Gold(I)-catalyzed 7-exo-dig Cyclization: A Key Step to Access the Bicyclo[4.2.1]nonane Skeleton of Vibsatin A, a Neurotrophic Diterpenoid

Inspired by Sawamura's works, the generation of the bicyclic backbone was smoothly achieved through a Conia-ene derived 7-exodig cyclization from an enantiomerically enriched TIPS-based silyl enol ether. The reaction, catalyzed by a sensitive gold(I) complex, was efficiently performed on large scale by glove-box free techniques. Furthermore, the shape of this system was exploited for subsequent installation of all the stereogenic centres.

Developing straightforward approaches towards the construction of various complex and polycyclic architectures in three dimensions is essential for the synthesis of biologically active products. In this context, the efficient synthesis of rigid bridged bicyclo [4.2.1]nonane skeletons encountered in significant natural alkaloids and terpenoids such as ajmaline, 1 isokopsine, 2 echinopines, [START_REF] Dong | Sesquiterpenoids Possessing an Unprecedented Skeleton from Echinops spinosus[END_REF] and vibsatins 4 can afford opportunities to investigate new methodologies and their limits.

Our group cultivated interest for developing an effective chemical route towards the 5/7 bridged core of vibsatin A 1. This vibsane-type diterpenoid, isolated in 2014 from the Chinese plant Viburnum tinus cv. variegatus by Zhao et al, was found to induce significant neurite outgrowth. [START_REF] Gao | Two New Tetranorvibsane-Type Diterpenoids from Viburnum tinus cv. variegatus[END_REF]5 Moreover, if different synthetic studies were accomplished towards the elaboration of various vibsane natural substances, 6 no synthesis of this new sub-type was reported to date. Herein, the asymmetric construction of the bicyclo [4.2.1]nonane skeleton of vibsatin A 1 and the stereocontrolled introduction of all the stereogenic centres is reported.

Taking advantage of the bicyclic shape of the molecule, the envisioned strategy is based on the enantioselective installation of a single stereocentre which would induce the stereocontrol of all others. Thus, accessing the bicyclo [4.2.1]nonane skeleton 4 of vibsatin A 1 in a stereoselective manner appeared of primary interest, although the synthesis of medium size carbocycles like 7-membered rings is renowned to be highly challenging (Scheme 1). In 2010, Sawamura's group developed a bulky semihollow-shaped phosphine which, in association with gold(I), allowed the unprecedented gold catalysed formation of 7-membered rings through a Conia-ene derived 7-exo-dig process, from alkyne-tethered silyl enol ethers. 7,8 Though this work represented a remarkable advance, it was achieved on a very small scale (0.1 mmol scale ≈ 20 mg) and required glovebox techniques, thus restricting its application field.

Hence, it has been foreseen to develop this carbocyclization in order to access bicyclic ketone 4, from enantiomerically enriched silyl enol ether 5. Although an adaptation of the procedure was required, this reaction would enable, in a single step, the construction of the desired [5-7]-bicyclic bridged backbone in the right configuration at C2 and C11. An asymmetric Michael addition from 2-methylcyclopentanone 6 would provide the (R) configuration at C11 required for the substrate 5 of the cycloaddition step. Then, oxidations and deoxygenation of the bicycle 4 would allow the formation of the α,βunsaturated ketone 3. Further 1,4-addition was expected to functionalize the Si-face of the double bond leading to a 10(S) configuration. Subsequent C-allylation would position an allyl chain at C5 in a trans-fashion leading to 2 and to the setting of the last stereogenic centre present in vibsatin A. The synthesis of vibsatin A 1 would then be completed by a Wacker oxidation at the double bond and a Wittig olefination for the installation of the lateral chain (Scheme 1).

Scheme 1. Synthetic strategy. The first goal was the synthesis of the alkyne-tethered silyl enol ether 5 from 2-methylcyclopentanone 6. In order to control the quaternary stereocentre at C11, we choose to rely on the works of d'Angelo and Desmaële. 9 The known enantioenriched keto ester (R)-7 was easily obtained in 93% yield and 91% e.e., by deracemizing Michael addition of the imine derived from 2-methylcyclopentanone 6 and (S)-1phenylethylamine on methyl acrylate. After protection of the ketone as a dioxolane group and reduction of the ester, 10 the corresponding alcohol 8 was transformed into its tosylate derivative 9. Subsequent displacement of this group by lithium-acetylide followed by acidic treatment provided ketone 10 in high yield. [START_REF] Schäfer | Intramolecular Reductive Ketone-Alkynoate Coupling Reaction Promoted by (η 2 -Propene)Titanium[END_REF] This seven step sequence from 2methylcyclopentanone 6 resulted in 50% overall yield thus delivering 24.6 grams (0.15 mol.) of ketone 10. At this stage, two different silyl groups were introduced with the formation of the TBS-based silyl enol ether 5a and the bulkier TIPSderivative 5b, in quantitative yields (Scheme 2).

