soxhlet extraction, the extract weight is even higher than the one extracted by Soxhlet extraction when same amount of sample were used for the extractions [START_REF] Caprioli | Lipid nutritional value of legumes: evaluation of different extraction methods and determination of fatty acid composition[END_REF]. Furthermore, there are no significant difference between the fatty acids composition of the extract extracted by both methods [START_REF] Caprioli | Lipid nutritional value of legumes: evaluation of different extraction methods and determination of fatty acid composition[END_REF].

It could be surmised that different extraction methods do not affect the lipids composition of the extracts. Extraction method can be chosen based on the nature of your samples. For nuts and seeds having high percentage of lipids, Folch extraction should be used since a small amount of sample (0.5 ge1 g) is sufficient to extract the highest amount of oil extract. For nuts and seeds having low percentage of lipids, Soxhlet extraction should be used in order to handle large amount of samples (>100 g) with less solvent.

Mass spectrometry measurement

In early reports, when IsoFs were first identified [START_REF] Fessel | Discovery of lipid peroxidation products formed in vivo with a substituted tetrahydrofuran ring (isofurans) that are favored by increased oxygen tension[END_REF], GC-MS was the tool of choice by researchers. It requires negative ion chemical ionization and monitors a single ion, 585 in the case of IsoFs and 609 for NeuroFs. However, there is a drawback with the application of GC-MS as it is unable to identify the isomeric forms therefore only the 'total' of the isomers could be determined. Nevertheless, due to the increasing interest in other isofuranoids such as PhytoFs from ALA, dihomo-IsoFs from AdA and NeuroFs from DHA as well as the isomers, the application of LC-MS/MS is currently preferred [START_REF] Leung | Current development in nonenzymatic lipid peroxidation products, isoprostanoids and isofuranoids, in novel biological samples[END_REF][START_REF] Vigor | Non-enzymatic lipid oxidation products in biological systems: assessment of the metabolites from polyunsaturated fatty acids[END_REF]. Multiple reaction monitoring (MRM) is highly sensitive and commonly employed in LC-MS/MS. The technical aspects of GC-MS and LC-MS/MS are well described in several reviews [START_REF] Leung | Current development in nonenzymatic lipid peroxidation products, isoprostanoids and isofuranoids, in novel biological samples[END_REF][START_REF] Vigor | Non-enzymatic lipid oxidation products in biological systems: assessment of the metabolites from polyunsaturated fatty acids[END_REF]. LC-MS/ MS approach however, requires the infusion of pure standards to identify the corresponding transition mass ions for the lipid of interest (Table 2). Currently, there are no commercialized standards for isofuranoids ready for researchers, hence the syntheses by organic chemists [9,10,12e16].

Implications of isofuranoids

Initially, the measurement of IsoFs and NeuroFs were used to determine the extent of oxidative stress with variable oxygen tension in different models such as fish, rodents, pig, lamb neonates and human (Table 3) and thereafter as potential biomarker (Table 4) related to the function of vital organs. As described below, we investigated the types of isofuranoid measurements conducted related to the vital organs and its potential role in the future.

Oxygen tension.

In vitro peroxidation of AA revealed a plateau from 21% to 100% of oxygen tension for the generation of IsoPs, contrary to the constant increase of IsoFs. Concentration of IsoFs was two-fold higher that IsoPs, and the ratio of IsoFs to IsoPs in brain and kidney (two highly oxygenated organs) was the inverse of that of liver, with levels of IsoFs exceeding those of IsoPs by 2.0to 2.3-fold. These early data suggests that the ratio levels of esterified IsoFs and F 2 -IsoPs may be able to serve as an index of tissue oxygenation in various organs (Fig. 2).

Complications can arise with more unsaturated precursors like DHA. Indeed, Song et al. showed no difference in NeuroFs concentration at 100% oxygen tension, probably due to the greater capabilities of the hydroperoxide NeuroF intermediates to generate dioxolane derivatives [START_REF] Yin | Identification of a novel class of endoperoxides from arachidonate autoxidation[END_REF] therefore limiting the total amount of NeuroF.

It is evident that a dynamic augmentation or surgical intubation of oxygen in the biological systems potentially increase the formation of isofuranoids compared to isoprostanoids. Fessel et al. [START_REF] Fessel | Discovery of lipid peroxidation products formed in vivo with a substituted tetrahydrofuran ring (isofurans) that are favored by increased oxygen tension[END_REF] found that IsoFs were predominantly generated above 21% oxygen tension in bronchoalveolar lavage (BAL) fluid (Table 3). The IsoFs levels were also high in lung tissues of hyperoxic mice but was a transient phenomenon whereas the levels remained high in lung tissues of genetic mice lacking in Cyp1a1gene [START_REF] Lingappan | Mice deficient in the gene for cytochrome P450 (CYP)1A1 are more susceptible than wild-type to hyperoxic lung injury: evidence for protective role of CYP1A1 against oxidative stress[END_REF][START_REF] Wang | Disruption of cytochrome P4501A2 in mice leads to increased susceptibility to hyperoxic lung injury, Free Radic[END_REF]. However, no change in IsoFs was found in lung tissues of lamb exposed to hyperoxia [START_REF] Cotton | Inhaled nitric oxide attenuates hyperoxic lung injury in lambs[END_REF]. IsoFs and NeuroFs were elevated in neonates of low gestational age and preterm babies respectively when they were resuscitated at extremely high inspired oxygen level [START_REF] Vento | Preterm resuscitation with low oxygen causes less oxidative stress, inflammation, and chronic lung disease[END_REF][START_REF] Kuligowski | Urinary lipid peroxidation byproducts: are they relevant for predicting neonatal morbidity in preterm infants?[END_REF]. Moreover, in surgical patients requiring tourniquet procedure did not show alteration of IsoFs in the plasma at differential inspired oxygen concentrations [START_REF] Corcoran | Hemoglobin attenuates the effects of inspired oxygen on plasma isofurans in humans during upper-limb surgery[END_REF]. Differential oxygen tension was also found in the brain where certain parts of the brain have higher oxygen tension (white matter) than others (grey matter) [START_REF] Skinner | Differences in the fatty acid composition of the grey and white matter of different regions of the brains of patients with Alzheimer's disease and control subjects[END_REF][START_REF] Feng | Depth profile of local oxygen tension and blood flow in rat cerebral cortex, white matter and hippocampus[END_REF]. Noticeably this difference is described in Tables 2 and3 in which hyperoxic environment elevated IsoFs and NeuroFs in brain tissues of newborn pigs and IsoFs in cognitively unimpaired transgenic mice [START_REF] Solberg | Resuscitation with supplementary oxygen induces oxidative injury in the cerebral cortex[END_REF][START_REF] Arendash | Oxygen treatment triggers cognitive impairment in Alzheimer's transgenic mice[END_REF]. In the marine ecosystem, hypoxia-hyperoxia is a natural event but could be altered in abnormal environment such as climate change or pollution. It was found hyperoxia did not elevate IsoFs and NeuroFs in marine fish muscles despite its high abundance in DHA. Interestingly, hyperoxia acutely augmented dihomo-IsoFs in these fish and was predominant in male gender (Table 3) [START_REF] Chung | Hyperoxia elevates adrenic acid peroxidation in marine fish and is associated with reproductive pheromone mediators[END_REF]. [START_REF] Skinner | Differences in the fatty acid composition of the grey and white matter of different regions of the brains of patients with Alzheimer's disease and control subjects[END_REF][START_REF] Feng | Depth profile of local oxygen tension and blood flow in rat cerebral cortex, white matter and hippocampus[END_REF], different oxygen tension exists between the white and grey matter where the latter is higher. In relation to this, recently de la Torre et al. observed dihomo-IsoFs, namely 7(RS)-ST-D 8 -11-dihomo-IsoF and 17(RS)-10-epi-SC-D 15 -11-dihomo-IsoF had the most noticeable level in rat and preterm pig brains [START_REF] De La Torre | Synthesis, discovery, and quantitation of dihomo-isofurans: biomarkers for in vivo adrenic acid peroxidation[END_REF][START_REF] De La Torre | Total syntheses and in vivo quantitation of novel neurofuran and dihomo-isofuran derived from docosahexaenoic acid and adrenic acid[END_REF]. This also corresponded to a previous finding in which adrenic acid was more concentrated in the white matter compared to the grey matter. This finding is important as one needs to be cautious when measuring isofuranoids as it is assumed the brain being rich in DHA, NeuroFs would be the variable metabolite among the isofuranoids determined (Table 4).