Scheme 2. Synthesis of acetylenic silyl enol ethers 5a-b. With the substrates 5a-b in hands, the 7-exo-dig cyclization reaction was examined. For practical reasons, an adaptation of Sawamura's procedure 8b to glove-box free conditions for subsequent application on large scale constituted our main objective. Synthesis of the bulky triethynylphosphine 11 was realized in three steps starting from 30 grams of bromophenol derivative in an overall yield of 53% (see Supporting Information). The 7-exo-dig cyclization necessitated an important optimization process. In order to run these reactions on a simple vacuum line, tailor-made Schlenk tubes were designed (see Supporting Information). This specific glassware allowed the in situ generation of the active gold catalyst [( 11)Au]NTf2 without the need for a glovebox at the crucial ligand exchange step. Our study started by investigating the 7-exo-dig cyclization on 0.3 mmol of 5a and 5b; most significant results are combined in Table 1. The first attempt from 5a, at room temperature, in the presence of 5 mol% catalyst and one equivalent of tBuOH in dichloromethane, with a substrate concentration of 0.02 M, resulted in total cleavage of the silyl enol ether function, regenerating ketone 10 (100% conversion, 4/10 8:92, Entry 1). Unlike Sawamura's report, moving toward the TIPSbased silyl enol ether 5b provided better results. Hence, a first trial, at room temperature, resulted in the total conversion of the substrate into the bicyclic target molecule 4 and an isolated yield of 80% was reached (Entry 2). With this good result in hands, concentration was raised to 0.05 M, affording 4 in 92% yield (Entry 3). Further increase in the reaction concentration ([M] = 0.1 M) finally afforded the best result in terms of isolated yield (95%) (Entry 4). Ultimate application of these optimized conditions to a large scale cyclization reaction allowed the transformation to operate from more than 8 g (25 mmol) of TIPS-based substrate 5b, to provide desired bicyclic product 4 in 95% yield (Entry 5). Lastly, in order to investigate the mandatory utilization of the Sawamura phosphine 11, a reaction was carried out with a commonly employed phosphine in Au(I) catalysis, such as JohnPhos (Entry 6). However, a low 10% conversion was observed. Lengthening the reaction time and/or increasing the temperature did not improve the conversion. The functionalization of the bicyclic skeleton 4 constituted the next focus. Several strategies have been studied and we present herein the sequence having allowed the installation of all stereocentres in the most straightforward manner, by taking advantage of the cage bicyclic structure of the molecule, as previously mentioned. Ketone 4 was stereoselectively reduced to corresponding alcohol in 96% yield and the hydroxyl group was protected as a benzyl ether 12. This compound readily underwent allylic oxidation promoted by SeO2 and subsequent Dess-Martin oxidation, affording α,β-unsaturated ketone 13 in 70% yield over two steps. The dihydroxylation reaction at the C3-C18 double bond, performed in the presence of a catalytic quantity of potassium osmate dihydrate (K2OsO4•2H2O), NMO and pyridine, stereoselectively delivered the diol 14 in 90% yield. [START_REF] Hernando | Studies towards the Taxoid Diterpene ABC-Ring System: Practical Access to Highly Functionalized Enantiomerically Pure Analogues of Major Group Representatives[END_REF] The C3 configuration of 14 was elucidated by performing NOE NMR experiments. The excellent substrate stereocontrol in these functionalizations validated our hypothesis regarding the stereocontrolled set-up of the stereogenic centres from enantiomerically enriched bicyclo [4.2.1]nonane skeleton of 4. Acetonide 15 was then readily obtained in 95% yield from diol 14. The introduction of a phenylselenyl group at C10 of 15 and subsequent selenoxide elimination in the presence of H2O2 and pyridine, produced the desired α,βunsaturated ketone 16 in 78% yield over two steps (Scheme 3). 13 Scheme 3. Synthesis of the ketones 17 and 18.

We next focused on the conjugate addition step at C10. Initial attempts to introduce a MOMOCH2 group employing the protocol previously described by Williams in the total synthesis of vibsanin E, proved to be unsuccessful. [START_REF] Schwartz | Asymmetric [4 + 3] Cycloadditions between Vinylcarbenoids and Dienes: Application to the Total Synthesis of the Natural Product (-)-5-epi-Vibsanin E[END_REF] The addition of a cyanide anion was foreseen as a good alternative. Thus, α,βunsaturated ketone 16 was treated with 1.1 equiv. of Et2AlCN (1M sol. in toluene) [START_REF] Tanaka | Synthetic Study of Pyrrocidines: First Entry to the Decahydrofluorene Core of Pyrrocidines[END_REF] at -20 °C. Under these conditions, the desired -cyanoketone 17 was smoothly delivered in 70% yield as a single diastereomer. The configuration of the newly formed C10 stereocentre of 17 was confirmed to be 10(S), i.e. the same configuration as the vibsatins, through NOE NMR experiments. In parallel, a vinyl group was introduced by means of a Lipshutz's modified Gilman cuprate. [START_REF] Lipshutz | Higher Order" Cyanocuprates R2Cu(CN)Li2: Discrete Reagents or "Lower Order" LiCN-Modified Gilman Cuprates?[END_REF] Practically, a mixture of four equivalents of CuCN and eight equivalents of vinyllithium 17 was reacted with 16 at -78 °C for 2 h, affording the required -vinylketone 18 in 70% yield as a single diastereomer. The configuration of the C10 centre was established by NOE NMR experiments and the structure was unambiguously confirmed by X-ray crystallography (Scheme 3); interest of such compound was highlighted below. 18,19 In order to complete the installation of the five stereocentres of the vibsatin skeleton, our attention has been turned to the allylation reaction at the C5 position of ketones 17 and 18. This reaction revealed to be much more challenging than expected. Various trials were attempted to perform the reaction on ketones 17 and 18 and most significant results are presented in Table 2. Noteworthy, only the treatment of 17 with KHMDS (1.5 equiv.)/DMPU (30 equiv.) and quench with allyliodide, at -50 °C for five hours, delivered the desired product 24 albeit in very low yield (15%), along with unreacted starting material 17 (24%) and degradation products (Entry 1). Under more drastic conditions, the product 25 featuring the allyl chain at C5 position but in the opposite configuration was obtained in 30% yield (Entry 2). Only degradation was observed by treatment of 17 with NaHMDS (1.2 equiv.)/DMPU (15 equiv.) (Entry 3). Moving towards LiHMDS/DMPU or LDA/HMPA systems led to the compound 26 with the allyl chain at C10 in -position to the nitrile group (respectively in 70 and 63% yields) (Entries 4 and 5). 20 a: allyl-I (1.5 equiv.) b: [allyl-Br (7 equiv.), TBAI (0.5 equiv.)] instead of allyl-I (1.2 equiv.).

To circumvent these results, we considered vinyl ketone 18 as starting material. 21 Surprisingly, no conversion was observed in the presence of LiHMDS or NaHMDS/DMPU and allyliodide (Entries 6 and 7). Fortunately, by treatment of 18 with LDA/HMPA and allyliodide, the O-allyl enolate 19 was isolated in 53% yield (70% conversion) (Entry 8). 22 Subsequent microwave-assisted Claisen rearrangement in toluene at 190 °C stereoselectively afforded the desired C-allylated product 20 in 89% yield. [START_REF] Schwartz | End Game Strategies towards the Total Synthesis of Vibsanin E, 3-Hydroxyvibsanin E, Furanovibsanin A, and 3-O-Methylfuranovibsanin[END_REF] Noteworthy, this procedure set the presumably most favorable trans-relationship between the vinyl and allyl groups. Then, a Wacker oxidation under optimized conditions allowed the formation of the methyl ketone 21 in 80% yield along with aldehyde 22 (8%). [START_REF] Schwartz | Asymmetric [4 + 3] Cycloadditions between Vinylcarbenoids and Dienes: Application to the Total Synthesis of the Natural Product (-)-5-epi-Vibsanin E[END_REF]24 The configuration at the C5 stereocentre of both compounds 20 and 21 was validated by NOE NMR experiments. Furthermore, the structure of 21 was confirmed by X-ray crystallography which proves the configuration of all stereocentres. 25 Finally, hydrolysis of the acetonide group in acidic medium led to diol 23 in 89% yield (Scheme 4). 26 Scheme 4. Synthesis of the diol 23.

In conclusion, we have developed an efficient method for the asymmetric construction of the bicyclo[4.2.1]nonane core of vibsatin A 1, a new neurotrophic vibsane-type. Thus, a Coniaene derived 7-exo-dig cyclization catalysed by a gold(I) complex permitted the generation of the [5-7]-bicyclic bridged backbone, from an enantiomerically enriched TIPS-based silyl enol ether. The reaction was performed on large scale and constraints formerly associated to glovebox techniques were suppressed. Furthermore, the shape of this framework authorized the installation of all the stereogenic centres without requiring any chiral reagents.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

Experimental details as well as compound characterization data and copies of 1 H and 13 C{1H} NMR spectra are available in the Supporting Information.

Accession Codes

  Luca Allievi, † Sabrina Dhambri, † Rongyu Sun, Mohamed Selkti, Marie-Isabelle Lannou,* Geoffroy Sorin,* Janick Ardisson* UMR CNRS 8038, Faculté de Pharmacie, Université de Paris, 4 avenue de l'Observatoire, 75270 Paris cedex 06 Vibsatin A is a new neurotrophic vibsane-type diterpenoid comprising a bridged bicyclo[4.2.1]nonane skeleton.

Table 1 . 7-exo-dig Cyclization: generation of the catalyst, screening of the substrates and reaction parameters.

 1

	[(11)Au]NTf 2 (5 mol%) tBuOH, MS4Å CH 2 Cl 2 , rt, 5 min 11 (5 mol%) (Me 2 S)AuCl R = TBS 5a R = TIPS 5b (5 mol%) + In situ generation of the	O 4 CH 2 Cl 2 , rt, 30 min Me 2 S catalyst [(	+ (11)AuCl (7.5 mol%) O 10 CH 2 Cl 2 , rt, 10 min AgCl AgNTf 2	[(11)Au]NTf 2

11)Au]NTf 2 11

					P	Si tBu OMe tBu OMe tBu tBu tBu tBu OMe	3
	Entry	Subs-trate	[M]	Conver-sion a (%)	4/10 a	4 (Isol. yield %)
	1	5a b	0.02	100	8:92	-
	2	5b b	0.02	100	≥ 95:5	80
	3	5b b	0.05	100	≥ 95:5	92
	4	5b b	0.10	100	≥ 95:5	95
	5	5b c	0.6	100	≥ 95:5	95
	6 d	5b b	0.05	10	≥ 95:5	-

a: 1 H NMR conversion. b: 0.3 mmol.0 scale. c: 25 mmol. scale. d: JohnPhos was used instead of 11.

Table 2 . Synthesis of the allylated products 24 and 19: Screening of reaction parameters.

 2

ACKNOWLEDGMENT

Université de Paris is acknowledged for a Ph. D. fellowship to Dr. L. Allievi. The authors gratefully thank Dr. C. Meyer (ESPCI, ParisTech, Paris), Dr. C. Pichon and Dr. S. Sengmany (ICMPE, UPEC) for technical support. We would like to thank Dr. R. Guillot from single-crystal X-ray diffraction service ICMMO-UMR 8182 Orsay.

AUTHOR INFORMATION

Corresponding Author * E-mail: marie-isabelle.lannou@parisdescartes.fr. * E-mail: geoffroy.sorin@parisdescartes.fr. * E-mail: janick.ardisson@parisdescartes.fr.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. † Luca Allievi and Sabrina Dhambri contributed equally.

ORCID

Luca Allievi: https://orcid.org/0000-0001-7229-1087 Sabrina Dhambri: https://orcid.org/0000-0002-4742-7787. Marie-Isabelle Lannou: https://orcid.org/0000-0002-3042-2058. Geoffroy Sorin: https://orcid.org/0000-0001-9656-0771. Janick Ardisson: https://orcid.org/0000-0001-5009-1527.

Notes

The authors declare no competing financial interest.


C Biache

Catherine Lorgeoux

Stéfan Colombano

A Saada

P Faure

Multistep thermodesorption coupled with molecular analyses as a quick, easy and environmentally friendly way to measure PAH availability in contaminated soils

Keywords: Gas chromatography, Mass spectrometry, Flame ionization detection, Polycyclic aromatic hydrocarbon, Potassium permanganate oxidation, Microbial incubation

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

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants presenting toxic, mutagenic and carcinogenic properties. They were introduced in the priority pollutant list in the 70s [START_REF] Keith | Priority pollutants I -a perspective view[END_REF] and are, since then, monitored in food and different environmental compartments. The monitoring of PAHs in contaminated matrixes is either for risk assessment purpose or to anticipate the efficiency of remediation treatments. However, in aged materials such as historically contaminated soils, the total (16 listed) PAH concentration is not necessarily representative of the risk inherent to these materials or of the degradable fraction. Indeed, the contaminants can be strongly bound to the matrix through different physicochemical processes resulting in the contamination ageing [START_REF] Alexander | Biodegradation and Bioremediation, second[END_REF][START_REF] Hatzinger | Effect of ageing of chemicals in soil on their bioavailability and extractability[END_REF]. The fraction to consider in these cases is the available fraction as it represents the part of the contamination that is readily accessible for uptake or degradation by living organisms or causing adverse effects to biota [START_REF] Alexander | Aging, bioavailability, and overestimation of risk from environmental pollutants[END_REF][START_REF] Sabat´e | Bioavailability assessment and environmental ˜ fate of polycyclic aromatic hydrocarbons in biostimulated creosote-contaminated soil[END_REF]. The available fraction can also readily react with soil constituents or chemicals. Currently there is no guarantee that these two fractions are identical or if it would be relevant to make the distinction between the bioavailable and the chemoavailable fractions. Indeed, various methods exist to evaluate the bioavailable fraction but, to our knowledge, there is no study dealing with the chemoavailable fraction.