Brain. As described by Skinner and Feng

Regardless to the oxygen tension, IsoFs and NeuroFs were determined in brain tissues of neurodegenerative diseases as well as neurological related injuries (Table 4). It was shown that IsoFs were higher than the controls in the substantia nigra of Parkinson's disease and dementia but not in multiple system atrophy and Alzheimer's disease (AD) [START_REF] Fessel | Isofurans, but not F2isoprostanes, are increased in the substantia nigra of patients with Parkinson's disease and with dementia with Lewy body disease[END_REF]. However, IsoFs and NeuroFs were shown to be higher in the brain cortex of AD transgenic mice compared to the wild-type [START_REF] Song | Neurofurans, novel indices of oxidant stress derived from docosahexaenoic acid[END_REF]. Also kainic acid induced epilepsy seizure in adult rats elevated IsoFs levels in the hippocampus and dentate gyrus compared to baseline [START_REF] Patel | Seizure-induced formation of isofurans: novel products of lipid peroxidation whose formation is positively modulated by oxygen tension[END_REF]. IsoFs in the cerebrospinal fluid from traumatic brain injury and subarachnoid hemorrhage patients were also augmented compared to the controls [START_REF] Corcoran | Are isofurans and neuroprostanes increased after subarachnoid hemorrhage and traumatic brain injury?[END_REF] but melatonin supplementation appeared to reduce IsoFs in the cortex tissue of hypoxia-ischemic injury in rat [START_REF] Balduini | The use of melatonin in hypoxic-ischemic brain damage: an experimental study[END_REF]. Viewing these reports, there seem to be a lack of studies in association of diseases and injuries of the brain and the isofuranoid determinations. It would be valuable to determine the different isofuranoids in the brain tissues by differentiating the white and grey matters and specifically frontal, parietal, temporal and occipital cortexes, and cerebellum and brain stem in normal and disease models to see if there is any distinction between them.

Heart.

The heart pumps oxygenated blood for circulation and this efficacy depends on the rhythm and status of the heart muscle. Measurement of IsoFs related to the heart (Table 4) were mainly focused in plasma and urine of cardiac bypass (CBP) surgery patients [START_REF] Simpson | Acetaminophen attenuates lipid peroxidation in children undergoing cardiopulmonary bypass[END_REF] in which it was high compared to the controls. Among CBP patients, those with acute kidney injury showed higher IsoFs levels than just CBP patients. However, treatment with acetaminophen to CPB prior to surgery alleviated urinary IsoFs compared to baseline indicating reduction of oxidative stress by acetaminophen [START_REF] Billings | Perioperative intravenous acetaminophen attenuates lipid peroxidation in adults undergoing cardiopulmonary bypass: a randomized clinical trial[END_REF]. Recently, plasma and urine IsoFs were determined in postoperative atrial fibrillation patients (PoAF) and it was observed that mitochondrial abnormality appeared to favor the generation of IsoF in PoAF condition [START_REF] Wu | Oxidative stress biomarkers and incidence of postoperative atrial fibrillation in the Omega-3 fatty acids for prevention of postoperative atrial fibrillation (OPERA) trial[END_REF]. Only one study determined IsoFs, NeuroFs and dihomo-IsoFs in heart tissues [START_REF] De La Torre | Total syntheses and in vivo quantitation of novel neurofuran and dihomo-isofuran derived from docosahexaenoic acid and adrenic acid[END_REF]. It was found among the isofuranoids, dihomo-IsoFs in particular the isomer 7(RS)-ST-D 8 -11dihomo-IsoF predominated. Interestingly in the same report, NeuroFs namely 4(RS)-ST-D 5 -8-NeuroF was higher in the heart tissue compared to the brain whereas no difference was observed for IsoFs; perhaps this is due to greater dynamics of oxygen tension in the heart compared to the brain, and the higher unsaturation of DHA compared to AA which are more prone to peroxidation. Further, Roy et al. [START_REF] Roy | Non-enzymatic cyclic oxygenated metabolites of omega-3 polyunsaturated fatty acid: bioactive drugs?[END_REF] recently observed some anti-arrhythmic event in mice cardiac cells by 4(RS)-ST-D 5 -8-NeuroF at high dose, indicating its potential role in the cardiovascular system. [42]

Marine fish Male y : 5.0 (baseline) vs 10.0 (hyperoxia) ng/g muscle Female y : 10.0 (baseline) vs 4.0 (hyperoxia) ng/g muscle Hyperoxia did not alter NeuroFs concentration fish muscles. [START_REF] Chung | Hyperoxia elevates adrenic acid peroxidation in marine fish and is associated with reproductive pheromone mediators[END_REF] Values are mean ± SD; y estimate from graphical display; Cr: creatinine; Se: selenium; PD: postnatal day; UPLC-MS/MS: ultra performance tandem mass spectrometry; CSF: cerebrospinal fluid; CCl 4 : carbon tetrachloride.

Table 4

The generation of isofurans, dihomo-isofurans, phytofurans and neurofurans in different biological samples related to vital organs that were unintended to generate hyperoxia condition in the study.

Study Sample and concentration Outcome Reference

Brain IsoFs Parkinson's disease (PD), multiple system atrophy (MSA), dementia with Lewy body (DLB) disease and Alzheimer's disease (AD) patients.