This chemoavailable fraction is particularly relevant for remediation purpose. Indeed, chemical treatments, such as in-situ chemical oxidation, are often used to remediate contaminated soils [START_REF] Baciocchi | Principles, developments and design criteria of in situ chemical oxidation[END_REF]. It is known that the efficiency of such treatments depends on contaminant (chemo) availability and this availability differs according to the oxidant [START_REF] Biache | Effect of pre-heating on the chemical oxidation efficiency: implications for the PAH availability measurement in contaminated soils[END_REF][START_REF] Usman | Effect of thermal pre-treatment on the availability of PAHs for successive chemical oxidation in contaminated soils[END_REF]. The determination of the fraction of the contamination chemoavailable towards a specific oxidant, in a specific matrix, would then allow assessing the remediation potential of this treatment. This would be particularly pertinent for aged contaminated soils where the amount of quantified contaminants may strongly differ from the available contaminants [START_REF] Ouvrard | In situ assessment of phytotechnologies for multicontaminated soil management[END_REF]. The methods existing to predict the bioavailable fraction are classically biological methods [START_REF] Keatinge | Biological tools to assess contaminant bioavailability in soils[END_REF] such as biosensor, biodegradation tests and worm accumulation, however they present some limitations. The restriction of biosensors is linked to the hydrophobicity/low water solubility of PAHs as the tests are performed in liquid (aqueous) media. Organic solvent can be used to enhance the PAH dissolution but (i) the dissolved PAHs would not necessarily reflect the available fraction and (ii) organic solvents usually have negative effect on the biosensor [START_REF] Kohlmeier | Comparison of naphthalene bioavailability determined by whole-cell biosensing and availability determined by extraction with Tenax[END_REF]. An alternative is to use microbial incubation which is a good option to assess the bioremediation potential [START_REF] Braida | Indices for bioavailability and biotransformation potential of contaminants in soils[END_REF][START_REF] Chung | Differences in sequestration and bioavailability of organic compounds aged in dissimilar soils[END_REF]. For risk assessment purpose, the earthworm uptake tests are generally preferred [START_REF] Stroo | Environmentally acceptable endpoints for PAHs at a manufactured gas plant site[END_REF]. However these biological method are costly, timeconsuming and can be difficult to set up as the survival of the organisms will depend on several parameters, including the level of exposure which, if too high, can induce a toxicity and have lethal effect on the tested organisms [START_REF] Guo | Polycyclic aromatic hydrocarbons bioavailability in industrial and agricultural soils: linking SPME and Tenax extraction with bioassays[END_REF]. To overcome these limitations, chemical methods have been proposed. Among them are mild-solvent extractions using solvent and solvent mixture such as butanol, methanol or water/methanol [START_REF] Kelsey | Selective chemical extractants to predict bioavailability of soil-aged organic chemicals[END_REF][START_REF] Liste | Butanol extraction to predict bioavailability of PAHs in soil[END_REF]. Passive samplers are also used as bioavailability measurement [START_REF] Hale | Changes in polycyclic aromatic hydrocarbon availability in River Tyne sediment following bioremediation treatments or activated carbon amendment[END_REF][START_REF] Tao | Predicting bioavailability of PAHs in field-contaminated soils by passive sampling with triolein embedded cellulose acetate membranes[END_REF] as they are supposed to mimic the biomembrane and measure the freely dissolved compounds without significantly affecting the soil-water or sediment-water equilibrium [START_REF] Macrae | Comparison of methods used to determine the availability of polycyclic aromatic hydrocarbons in marine sediment[END_REF]. Other methods are based on the partitioning between sediments or soils and water with an extractant acting as infinite sink such as cyclodextrin and Tenax [START_REF] Puglisi | Non-exhaustive extraction techniques (NEETs) for bioavailability assessment of organic hydrophobic compounds in soils[END_REF]. Cyclodextrin has hydrophilic surface and an internal hydrophobic cavity that makes the molecule fairly stable in water and able to entrap hydrophobic molecules in the cavity. Tenax is a resin that adsorbs the PAHs from the water phase. Both cyclodextrin and Tenax based methods were standardized for the determination of the environmental availability for non-polar organics [START_REF]-Qualité du sol -Disponibilité environnementale des composés organiques non polaires -Détermination de la fraction potentiellement biodisponible et de la fraction non biodisponible en utilisant un agent adsorbant fort ou un agent complexant[END_REF]. Some studies comparing bioavailability measurements reported consistent results obtained with both chemical and biological methods [START_REF] Kohlmeier | Comparison of naphthalene bioavailability determined by whole-cell biosensing and availability determined by extraction with Tenax[END_REF][START_REF] Liste | Butanol extraction to predict bioavailability of PAHs in soil[END_REF][START_REF] Reid | Nonexhaustive cyclodextrin-based extraction technique for the evaluation of PAH bioavailability[END_REF] but they were performed on spiked soils or sediments and considered only a limited number of PAHs. Consequently, they were not representative of real contaminated soils and sediments that (i) are often contaminated by a wide range of PAHs and (ii) underwent ageing process that influences the behavior of PAHs during these bioavailability tests.

Other studies aiming at comparing several bioavailability determination methods on real contaminated soils and sediments showed that results were not consistent [START_REF] Macrae | Comparison of methods used to determine the availability of polycyclic aromatic hydrocarbons in marine sediment[END_REF][START_REF] Barthe | Passive samplers versus ´ surfactant extraction for the evaluation of PAH availability in sediments with variable levels of contamination[END_REF][START_REF] Bergknut | Comparison of techniques for estimating PAH bioavailability: uptake in Eisenia fetida, passive samplers and leaching using various solvents and additives[END_REF][START_REF] Swindell | Comparison of selected non-exhaustive extraction techniques to assess PAH availability in dissimilar soils[END_REF]. It is not surprising as the available fraction differs according to the targeted organisms or even species as they are exposed from different routes [START_REF] Reid | Bioavailability of persistent organic pollutants in soils and sediments-a persective on mechanisms, consequences and assessment[END_REF], so one chemical method cannot measure several bioavailabilities. However, the discrepancies in the results presented in these studies, especially the ones confronting biological and chemical methods, underline the necessity to propose a standardized way to measure availability. In this study we propose a new method based on thermodesorption (Td) coupled to molecular analyses to determine PAH availability in soils. Few studied explored the potential of thermal methods to detect PAHs in environmental matrices [START_REF] Rumpel | Composition and reactivity of morphologically distinct charred materials left after slash-and-burn practices in agricultural tropical soils[END_REF][START_REF] Quenéa | Double-shot pyrolysis of the non-hydrolysable organic fraction isolated from a sandy forest soil (Landes de Gascogne, South-West France): comparison with classical Curie point pyrolysis[END_REF][START_REF] Gonzalez-Pérez | Appraisal of polycyclic aromatic hydrocarbons (PAHs) in environmental matrices by analytical pyrolysis (Py-GC/MS)[END_REF] but these methods did not enable PAH quantification. More recently, a study proposed a new method for PAH quantification using pyrolysis-gas chromatography-mass spectrometry/flame ionization detection (Py-GC-MS/FID) coupling [START_REF] Biache | Fast method to quantify PAHs in contaminated soils by direct thermodesorption using analytical pyrolysis[END_REF]. This method consists in direct thermodesorption at 450 • C, which is a good compromise between a temperature high enough to desorb heavier PAHs or PAHs associated with matrices with high retention properties and a temperature low enough to prevent thermal cracking of the soil organic matter. In this study, a link between the aging of the contaminated soil and the ability to thermodesorb PAHs is suspected. Indeed, it can be admitted that there is a link between the availability of a compound, the strength to which this compound is retained to the matrix and the energy required to break this bond [START_REF] Biache | Behavior of PAH/mineral associations during thermodesorption: impact for the determination of mineral retention properties towards PAHs[END_REF]. The increase in the temperature would bring higher energy capable of desorbing molecules bound with increasing strength. Therefore, it could be possible to define for a given molecule the desorption temperature corresponding to different levels of availabilities (e.g. bioavailability and chemo availability). The aim of this study was then to determine the PAH thermodesorption profiles of several contaminated soil using the Td-GC-MS/FID coupling with several desorption steps and to compare them to the efficiency of several remediation treatments (microbial incubations, chemical oxidations) in order to define the temperatures corresponding to the PAH available fractions. This work will allow (i) determining if there are several availabilities according to the treatment (biological or chemical) and (ii) proposing a method able to predict the efficiency of different treatments in one set of analysis.