Brain substantia nigra tissue y: 5 (control), 10 (PD), 10 (DLB), 4 (MSA) and 3 (AD) ng/g.

IsoFs increased preferentially in PD and DLB patients and may be related to the relative intracellular hyperoxia due to mitochondrial dysfunction.

[49]

Alzheimer disease Tg 2576 transgenic mice Cortex tissues: 10.5 (control) vs 14.3 (Tg 2576) ng/g. Cerebellum y : 80 (control) vs 100 (Tg 2576) ng/g.

IsoFs are elevated in Tg 2576 brain. [START_REF] Song | Neurofurans, novel indices of oxidant stress derived from docosahexaenoic acid[END_REF] Kainic acid-induced with seizure (status epilepticus) in adult rats.

Hippocampus y : 1.25 ng/g tissue before seizure, and 4.5 ng/g tissue (16 h) and 3.25 (48 h) ng/g tissue after seizure. Dentate gyrus y : 1.0 ng/g tissue before seizure, and 6.75 ng/g tissue (16 h) and 3.25 (48 h) ng/g tissue after seizure. Cerebellum y : 0.75 ng/g tissue before seizure, and 1.0 ng/g tissue (16 h) and 1.25 (48 h) ng/g tissue after seizure.

Increased IsoFs after seizure induction in hippocampus and dentate gyrus. No change in cerebellum. The hippocampal IsoFs correlated with mitochondrial oxidative stress and reoxgyenation after a transient hypoxia.

[50]

Comatose severe aneurysmal subarachnoid hemorrhage (aSAH) and traumatic brain injury (TBI) patients.

CSF y : 0.50 (control) and 2.10 nmol/L (aSAH) patients; 0.50 (control) and 1.80 nmol/L (TBI patients.

IsoFs measurement could assist in management of comatose-neurological care patients. [START_REF] Corcoran | Hemoglobin attenuates the effects of inspired oxygen on plasma isofurans in humans during upper-limb surgery[END_REF][START_REF] Corcoran | Are isofurans and neuroprostanes increased after subarachnoid hemorrhage and traumatic brain injury?[END_REF] Rat with cerebral hypoxia-ischemia (HI) injury and melatonin (Met) treatment.

Cerebral right cortex tissue: 0.11 (control), 1.56 (HI) and 0.84 HI-Met ng/mg.

Melatonin protected brain injury. [START_REF] Balduini | The use of melatonin in hypoxic-ischemic brain damage: an experimental study[END_REF] Preterm pig brain cortex Cortex tissue y : 2 ng/g (prefrontal) and 6 ng/g (medial prefrontal cortex)

Higher IsoFs concentration in medial prefrontal cortex tissues.

[9]

Rat brain Brain tissue y : 3.5 ng/g No difference in the level of IsoFs between brain and heart.

[16]

Dihomo-IsoFs

Preterm pig brain cortex Cortex tissue y : 11 (prefrontal) and 16 (medial prefrontal cortex) ng/g of 17(RS)-10-epi-SCD 15 -11-dihomo-Isofuran. 17(RS)-10-epi-SCD 15 -11-dihomo-IsoF was identified in brain tissue.

[

Rat brain Brain tissue y : 280 ng/g of 7(RS)-ST-D 8 -11dihomo-Isofuran and 3 ng/g of 17(RS)-10-epi-SCD 15 -11-dihomo-Isofuran.

Among the isofuranoids, 7(RS)-ST-D 8 -11dhihomo-IsoF predominated in the brain tissue.

[16]

NeuroFs

Alzheimer disease Tg 2576 transgenic mice.

Cortex tissues: 156.2 (wild-type) vs 99.3 (p47 phox ) ng/g; 109.1 (wild-type) vs 173.2 (Tg 2576) ng/g. Elevated levels of NeuroFs are found in Tg 2576 brain cortex.

[8]

Preterm pig brain cortex Cortex tissue y : 3 ng/g (prefrontal) and 5 ng/g (medial prefrontal cortex).

No difference between brain tissue types. [START_REF] De La Torre | Synthesis, discovery, and quantitation of dihomo-isofurans: biomarkers for in vivo adrenic acid peroxidation[END_REF] Rat brain Brain tissue y : 0.2 ng/g (total neurofuran) and 0.02 ng/g (4(RS)-ST-D 5 -8-Neurofuran).

Neurofuran was synthesized and identified in heart and brain tissues. Hemoprotein induced in AKI increased lipid peroxidation.

[74]

Elective cardiac bypass (CPB) surgery children patients treated with acetaminophen or placebo prior to surgery.

Acetaminophen plasma y :85 (baseline) and 120 (post CPB) pg/mL. Acetaminophen urine y :15 pg/mg Cr (baseline) and 30 (post CPB) ng/mg Cr.

Compared to placebo (data not provided), acetaminophen attenuated the IsoFs increase in plasma during CPB.

[53]

Rat heart

Heart tissue y : 3.0 ng/g No difference in the level of IsoFs between brain and heart.

[16]

Cardiac bypass (CPB) surgery patients treated with acetaminophen or placebo prior to surgery. Heart tissue y : 140 ng/g of 7(RS)-ST-D 8 -11dihomo-Isofuran and 3 ng/g of 17(RS)-10-epi-SCD 15 -11-dihomo-Isofuran).

Placebo plasma

Among the isofuranoids, 7(RS)-ST-D 8 -11dhihomo-IsoF predominated in the heart tissue.

[16]

NeuroFs

Rat heart

Heart tissue y : 0.2 ng/g (total neurofuran) and 0.11 ng/g (4(RS)-ST-D 5 -8-Neurofuran).

Higher 4(RS)-ST-D 5 -8-neurofurans level in heart tissue than the brain was found.

[16]

Arrhythmia of mice cardiac cells 4(RS)-ST-D 5 -8-Neurofuran reduced arrhythmia.

Higher concentration (1 mM) of 4(RS)-ST-D 5 -8-Neurofuran compared to lower concentration (0.1 mM) showed anti-arrhythmic effect in cardiac cells.

[56]

Kidney

IsoFs

Severe sepsis patients with and without renal failure or hepatic failure or coagulation failure.

Plasma without renal failure y : 250 pg/mL. Plasma with renal failure y : 1250 pg/mL. Plasma with coagulation failure y : 1500 pg/mL.

IsoFs are highly associated with renal in critically ill patients with severe sepsis.

[61]

Maintenance hemodialysis therapy patients supplemented with or without antioxidant (mixed tocopherol þ a-lipoic acid) for 6 months.

Placebo plasma: 0.89 ± 1.9 (baseline) and 0.79 ± 1.36 (6 months) ng/mL. Antioxidant plasma: 0.69 ± 1.31 ng/mL (baseline) and 0.65 ± 0.90 (6 months) ng/mL.

No improvement of oxidative stress by antioxidant supplementation to hemodialysis patients.

[57]

End-stage renal disease patients receiving hemodialysis.

Baseline:<0.25 -! 0.73 ng/mL plasma. 6-month after hemodialysis:<0.26 -! 0.73 ng/ mL plasma.

Higher level of IsoFswas associated with increased resistance to erythropoiesis stimulating agents. [START_REF] Rivara | Association of plasma F2-isoprostanes and isofurans concentrations with erythropoiesisstimulating agent resistance in maintenance hemodialysis patients[END_REF][START_REF] Rivara | A pilot randomized crossover trial assessing the safety and short-term effects of pomegranate supplementation in hemodialysis patients[END_REF] End-stage renal disease patients receiving hemodialysis supplemented with 4 weeks of pomegranate juice and 4 weeks of pomegranate extract.

Plasma: Not reported. Supplementation had no effect on the level of IsoFs.

[ [START_REF] Rivara | Association of plasma F2-isoprostanes and isofurans concentrations with erythropoiesisstimulating agent resistance in maintenance hemodialysis patients[END_REF][START_REF] Rivara | A pilot randomized crossover trial assessing the safety and short-term effects of pomegranate supplementation in hemodialysis patients[END_REF] Supplementation of coenzyme Q10 to hemodialysis patients.

Plasma: 141 ± 67.5(before) and 72.2 ± 37.5 (after) pg/mL supplementation.

Plasma IsoFs concentrations decreased after coenzyme Q10 supplementation.

[60]

Liver IsoFs Rat, mouse and human. 2 (-CCl 4 ) vs 30 ng/g (þCCl 4 ) liver tissue. The formation of IsoFs by CCl 4 in liver.

[7] Severe sepsis patients with and withouthepatic failure.

Plasma without hepatic failure y : 500 pg/mL. Plasma with hepatic failure y : 1300 pg/mL.

IsoFs are highly associated with hepatic failure in critically ill patients with severe sepsis.

[61] [62]

NeuroFs

Lung transplant recipients with primary graft dysfunction (PGD) and controls (non-PGD).

Non-PGD plasma: 31.9 pg/mL. PGD plasma: 39.7 pg/mL. PDG with non-smoker lung plasma: 34.6 pg/mL. PDG with smoker's lung plasma: 66.9 pg/mL.

IsoFswas higher in patients with PGD after smoker lung transplantation compared with patients without PGD development.

[63]

Others IsoFs Rat and human. Rat: 3.3 ± 0.3 ng/mL urine; 334 ± 80 pg/mL plasma; 0.32 (control) vs 0.05 (nacetylcysteine) vs 0.12 (a-lipoic acid) ng/mL plasma y ; Human: 5.8 ± 1.0 ng/mL urine; 71 ± 10 pg/mL plasma.

IsoFs detectable in different samples. [START_REF] Fessel | Discovery of lipid peroxidation products formed in vivo with a substituted tetrahydrofuran ring (isofurans) that are favored by increased oxygen tension[END_REF] Mice low-high iron proximal colon after azoxymethane (AOM) or saline (control) treatment.

Control y : 5.5 (low iron) and 2.5 (high iron) ng/g. AOM y : 2.8 (low iron) and 3.0 (high iron) ng/g. Iron overload did not affect colon tissue levels of IsoFs.

[65]

Severe sepsis patients with and coagulation failure.

Plasma without coagulation failure y : 300 pg/mL. Plasma with coagulation failure y : 1500 pg/mL.

IsoFs are highly coagulation failure in critically ill patients with severe sepsis. IsoFs were lower in patients undergoing SA compared with patients undergoing GA.

[66]

Normal pregnancy versus pre-eclampsia. Plasma from maternal blood y : 2.00 (normal) and 2.50 (pre-eclampsia) nmol/L. Plasma from cord blood y : 12.00 (normal) and 14.00 (pre-eclampsia) nmol/L.

Significant elevation of IsoF in maternal plasma but not cord blood. [START_REF] Barden | Is there a role for isofurans and neuroprostanes in pre-eclampsia and normal pregnancy?[END_REF] mainly focused on hemodialysis patients (Table 4) in particular to reduce oxidative stress. It was shown antioxidant supplementation of mixed tocopherols with a-lipoic acid or pomegranate juice had no effect on the IsoFs level in plasma but supplementation of coenzyme Q10 decreased plasma IsoFs concentration in hemodialysis patients [57e60]. However, in critically ill patients with sepsis and renal failure showed multiple increase in IsoFs concentration in plasma compared to severe sepsis patients [START_REF] Ware | Plasma biomarkers of oxidant stress and development of organ failure in severe sepsis[END_REF]. To date, no reports on dihomo-IsoFs and NeuroFs levels in relation to kidney disorders were found.

5.2.1.5. Liver. Liver is the main organ for the synthesis, metabolism and storage of PUFA. Despite this, only a handful of studies on isofuranoids and liver function were found in the literature (Table 4), probably because the liver oxygen tension is the lowest of all organs, therefore the isoprostanoids should predominate (ref [START_REF] Fessel | Discovery of lipid peroxidation products formed in vivo with a substituted tetrahydrofuran ring (isofurans) that are favored by increased oxygen tension[END_REF]. It was shown severe sepsis patients with hepatic failure had higher levels of plasma IsoFs compared to severe sepsis patients [START_REF] Ware | Plasma biomarkers of oxidant stress and development of organ failure in severe sepsis[END_REF]. Moreover, an acute increase of NeuroFs was identified in mice liver when treated with carbon tetrachloride [START_REF] Song | Neurofurans, novel indices of oxidant stress derived from docosahexaenoic acid[END_REF]. Age macular degeneration (AMD) patients before and after antioxidant supplementation.

Non-AMD plasma: 0.20 ± 0.072 (before) and 0.17 ± 0.048 (after) ng/mL. AMD plasma:0.24 ± 0.20 (before) and 0.25 ± 0.29 (after) ng/mL.

Antioxidant failed to reduce IsoFs in AMD. [START_REF] Brantley | Plasma biomarkers of oxidative stress and genetic variants in age-related macular degeneration[END_REF][START_REF] Brantley | The short-term effects of antioxidant and zinc supplements on oxidative stress biomarker levels in plasma: a pilot investigation[END_REF] Canadian Inuits Plasma:20.86 (18.90e23.02 range) pg/mL IsoFs correlated with visceral adiposity and smoking was related to lower plasma levels of IsoFs due to lower ambient levels of tissue oxygen among smokers.

[70]

Canadian Inuits

Plasma from blood with: <200 Se mg/L, 29.98 pg/ mL IsoFs; !200 and < 340 Se mg/L, 17.53 pg/mL IsoFs; !340 Se mg/L, 18.14 pg/mL IsoFs.

Plasma IsoFs decreased with increasing tertiles of blood Se.

[71]

Storage of leukoreduced human red blood cells (RBC).

Supernatant of stored RBC y : 5 days: 75 pg/mL; 12 days: 90 pg/mL; 40 days: 170 pg/mL; 47 days: 190 pg/mL. Supernatant washing of RBC y : 55 pg/mL (prewash) vs 17 pg/mL (post-wash).