Materials and methods

Soil samples

Soils were collected in (i) three former coking plants, Cok.P-1 (Neuves-Maisons, France), Cok.P-2 and Cok.P-4 (Homécourt, France) and Cok.P-3 in confidential site (Lorraine Region -France), (ii) one former gas plant: GasP (Rennes -France) and (iii) one active wood-treating facility: WoodTF (Midi-Pyrenées Region -France). After sampling, soils were stored at -18 • C before being freeze-dried. Samples were then sieved (2 mm) and the undersize was crushed (<500 μm). After homogenization, a sub-fraction of the crushed samples was grinded more finely for Td analyses (<100 μm). Indeed, as shown in our previous study [START_REF] Biache | Fast method to quantify PAHs in contaminated soils by direct thermodesorption using analytical pyrolysis[END_REF], grinding the sample to such particle size allow to greatly decrease the variability in the measure.

Thermodesorption (Td) analyses

Thermodesorption were carried out on a Frontier Lab Multi-shot pyrolyzer EGA/PY-3030D and a Micro Jet Cryo-Trap MJT-1030Ex installed on an Agilent GC-FID 7890B coupled with a 5977A MS detector. The GC was equipped with an Agilent DB-5MS column (20 m × 0.18 mm i.d. × 0.18 μm film thickness). The carrier gas was helium at a constant flow of 1.6 mL/min. Deactivated fused silica columns connected both detectors to a dean switch allowing the column flow split between the detectors with a FID/MS flow separation of 1/3. The pulsed split mode was used with pulsed pressure of 50 psi until 0.5 min and a split ratio of 20:1. The inlet and transfer line temperatures were set at 320 and 340 • C, respectively and the MS quadrupole and ion source were set at 150 and 230 • C, respectively. For all analyses the MS was used in the combined selected ion monitoring (SIM)/full scan mode (Table S1). The FID temperature was set at 320 • C and the air, hydrogen and makeup (He) flows were 400 mL/min, 30 mL/min and 25 mL/min, respectively. The FID was calibrated for the quantification of 15 PAHs (Table 1) according to Biache et al. [START_REF] Biache | Fast method to quantify PAHs in contaminated soils by direct thermodesorption using analytical pyrolysis[END_REF]. Briefly, solventwashed pieces of glass fiber filter were placed into stainless steel cups and impregnated with 1 μL of calibration solutions at nine concentrations ranging from 0.5 to 100 μg/mL. The cups were then inserted in the Td oven set at 450 •C. The temperature of the pyrolyzer interface was maintained at 320 •C. Compounds were concentrated at the head of the column thanks to a cryo trap held at -180 •C during 5 min before the beginning of the GC analysis according to the following oven temperature program: For the soil Td analyses, samples (0.2-5 mg according to the PAH content) were placed into stainless steel cups which were inserted into the Td oven. To obtain a reference thermodesorption profile corresponding to the maximum available fraction, the extractable organic matter (EOM) of the untreated soil were analyzed as well by impregnating glass fiber filters placed in stainless steel cups with 1-4 μL of EOM. Samples were then heated according to seven successive 50•C-Td steps from 100 to 450 •C (i.e. from 100 to 150 •C, then from 150 to 200 •C, then from 200 to 250 •C, and so on) at 10 •C/min.

As discussed in a previous study [START_REF] Biache | Fast method to quantify PAHs in contaminated soils by direct thermodesorption using analytical pyrolysis[END_REF], the selection of the maximum temperature was done in order to allow the vaporization of higher boiling point compounds but to avoid thermal cracking and aromatization reactions. Maximal temperature of 450 •C happened to be a good compromise. Even if 450 •C is consistent with pyrolysis, the aim of using such temperature was not to induce thermal cracking but the desorption of HMW PAH, that is why the term for the used technique is referred as "thermodesorption" throughout this paper.

The cryo trap was maintained at -180 •C during the Td steps, which were each followed by GC analysis runs according to the parameters described above. Finally, seven chromatograms were generated for each sample, each chromatogram corresponding to a 50 •C-temperature range. Td were done in triplicate for each soil sample.

Compound identification was confirmed by the mass spectra obtained with the MS detector in comparison with the NIST08 and Wiley database spectra, and calibrated PAHs were quantified on the FID response.

KMnO4 oxidation

Procedures for chemical oxidation were detailed in previous studies [START_REF] Biache | Effect of pre-heating on the chemical oxidation efficiency: implications for the PAH availability measurement in contaminated soils[END_REF][START_REF] Ranc | Comparison of the effectiveness of soil heating prior or during in situ chemical oxidation (ISCO) of aged PAH-contaminated soils[END_REF]. Briefly, KMnO4 treatments were applied on sieved and crushed (<500 μm) samples in order to reach at least twice the stoichiometric oxidant demand (SOD) based on the extractable organic matter (EOM) content according to Ranc et al. [START_REF] Ranc | Selection of oxidant doses for in situ chemical oxidation of soils contaminated by polycyclic aromatic hydrocarbons (PAHs): a review[END_REF]. The following amounts of KMnO4 were used for 1 g of soil: 0.3340 g for Cok-P.1, 0.4451 for Cok.P-3, 3.112 for Cok-P4, 0.3340 g for Gas-P and 7.4300 g for WoodTF. Samples were agitated (200 rpm) in the dark for one week.

Bioremediation experiment

Bioslurry experiments were described in detail in previous studies [START_REF] Biache | Bioremediation of PAH-contaminated soils: consequences on formation and degradation of polar-polycyclic aromatic compounds and microbial community abundance[END_REF][START_REF] Biache | Biodegradation of the organic matter in a coking plant soil and its main constituents[END_REF]. Briefly, inocula obtained from the "fresh" WoodTF, Cok.P-1, Cok.P-2 and GasP soils were added to their respective freeze-dried and crushed (<500 μm) soils with a nutritive solution (Bushnell Hass). The flasks containing the slurry were placed in an incubation chamber at 28 • C in the dark under continuous stirring (120 rpm). Samples were collected after 5 months (Cok.P-1 soil, GasP and WoodTF soils) and 9 months of experiment (Cok.P-2 soil) and were freeze-dried before analysis.