Linear increase in IsoFs in RBC supernatant with storage time but decreased if washed.

[72]

Infants with normal gestational period Serum: 0.015e147 nmol/L range. No outcome.

[73] Dihomo-IsoFs Pine nut, walnut, chia seed and flax seed Nuts and seeds y : 0.3 (pine), 9 (walnut), 6 (chia) and 0.7 (flax) ng/g of ent-16-(RS)-13-epi-ST-D 14 -9-Phytofuran.

ent-16-(RS)-13-epi-ST-D 14 -9-PhytoF predominated in walnut and chia seeds.

[10]

NeuroFs

Infants with normal gestational period Serum: 0.013e14.5 nmol/L range. No outcome. [START_REF] Ch Afer-Peric As | Ultra high performance liquid chromatography coupled to tandem mass spectrometry determination of lipid peroxidation biomarkers in newborn serum samples[END_REF] Values are mean ± SD; y estimate from graphical display; Cr: creatinine; Se: selenium; PD: postnatal day; UPLC-MS/MS: ultra performance tandem mass spectrometry; ICU: intensive care unit.

Fig. 2. Summary of isoprostanoids and isofuranoids identified and its association to diseases: Isofuranoids formation from arachidonic acid shows constant increase with oxygen tension (up to 100%) compared to the plateau reached by isoprostanoids at 21% of oxygen tension, in vitro experiment. Can this fact be translated to specific tissues, organs, diseases and pathologies for more accurate biomarkers of oxidative stress?.

5.2.1.6. Lung. When determining isofuranoids in the biological systems, one area of interest is undoubtedly the lung since it is the main organ for oxygen delivery to the circulatory system. It was found that alveolar hemorrhage induces IsoFs formation in human bronchoalveolar lavage [START_REF] Bastarache | Low levels of tissue factor lead to alveolar haemorrhage, potentiating murine acute lung injury and oxidative stress[END_REF], and that plasma of patients undergoing lung transplant with primary graft dysfunction (PGD) had higher IsoFs level especially those receiving a smoker's lung compared to non-PGD and PGD [START_REF] Diamond | The relationship between plasma lipid peroxidation products and primary graft dysfunction after lung transplantation is modified by donor smoking and reperfusion hyperoxia[END_REF]. Lung tissues of transgenic mice with low levels of tissue factor had higher IsoFs compared to the wild-type when exposed to inflammation such as lipopolysaccharide (LPS) [START_REF] Bastarache | Low levels of tissue factor lead to alveolar haemorrhage, potentiating murine acute lung injury and oxidative stress[END_REF] and mice with cytoplasmic domain mutation had higher IsoFs in the lung tissue compared to the controls [START_REF] Lane | Oxidative injury is a common consequence of BMPR2 mutations[END_REF]. Surprisingly, no reports were identified for dihomo-IsoFs and NeuroFs in relation to biological systems where oxygen concentrations were not intentionally induced as explained above.

5.2.1.7. Others. Aside from the function of vital organs, reports of isofuranoids levels were made in different systems. It was shown iron overload in mice did not alter IsoFs in the colon tissues compared to controls [START_REF] Ilsley | Dietary iron promotes azoxymethane-induced colon tumors in mice[END_REF]. Moreover, the use of spinal anesthesia reduced plasma IsoFs whereas general anesthesia augmented IsoFs level in surgical procedure for leg ischemic-reperfusion compared to baseline [START_REF] Mas | Effects of spinal or general anesthesia on F2-isoprostanes and isofurans during ischemia/reperfusion of the leg in patients undergoing knee replacement surgery[END_REF]. High blood pressure showed an effect on IsoFs concentration, where maternal blood of pre-eclampsia showed higher IsoF levels compared to normal pregnancy [START_REF] Barden | Is there a role for isofurans and neuroprostanes in pre-eclampsia and normal pregnancy?[END_REF]. It was also found plasma IsoFs may have potential role in the development of age macular degeneration (AMD) in adults [START_REF] Brantley | Plasma biomarkers of oxidative stress and genetic variants in age-related macular degeneration[END_REF] but, antioxidant supplementation was unable to reduce IsoFs in AMD patients [START_REF] Brantley | The short-term effects of antioxidant and zinc supplements on oxidative stress biomarker levels in plasma: a pilot investigation[END_REF]. Plasma IsoFs was found to have an association with visceral adiposity and smoking in Canadian Intuits but appears to decrease in plasma IsoFs with increased blood selenium concentration [START_REF] Alkazemi | Isoprostanes and isofurans as non-traditional risk factors for cardiovascular disease among Canadian Inuit[END_REF][START_REF] Alkazemi | New insights regarding tissue Se and Hg interactions on oxidative stress from plasma IsoP and IsoF measures in the Canadian Inuit population[END_REF]. In a more recent study, IsoFs levels in red blood cells increased with storage time but interestingly a washing procedure of the red blood cells reduced the IsoFs level and were not further induced during storage [START_REF] Spinelli | Isoprostane and isofuran lipid mediators accumulate in stored red blood cells and influence platelet function in vitro[END_REF]. Serum NeuroFs of infants of normal gestational period was determined lately [START_REF] Ch Afer-Peric As | Ultra high performance liquid chromatography coupled to tandem mass spectrometry determination of lipid peroxidation biomarkers in newborn serum samples[END_REF] and PhytoFs in nuts and seed were measured, which showed some variable levels depending on the type [START_REF] Cuyamendous | Synthesis and discovery of phytofurans: metabolites of a-linolenic acid peroxidation[END_REF].

Conclusion

It is clear from this report (Fig. 2) that isofuranoids are an important group of compounds in biological systems. They exist due to increased or dynamic change of oxygen tension in vivo in the vital organs, and the gauging of isofuranoids may be necessary in critical illnesses or surgical procedures, and monitoring neurodegenerative diseases for better management of the patients e an elevation or reduction by intervention could indicate the health status of the patients. Isofuranoids are a group of novel compounds which in the future could be promising oxidative stress biomarkers due to their stability and abundance especially in the brain and heart but may also have a biological role in particular ones from n-3 PUFA that needs to be studied explicitly.

Scheme 1 .

 1 Scheme 1. Mechanistic explanation for the formation of IsoPs and IsoFs (only one isomer of one series is shown).

Fig. 1 .Scheme 2 .Scheme 3 .

 123 Fig. 1. Nomenclature of the furan derived from PUFA.

Scheme 4 .

 4 Scheme 4. Galano-Oger first strategy.

5. 2 . 1 . 4 .