Extraction of OM and PAH quantification

In order to determine the efficiency of the oxidations and bioremediation experiments, PAHs were quantified before and after treatments.

First, EOM was obtained by extracting 1.5-2 g of dw soil using an accelerated solvent extractor (ASE 350, Dionex) with dichloromethane (130 • C, 100 bars, 10 min). Molecular sulfur and residual water were removed by adding to the extraction cells activated copper powder (2 g) and sodium sulfate (2 g), respectively. The extract volume was adjusted to 20 mL in volumetric flask after solvent evaporation under a gentle nitrogen stream. An aliquot (3 mL) was transferred into pre-weighed vials. The EOM content was determined by weighing the vial after solvent evaporation to dryness.

Quantification of 15 regulatory PAHs was reported in previous studies [START_REF] Biache | Effect of pre-heating on the chemical oxidation efficiency: implications for the PAH availability measurement in contaminated soils[END_REF][START_REF] Biache | Bioremediation of PAH-contaminated soils: consequences on formation and degradation of polar-polycyclic aromatic compounds and microbial community abundance[END_REF]. Briefly, it was carried out using internal calibration. An internal PAH standard mix of [ 2 H8]naphthalene, [ 2 H10]acenaphthene, [ 2 H10]phenanthrene, [ 2 H12]chrysene, [ 2 H12]perylene, (Cluzeau®, 20 μL at 16 μg/mL) was added to 80 μL of the EOM before being injected in a gas chromatograph coupled with a mass spectrometer (GC-MS). For each quantified compound, internal calibration curve was drawn with six concentrations (0.3, 0.9, 1.5, 3, 6 and 9 μg/mL). Two GC-MS instruments were used: Shimadzu system for samples from bioslurry experiment and Agilent system for samples from KMnO4 oxidation. Untreated samples were analyzed with both systems. The gas chromatographs (GC) were a Shimadzu GC-2010 plus and an Agilent 6890, equipped with a capillary column in silica glass DB-5MS (60 m × 0.25 mm i.d. × 0.1 μm film thickness and 60 m × 0.25 mm i. d. × 0.25 μm film thickness, respectively) coupled to a QP2010-Ultra (Shimadzu) electronic ionization mass spectrometer (MS) and an Agilent 5973 Inert Mass Selective Detector, respectively, operating at 70 eV. The Shimadzu system operated in alternated fullscan/selected ion monitoring mode and the Agilent system operated in fullscan mode, and both had their transfer line heated at 300 • C. The oven temperature program was as follow: 70 • C for 2 min, from 70 to 130 • C at 15 • C/min, then from 130 to 315 • C at 3 • C/min and then a 15 min hold at 315 • C. The carrier gas was helium at 1.4 mL/min constant flow. Injections were done with one microliter in split mode with a split ratio of 1:5 for the Shimadzu and in splitless mode for the Agilent system.

Statistical analyses

The PAH concentrations obtained during the thermodesorption steps and degraded by the remediation treatments were subjected to principal component analyses (PCA) to investigate their relationships and similarities. PCA calculates new variables or principal components, which are linear combinations of the original variables accounting for as much of the variance of the original data as possible [START_REF] Ramette | Multivariate analyses in microbial ecology[END_REF]. Principal component analysis was realized using the software XLStat (Addinsoft, 2018) with the PAH concentrations obtained during the different thermodesorption steps and the amount of PAHs degraded during the microbial incubation and chemical treatment. The PCA was performed using a covariance matrix. A PCA was obtained for each of the studied 15 PAHs, the different soil samples constituted the variables and the thermodesorbed concentrations and amount of PAHs degraded during the remediation treatments were the individuals used for the PCA.

Results and discussion

Are the thermodesorption profiles dependent on the solid matrix?

In order to confirm that the PAH desorption temperatures are related to the bond between the compound and the matrix, thermodesorption profiles of the studied soils were compared to their corresponding EOM from which PAHs are classically quantified (Fig. 1). For both types of sample, PAHs with higher boiling points (and molecular weights) are desorbed at higher temperatures (boiling points of the studied PAHs are reported in Table S1). For the EOM, thermodesorption profiles were similar for the studied samples. Most of the PAHs were desorbed at 150-200 • C and 250 • C for the PAHs with molecular weights up to 202 g/mol and higher than 228 g/mol, respectively. These desorption temperatures were comparable to those obtained with a standard PAH mixture (i.e. solution used for calibration; Figure S1). These observations seem to indicate that whatever the initial soil composition, the PAH desorption temperatures from the EOM are similar. However, Cok.P-2 and Cok.P-3 EOM thermodesorption profiles showed some particularities as low molecular weight (LMW) PAH kept being released at higher temperatures, especially for Cok.P-2. This behavior could indicate a cracking of heavy compounds/macromolecules. This hypothesis tends to be supported by the fact that Cok.P-2 EOM present a high proportion of asphaltenes (69%) compared to the other samples (from 25 to 43%) that could be responsible for the LMW PAH release at high temperature (Table S2) due to thermal cracking. This assumption if confirmed by the fact that the amounts of these LMW PAHs were higher for EOM Td analysis than for classical EOM GC-MS analysis (Table S3).

Are the PAH thermodesorption profiles indicators of the contaminant status?