 214 Kidney. Reports of IsoFs detection in the renal function

Table 2

 2 Precursor and product ions of isofuranoids determined by gas chromatographymass spectrometry (GC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS): mass-ion-ratio; Q1: precursor ion; Q3: transition ion.

  with developed acute kidney injury (AKI) Plasma: 32 (baseline), 58 (post cardiac bypass), 57 (ICU) and 30 (day 3 post-operation) pg/mL y ; 37.7 ± 5.7 (control) vs 27.4 ± 5.4 (AKI) pg/mL. Urine: 2.7 (baseline), 5.5 (post cardiac bypass), 8.5 (ICU) and 4 (day 3 post-operation) ng/mL Cr y ; 4.2 ± 0.5 (smokers) vs 2.5 ± 0.3 (nonsmokers) ng/mL Cr among the patients; 3.3 ± 0.5 ng/mL Cr (control) vs 2.2 ± 0.3 (AKI) ng/mL Cr.

[ 61 ]

 61 Spinal (SA) or general (GA) anesthesia in ischemia/reperfusion of the leg surgery Plasma GA: 8.80 (baseline), 8.70 (2 h), 8.80 (3.5 h) and 5.80 (24 h) nmol/L. Plasma SA: 8.80 (baseline), 8.30 (2 h), 5.50 (3.5 h) and 5.40 (24 h) pmol/L.

Table 1

 1 Summary of the number of oxygenated furan metabolites of PUFAs generated.

	PUFA	Metabolite abbreviation	Number of lateral chain structures	Number of regioisomers	Number of isomers
	AA	IsoF	4	8	256
	AdA	Dihomo-IsoF	4	8	256
	ALA	PhytoF	4	4	128
	DHA	NeuroF	8	16	512

  It is noted that no matter which method is used for lipid extraction, ground dry nuts and seeds samples should be used since ground samples have a higher surface area which gives better yield and also that dry weight is a better reference for comparisons between different samples [31e34].

was found that PhytoFs are detected in nuts and seeds by LC-MS/MS recently

[START_REF] Cuyamendous | Synthesis and discovery of phytofurans: metabolites of a-linolenic acid peroxidation[END_REF]

. It is essential for us to extract lipids from the samples properly in order to measure PhytoFs accurately. Three different methods, Soxhlet extraction and solvent extraction have been commonly used by researchers to extract lipids from the nuts and seeds.

Table 3

 3 Concentration of isofurans, dihomo-isofurans and neurofurans in different biological samples intentionally induced with hyperoxia.

	Study	Sample and concentration	Outcome	Reference
	Isofurans (IsoFs)
	Brain
	Cognitively unimpaired Alzheimer's transgenic	Hippocampus non-TG y : 8 (A) and 10 (H) ng/g	IsoFs increased in hippocampus and	[47]
	(TG) mice exposed to air (A) or hyperoxia (H).	after 3 h; 5 ng/g (A) and 8 ng/g (H) after 24 h.	neocortex of TG mice at 24 h after a
		Hippocampus TG y : 10 (A) and 10 ng/g (H) after	single hyperoxia exposure.
		3 h; 8 (A) and 28 (H) ng/g after 24 h.
		Neocortex non-TG y : 4 (A) and 10 (H) ng/g after
		3 h; 5 (A) and 5 (H) ng/g after 24 h.
		Neocortex TG y : 7 (A) and 6 (H) ng/g after 3 h; 8
		(A) and 27 (H) ng/g after 24 h.
	Newborn pigs exposed to 0, 21, 40 and 100%	Prefrontal cortex tissue: 14.69 ± 3.0 (0%),	Dose-dependent increase of IsoFs in	[46]
	oxygen.	36.70 ± 10.5 (21%), 48.99 ± 21.1(40%) and	brain.
		55.99 ± 22.6 (100%) ng/g.
	Lung
	Mice	Mouse: 210 ± 30 pg/mL bronchoalveolar lavage	The formation of IsoFs becomes	[7]
		fluid; 24 (air) vs 90 (>98% O 2 ) pg mg protein	increasingly favored as oxygen tension
		lung tissue.	increasedabove 21%.
	Lamb exposed to room air or 95% O 2 or 95%	Lung tissue: no data shown.	There was no difference in lung tissue	[40]
	O 2 plus nitric oxide (5e10 ppm).		IsoFs between treatments.
	Extremely low gestational age neonate (ELGAN)	30% resuscitation: no data provided.	Oxidative stress increased significantly	[41]
	resuscitated with a lower (30%) or higher	90% resuscitation: 8 ng/mg Cr (BPD) and 4 ng/mg	in the 90% oxygen group compared to
	(90%) fraction of inspired oxygen (F IO2 ) with	Cr (no BPD) in urine day 7 after birth.	30% oxygen group first week after birth.
	and without bronchopulmonary dysplasia
	(BPD).
	Mice lacking Cyp1a1 gene (more susceptible to	Lung tissue WT y : 0.85 (air) and 1.8 (24 h) ng/mg	WT lung had increased IsoFs after 24 h	[38]
	hyperoxic lung injury) and C57BL/6J wild-	protein.	hyperoxia exposure and Cyp1a1( À/À )
	type (WT) mice under hyperoxia	Lung tissueCyp1a1 À/Ày : 0.75 (air), 1.1 (24 h) and	mice lung had increased IsoFs after 48
	environment for 24e72 h.	0.95 (48 h) ng/mg protein.	e72 h.Thereafter, the concentrations
			returned to 'air' level.
	Mice lacking Cyp1a1 gene (more susceptible to hyperoxic lung injury) and C57BL/6J wild-type (WT) mice in room air (A) or exposed to >95% oxygen for 24, 48, or 72 h	Lung tissue WT Liver tissueCyp1a1 À/Ày : 0.7 (A), 0.4 (24 h), 0.8	transient in WT but not in Cyp1a1 À/À mice. Hyperoxia leads to lung injury but it is	[39]
		(48 h) and 0.82 (72 h) ng/mg protein.
	Others
	Planned surgical patients with isolated upper-	Plasma: 6.11 (30%), 6.65 (50%) and 5.94 (80%)	Different concentration of inspired	[43,51]
	limb trauma requiring the pneumatic arterial	nmol/L prior oxygen inspiration i.e. baseline;	oxygen did not alter IsoFs.
	tourniquet procedure and inspired with 30,	6.02 (30%), 5.70 (50%) and 4.62 (80%) nmol/L
	50, or 80% oxygen concentrations.	15 min after inspired oxygen.
	Marine fish muscles	Male y : 0.75 (baseline) vs 1.8 (hyperoxia) ng/g	Hyperoxia did not alter IsoFs	[48]
		muscle	concentration in fish muscles.
		Female y : 3.0 (baseline) vs 1.8 (hyperoxia) ng/g
		muscle
	Preterm babies resuscitated with inspired	Control urine: 211 ± 239 (PD 1), 547 ± 651 (PD7)	Increased IsoFs during the first week	[42]
	oxygen (F IO2 ).	and 167 ± 188 (PD 28) signal units of UPLC-MS/	after birth.
		MS per mL
		BPD urine: 455 ± 188 (PD 1), 612 ± 548 (PD7)
		and 198 ± 219 (PD 28) signal units of UPLC-MS/
		MS per mL
	Dihomo-isofurans (Dihomo-IsoFs)
	Others
	Marine fish	Male y : 0.80 (baseline) vs 3.1 (hyperoxia) ng/g	Hyperoxia significantly elevated	[48]
		muscle.	dihomo-IsoFs, namely 10-epi-17(RS)-
		Female y : 0.5 (baseline) vs 0.8 (hyperoxia) ng/g	SC-D 15 -11-dihomo-isofuran in male
		muscle.	fish muscle.
	Neurofurans (NeuroFs)
	Brain
	Newborn pigs exposed to 0, 21, 40 and 100%	Prefrontal cortex tissue: 6.07 ± 2.0 (control),	Dose-dependent increase of NeuroFs in	[46]
	oxygen.	6.84 ± 2.7 (21%), 13.98 ± 7.8 (40%) and	brain.
		15.81 ± 10.1 (100%) ng/g.
	Others
	Preterm babies resuscitated with inspired	Control urine: 16 ± 26 (PD 1), 44 ± 38 (PD7) and	Augmented NeuroFs by hyperoxia was
	oxygen (F IO2 ).	23 ± 28 (PD 28) signal units of UPLC-MS/MS per	reduced after one-week PD.
		mL
		BPD urine: 20 ± 18 (PD 1), 54 ± 44 (PD7) 34 ± 19
		(PD 28) signal units of UPLC-MS/MS per mL