Differences between soil behaviors were observed with contrasted profiles obtained with successive thermodesorptions (Fig. 1). In some cases (e.g. GasP, WoodTF, Cok.P-4), PAHs were mostly desorbed between 100 and 250 •C, and between 250 and 350 •C for LMW and high molecular weight (HMW) compounds, respectively. In other cases (e.g. Cok.P-1, Cok. P-2, Cok.P-3), LMW PAHs were desorbed mostly between 200 and 350 •C and HMW PAHs between 300 and 450 •C. These differences in thermodesorption temperatures can be linked to PAH status, i.e. their availability. Indeed, availability level can be associated with the level of energy necessary to break the bond between the PAH and the matrix and therefore to release it, and this energy can be brought by increasing temperature. The studied soils are known to exhibit contrasted availability levels as they responded differently to various remediation treatments. The contrasted thermodesorption profile can then be linked to the contrasted response to treatments according to the soil sample. For instance, when applied to Cok.P-1, bioremediation treatment [START_REF] Biache | Bioremediation of PAH-contaminated soils: consequences on formation and degradation of polar-polycyclic aromatic compounds and microbial community abundance[END_REF] and Fenton-like oxidation [START_REF] Biache | Effect of pre-heating on the chemical oxidation efficiency: implications for the PAH availability measurement in contaminated soils[END_REF][START_REF] Usman | Effect of thermal pre-treatment on the availability of PAHs for successive chemical oxidation in contaminated soils[END_REF][START_REF] Usman | Remediation of PAH-contaminated soils by magnetite catalyzed Fenton-like oxidation[END_REF] were not effective to remove PAH contamination as they resulted in low PAH degradation in both cases (34% and 0% of PAH degradation, respectively). On the contrary, PAHs from GasP were easily degraded by microbial incubation (76% PAH degradation) and Fenton-like oxidation (78% PAH degradation). From these studies, it can be established that the response of the soil contamination towards the remediation treatment also depends on the type of treatment applied. Indeed, for the same soil, different treatments lead to different levels of contamination degradation. For instance, application of potassium permanganate to Cok.P-1 soil lead to a PAH degradation of 52% whereas no PAH decrease was observed with Fenton-like treatment [START_REF] Biache | Effect of pre-heating on the chemical oxidation efficiency: implications for the PAH availability measurement in contaminated soils[END_REF]. Therefore, the fraction of the contaminants available for potassium permanganate oxidation is much more important than the fraction available for Fenton-like treatment. Consequently, there are as many availabilities as remediation treatments; in this example we can consider the fraction available for microbial degradation, the fraction available for Fenton-like oxidation and the fraction available for potassium permanganate. The soil showing lower PAH desorption temperature (i.e. gas plant soil) also shows higher contaminant availability towards the considered treatments.

Determination of the desorption temperatures corresponding to the available fractions

Principal component analyses were done for each compound and each treatment to compare the cumulated PAH concentrations obtained during the thermodesorption steps and the PAHs actually degraded during the treatments in order to determine the desorption temperature leading to the release of the bio-and chemoavailable PAHs. For all PCAs the first two components explained more than 90% of the total variance (Table S4), with the first component (F1) contributing for more than 77% on average. A regression analysis was performed after plotting the desorption temperatures as a function of F1 coordinates of the thermodesorption individuals (Figs. 2 and3) and the curve equation with the determination coefficient determined. The curve equation was then used to calculate the desorption temperature corresponding to the F1 coordinates of the individuals from the remediation experiments. In other words, for each PAH and each treatment, the x variable of the equation was replaced by the F1 coordinates of the remediation treatment in order to obtain the desorption temperature corresponding to the degraded fraction of the PAH during the remediation treatments (Table 1). As thermodesorptions were performed according to 50 • Csteps, desorption temperatures corresponding to the available fractions were given as 50 • C-windows which will be further referred as low and high availability estimations (Table 1).

Overall, the temperatures corresponding to the bioavailability estimation were higher than those of the chemoavailability estimation for LMW compounds, and lower for HMW compounds. It is consistent with the fact that (i) microorganisms degrade more easily LMW compounds than HMW compounds [START_REF] Cerniglia | Biodegradation of polycyclic aromatic hydrocarbons[END_REF] and (ii) KMnO4 degrade nonselectively LMW and HMW compounds [START_REF] Biache | Effect of pre-heating on the chemical oxidation efficiency: implications for the PAH availability measurement in contaminated soils[END_REF].

Method application

The different availabilities were determined for the six studied soils according to the temperature ranges presented in Table 1 and compared to the PAH amounts actually degraded by biological (microbial incubation) (Fig. 4) and chemical (KMnO4 oxidations) treatments (Fig. 5). fractions are reported as ranges with low (in grey) and high (in black) estimations obtained with the thermodesorption temperatures reported in Table 1. See Table 1 for abbreviation meanings. The error bars represent the SD (n = 3).

Overall the thermodesorption method gave satisfactory results for the estimation of PAH availability. Indeed, using the high availability range values, the Td methods gave a good estimation, i.e. 86% and 88%, of what was actually degraded during microbial incubation of the GasP and WoodTF soils, respectively. In the same way, Td method estimation for KMnO4 treatment efficiency reach 80% and 95% for the same samples, with the high availability estimation.

The low availability estimation fitted better with the chemically degraded and biodegraded PAHs for Cok.P-3 (146% of the degraded fraction) and Cok.P-2 soils (179% of the degraded fraction), respectively. The overestimation of the Cok.P-2 bioavailability is mainly due to high values obtained for LMW compounds, probably due to a contribution of thermal degradation of asphaltenes and heavier fractions. Indeed, as previously mentioned, this sample exhibit a high content of asphaltenes (69% of the EOM). When considering PAHs with ring number of four or higher, the estimation satisfactorily reached 112% of the biodegraded fraction. For the Cok.P-4 sample, the fraction available for KMnO4 oxidation was overestimated as less PAHs were degraded by KMnO4 oxidation than those predicted by the Td method, even when considering the low availability estimation.

Using the Td method to predict the chemo-and bioavailable PAHs of Cok.P-1 led to an underestimation of this fraction in both cases. During the validation of a method for PAH quantification by flash-thermodesorption (450 • C) coupled with molecular analysis [START_REF] Biache | Fast method to quantify PAHs in contaminated soils by direct thermodesorption using analytical pyrolysis[END_REF], issues were already encountered with this soil. Indeed, the PAHs quantified by flash-Td were lower than the PAHs quantified using traditional method (solvent extraction and GC-MS analyses). As already mentioned, Cok.P-1 soil is an aged contaminated soil and is known to present low PAH availability [START_REF] Biache | Effect of pre-heating on the chemical oxidation efficiency: implications for the PAH availability measurement in contaminated soils[END_REF][START_REF] Usman | Effect of thermal pre-treatment on the availability of PAHs for successive chemical oxidation in contaminated soils[END_REF][START_REF] Ouvrard | In situ assessment of phytotechnologies for multicontaminated soil management[END_REF][START_REF] Biache | Bioremediation of PAH-contaminated soils: consequences on formation and degradation of polar-polycyclic aromatic compounds and microbial community abundance[END_REF][START_REF] Usman | Remediation of PAH-contaminated soils by magnetite catalyzed Fenton-like oxidation[END_REF][START_REF] Cébron | Experimental increase in availability of a PAH complex organic contamination from an aged contaminated soil: consequences on biodegradation[END_REF][START_REF] Pernot | Protective role of fine silts for PAH in a former industrial soil[END_REF] probably because of strong PAH-matrix bonds resulting in limited PAH thermodesorption, even at 450 • C. Because of its persistence, KMnO4 can penetrate deeper into the soil particles [START_REF] Huling | In-situ Chemical Oxidation[END_REF], access and degrade PAHs that cannot be released with Td, even at 450 • C. The high level of PAH sequestration can also affect the bioavailability estimation. However, in previous studies bioavailability measurements were carried out on Cok.P-1 using both standardized chemical methods (i.e. Tenax and cyclodextrin extractions) Cebron et al. [´ 40] reported a value of 12 mg/kg for the sum of 15 PAHs (the listed PAHs without the naphthalene) obtained after Tenax extraction. In the same way, Ouvrard et al. [START_REF] Ouvrard | In situ assessment of phytotechnologies for multicontaminated soil management[END_REF] measured a PAH available fraction (sum of the16 listed PAHs) of 16 mg/kg using also Tenax extraction. Finally, Bourceret et al. [START_REF] Bourceret | Mapping the centimeter-scale spatial variability of PAHs and microbial populations in the rhizosphere of two plants[END_REF] estimated the PAH bioavailable fraction using cyclodextrine extraction and obtained a value of 40 mg/kg for the sum of 15 PAHs (listed PAHs without the naphthalene). With the Td method, the amount of bioavailable PAHs (16 listed PAHs without naphthalene) was estimated at 165 and 217 mg/kg, for low and high availability, respectively. If these values remained lower than the actually biodegraded PAHs (413 mg/kg), they are still more realistic than the one obtained with Tenax and cyclodextrine that represented only 3% and 10% of biodegraded PAHs, respectively.