y : 0.9 (A), 1.4 (24 h), 0.5 (48 h) and 0.7 (72 h) ng/mg protein. Lung tissueCyp1a1 À/Ày : 0.8 (A), 0.7 (24 h), 1.2 (48 h) and 1.0 (72 h) ng/mg protein.

Liver tissue WT y : 0.5 (A), 0.6 (24 h), 0.5 (48 h) and 0.75 (72 h) ng/mg protein.

Liver tissue WT y : 0.5 (A), 0.6 (24 h), 0.5 (48 h) and 0.7 (72 h) ng/mg protein.

Table 4

 4

	(continued )
	Study	Sample and concentration	Outcome	Reference
		and 2.5 (day 2 post operation) ng/mg Cr.
		PoAF urine y :2 (baseline), 9 (end of surgery) and
		2.8 (day 2 post operation) ng/mg Cr.
	Dihomo-IsoFs
	Rat heart

Table 4

 4

	(continued )
	Study	Sample and concentration	Outcome	Reference
	Age macular degeneration (AMD) patients.	Non-AMD plasma: 0.15 ± 0.18 ng/mL.	Potential role of IsoFs in AMD pathogenesis.	[68,69]
		AMD plasma: 0.22 ± 0.38 ng/mL.


Mathieu Candy

Thierry Durand

Jean-Marie Galano
email: jean-marie.galano@umontpellier.fr

Camille Oger
email: camille.oger@umontpellier.fr

Total Synthesis Total Synthesis of the Isoketal 5-D 2 -IsoK Natural Product Based on Organocatalysis

Keywords: Fatty acids, Isoketals, Olefination, Organocatalysis, Total synthesis

The enantioselective total synthesis of the highly reactive isoketal 5-D 2 -IsoK was accomplished. The synthesis involved construction of the isoketal core by an organocatalyzed Michael addition between ideally functionalized aldehyde and nitroolefin partners with good enantioselectivity. The lateral

Introduction

Isoprostanes (IsoPs) are prostaglandin-like compounds discovered in the 1990s as oxidative-stress-induced nonenzymatic peroxidation products of arachidonic acid (C20:4 n-6). [START_REF] Morrow | Proc. Natl. Acad. Sci[END_REF] They are formed in the human body from phospholipids through bicyclic endoperoxide intermediates (G-IsoPs), and they can be partially or completely reduced to produce the well-known D-, E-or F-types of IsoPs (Scheme 1). [START_REF] For A Review | [END_REF] The same endoperoxides may also undergo rearrangement in vivo to produce isoketal derivatives (IsoKs), also called isolevuglandins. [3] IsoKs react with the primary amine groups of proteins (lysine residues) or phosphatidylethanolamine to form the corresponding pyrroles, which are readily oxidized over time into highly stable lactam and hydroxylactam adducts. [4] Much more than just a dosimeter of oxidative stress, [5] IsoKs appear to be disease biomarkers in pathological processes, as exogenous IsoKs on cultured cells include induction of inflammatory pathways, immune responses, and cell death, as well as the inhibition of ion-channel function. [6] From a structural point of view, IsoKs possess two aliphatic side chains containing two double bonds (E and Z), fixed onto a methyl γ-keto aldehyde. However, despite their relatively simple chemical structures, this class of compounds represents a challenging target for the synthetic community. First, endoperoxide rearrangements could give rise to the E and D isomers that bear three stereogenic centers, which would result in 64 possible IsoK isomers. Second, the construction of the highly reactive γketo aldehyde motif is not a trivial task, as the C8 and C12 stereogenic centers can readily racemize under acidic or basic conditions; third, the β,γ-unsaturated aldehyde may easily chains were branched through Horner-Wadsworth-Emmons and Wittig olefinations to provide flexibility, whereas the final deprotection and oxidation steps were performed under mild conditions to avoid known racemization and/or degradation of the very sensitive isoketal. go through conjugation or conjugation/dehydration of the hydroxy group at C5.

To the best of our knowledge, only the group of Salomon has attempted this synthetic challenge. More particularly, they reported the synthesis and biological investigations of two IsoK derivatives, namely, 5-D 2 -IsoK and 12-E 2 -IsoK, and 17-E 4 -NeuroK derived from docosahexaenoic acid. [7] The key features of their synthetic route include: (1) diastereoselective 1,4-addition of an elaborated vinylcyanocuprate to a chiral γ-alkoxyenone to build the central core; (2) deprotection of the sensitive aldehyde function (protected as acetonide) in the last step under acidic conditions (AcOH, H 2 O) followed by mild oxidative cleavage of a vicinal diol. However, this elegant strategy has some disadvantages that limits its practicality, such as the low control of the stereochemical outcome of the 1,4-addition (d.r. 1.5:1), combined with epimerization of the acidic centers next to the keto and aldehyde functionalities.

Our interest in lipid peroxidation prompted us to develop a new strategy allowing the synthesis of IsoK derivatives by taking into account the inherent epimerization problems and also a more flexible approach for introduction of the side chains. Herein, we report our efforts in the enantioselective synthesis of 5-D 2 -IsoK (1) by using an organocatalyzed Michael addition between a nitroolefin and an aldehyde as a key step. of aldehyde 8 [8] and nitroolefin 7 (1.15:1 ratio aldehyde/olefin) by using Jørgensen-Hayashi's catalyst 9 (5 mol-%) and p-nitrophenol as a co-catalyst (10 mol-%), which afforded the expected 1,4-nitroaldehyde in excellent yield (97 %) with good enantiomeric excess (90 %) and diastereomeric ratio (3:1 dr), clearly in the range of that previously report by Ruiz et al. [9] The C10 methyl group was then introduced by mild alkylation of aldehydes 6a/6b by using trimethylaluminum, avoiding epimerization of the base-sensitive aldehyde. The resulting nitro compound was subjected to the Nef reaction, [10] which proved to be delicate and unsuccessful following standard protocols. The milder conditions developed by McMurry [11] also led to degradation of the expected aldehyde. Finally, the use of sodium nitrite and acetic acid as reported by Mioskowski's group proceed smoothly. [12] These reaction conditions known to convert primary nitro groups into carboxylic acids led, in our case, to lactone cis/trans-10 in 64 % yield (over two steps) as a mixture of four diastereoisomers, probably through intermolecular trapping of the known nitrile oxide intermediate formed. [13] Gratifyingly, treatment of the mixture of cis/trans-10 with a catalytic amount of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) induced full epimerization of cis-lactone 10 into the thermodynamically more stable and desired trans-lactone 10. By this way, the unwanted diastereoisomer could be easily converted into the desired one. This strategy fully overcame the low diastereoselectivity of the first Michael addition, thus avoiding loss of material. Next, trans-lactone 10 was reduced with lithium aluminum hydride to the corresponding diol prior to selective protection of the primary alcohol as a triethylsilyl ether and secondary hydroxy group as its acetate to furnish intermediate 5 in excellent yield (81 % yield over four steps) and a unique purification step. At that stage, flash column chromatography allowed the separation of the two C11 diastereomers of 5, which was not necessary for completion of the synthesis but quite handy. Therefore, the synthesis proceeded with the major diastereomer of compound 5 (configuration of C11 not determined), for NMR spectroscopy simplification and attribution of the absolute configuration of the allylic alcohol that remained to be introduced.

Results and Discussion

With requisite intermediate 5 in hand, we next focused on the construction of the lateral chains (Scheme 4). Exposing TESprotected primary alcohol 5 to Swern conditions cleanly furnished, without purification, the corresponding aldehyde. The α-chain (bearing the acid moiety) was then introduced by using HWE olefination with phosphonate 11 [14] and barium hydroxide as base to prevent epimerization at C8, [15] and desired ester 12 was obtained in 78 % yield. Luche reduction of the α,βunsaturated ketone gave a 3:1 diastereomeric mixture of the corresponding allylic alcohols in 98 % yield. The C5 epimers 5815 were separated by column chromatography, and the configuration of the major epimer was unambiguously attributed as (5S) by NMR spectroscopy analysis of the corresponding mandelate derivatives. [16] The ω-chain was introduced from the (5S) diastereoisomer by protection as tert-butyldimethylsilyl (TBS) ether 13 prior to cleavage of the p-methoxybenzyl (PMB) ether by using 2,3-dichloro-5,6-dicyanobenzo-1,4-quinone (DDQ) to release the primary alcohol. Dess-Martin periodinane (DMP) oxidation and Wittig olefination by using phosphonium salt 14 and sodium hexamethyldisilazane (NaHMDS) as a base furnished desired (E,Z)-diene 4 in 71 % yield over three steps.

Having succeeded in the stereoselective installation of the olefin lateral chains, we next conducted compound 4 through deprotection and oxidation steps to achieve the synthesis of desired 5-D 2 -IsoK (1, Scheme 5). The ester and acetate functions were simultaneously cleaved by using lithium hydroxide, but whereas the methyl ester reacted rapidly, the acetate group was found to be surprisingly more stable and 5 days was required to achieve full cleavage. Notably, the classical use of potassium carbonate in methanol was totally ineffective to remove the acetate group. The resulting secondary alcohol was readily oxidized by using Dess-Martin periodinane to give ketone 15 in 67 % yield over two steps. At that stage, our plan was to convert intermediate 15 into isoketal 1 by concomitant acidic deprotection of both the TBS ether and dimethyl acetal groups. Despite our efforts, under all conditions tested (Amberlyst 15, BiBr 3 , AcOH/H 2 O) elimination product 18 was formed in a large amount along with desired product 1 and other undetermined byproducts. We thus decided to remove the TBS group, as corresponding alcohol 16 would be less prompt to elimination. The use of a large excess amount of tetrabutylammonium fluoride (TBAF) buffered with ammonium chloride [17] afforded alcohol 16 in 46 % yield (81 % based on recovered starting material) without epimerization at C12 and a trace amount of a lactonization product (separable if desired). Only one step from the desired and sensitive IsoK, we had further problems to deal with, as the right balance between conversion into 1, minimum formation of lactone 17, and elimination product 18, in addition to other potential double epimerization. Furthermore, target 1 was not suitable for silica gel purification. Finally, subjecting acetal 16 to a mixture of acetic acid and water at 15 °C for 5 h gave the best possible outcome by avoiding any purification and limiting the formation of side products. As a result, 5-D 2 -IsoK (1) was thus obtained in 79 % yield reproducibly with up to 10-20 % of lactonization product 17 and 5-10 % of compound 18. However, despite this fact, the purity of IsoK 1 is significantly improved relative to that of the first synthesis reported. [7b] Finally, because IsoK 1 is a bioactive compound of high interest but clearly quite unstable, we decided to provide a sub-micromolar scale access to IsoK 1 from a storable solution of compound 16. [18] Thus, simple treatment of a methanol solution of 16 with acetic acid and water at 15 °C provided desired compound 1 in good purity suitable for direct biological use.

Conclusion

In conclusion, we achieved a novel enantioselective synthesis of 5-D 2 -isoketal (1, 5-D 2 -IsoK) by using a unified strategy for the construction of the isoketal core. Multigram access to core intermediate 5, easiness of side-chain introduction, and a last step deprotection sequence may be employed for the synthesis of other polyunsaturated fatty acid isoketal derivatives. Furthermore, a sub-micromolar scale for the last step of the synthesis

Scheme 1 .

 1 Scheme 1. Biosynthesis of IsoKs and pyrrole derivatives.

Scheme 2

 2 Scheme 2 reveals the essential features of our retrosynthetic disconnections. First, we planned to uncover the sensitive aldehyde function of 5-D 2 -IsoK (1) in the last stage under mild conditions for dimethyl acetal hydrolysis of compound 4. More classical and, therefore, versatile introduction of the two lateral chains would proceed through Wittig and Horner-Wadsworth-Emmons (HWE) olefinations from 5. Interconversion of the triethylsiloxy (OTES) and OAc groups of 5 by Nef reaction and simple methyl alkylation would reveal intermediate 6, which could be obtained through an organocatalyzed Michael addition between simple aldehyde 8 and nitroolefin 7.

Scheme 2 .

 2 Scheme 2. Retrosynthetic approach.

www.eurjoc.org © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Acknowledgments

We gratefully thank the University of Montpellier (M.C. postdoctoral fellowship), the Centre National de la Recherche Scientifique (CNRS), and Dr. Stellios Arseniyadis (ESPCI ParisTech, France) for measuring the enantiomeric excess values by chiral super fluidic chromatography.