Conclusion

A new method, based on Td coupled with molecular analyses, was proposed to estimate the availability of PAHs in several contaminated soils towards chemical (KMnO4) and biological (microbial incubation) treatments. As mentioned and already demonstrated in previous studies (aforesaid references), the efficiency of chemical (KMnO4 oxidation) and biological (microbial incubation) remediation treatment differs. Consequently, a distinction can be made between the bio -and the chemo-availabilities.

The comparison between the chemo-available fraction for permanganate potassium treatments determined by Td and the PAH fractions actually degraded during these treatments showed a good match except for two soils. In one case, Cok.P-4, the estimated available fractions were higher than the degraded fraction. For the other sample, Cok.P-1, the chemoavailability measurements underestimated the fraction really degraded, and it was also the case for the bioavailability estimation performed on this soil. PAHs in this soil are probably highly sequestrated and the maximum desorption temperature was not able to released them from the matrix, leading to this underestimation. However, comparison with bioavailability measurements performed with other methods (cyclodextrin and Tenax extractions) showed than even if the Td method underestimated the available fraction, the obtained results remained much closer to reality than the one obtained using "traditional" methods. For the other tested soils, the estimation of bioavailable PAHs by thermodesorption matched the amount of biodegraded PAHs. From these results we can conclude that this method was proven satisfactory for the estimation of PAH bio-and chemo-availability. It could then be used (i) prior the application of remediation treatments in order to determine the best way to manage the contamination, (ii) during the application of remediation treatment to monitor the degradation of available PAHs, and (iii) after remediation to ensure that the degradation of the available fraction is complete and/or the applied dose of oxidant was sufficient to degrade it.

The main advantages of this method are its quickness as it takes less than 4 h to obtain a thermodesorption profile, and the fact that one set of analysis allows to estimate at the same time the chemo-and the bio-availabilities. Another benefit is the fact that this method is environment-friendly since it is solvent free.

In this study, even if the concentration range varied from 917 to 13000 μg/g of 15 PAHs (16 listed PAHs without naphthalene -Table S3), these soils present relatively high contamination level. It would be now interesting to test our method on less impacted soils and to other contaminated matrixes such as sediments. It is also planned to broaden the comparison with other methods classically used to determine the available fraction on a wider range of soils. As this method is adapted even to compounds with high boiling points such as HMW PAHs (e.g. dibenzo[a,h]anthracene boiling point = 797 • C [44]), we can also consider extending and adapting this method to other organic contaminants such as petroleum hydrocarbons.

  1 min at 40 •C, from 40 to 195 •C at 25 •C/min, a hold-time of 2 min at 195 •C, from 195 to 315 •C at 20 •C/min, from 315 to 320 •C at 5 •C/min followed by a hold-time of 3 min at 320 •C.

Fig. 1 :

 1 Fig. 1: Thermodesorption profiles (i.e. cumulated PAHs quantified during the seven 50 • C-thermodesorption steps from 100 • C to 450 • C) for the six studied soil samples and their extractable organic matter (EOM). SeeTable 1 for PAH abbreviation meanings.

Fig. 2 :

 2 Fig. 2: Plot of the desorption temperature as a function of the F1 coordinate of the principal component analysis for the 15 PAHs degraded during microbial incubation. The grey areas have been added to facilitate the reading of the desorption temperature of the incubated samples.

Fig. 3 :

 3 Fig. 3: Plot of the desorption temperature as a function of the F1 coordinate of the principal component analysis for the 15PAHs degraded during KMnO4 oxidation. The grey areas have been added to facilitate the reading of the desorption temperature of the oxidized samples.

Fig. 4 .

 4 Fig. 4. Bioavailable PAHs estimated by thermodesorption compared to PAHs quantified in the initial samples and the actually degraded PAHs during microbial incubation for Cok.P-1, Cok.P-2, GasP and WoodTF soil samples. Available fractions are reported as ranges with low (in grey) and high (in black) estimations obtained with the thermodesorption temperaturesreported in Table1. See Table1for abbreviation meanings. The error bars represent the SD (n = 3).

Fig. 5 :

 5 Fig. 5: Chemoavailable (KMnO4 oxidation) PAHs estimated by thermodesorption compared to the initial samples and the actually degraded PAHs during microbial incubation for Cok.P-1, Cok.P-3, Cok.P-4, GasP and WoodTF soil samples. Availablefractions are reported as ranges with low (in grey) and high (in black) estimations obtained with the thermodesorption temperatures reported in Table1. See Table1for abbreviation meanings. The error bars represent the SD (n = 3).

Table 1 :

 1 List of the analyzed PAHs with their abbreviations and the minimal and maximal thermodesorption temperatures corresponding to the low and high available fractions towards microbial incubation and permanganate oxidation treatments.

			Biodegradation	KMnO4
	PAHs	Abbreviations Low	High	Low	High
	Acenaphtylene	Acy	250	300	250	300
	Acenaphthene	Ace	250	300	200	250
	Fluorene	Flu	400	450	300	350
	Phenanthrene	Phe	400	450	200	250
	Anthracene	Ant	350	400	300	350
	Fluoranthene	Fluo	400	450	250	300
	Pyrene	Py	400	450	350	400
	Benz[a]anthracene	B(a)A	400	450	400	450
	Chrysene	Chry	350	400	300	350
	Benzo[b]fluranthene	B(b)F	300	350	300	350
	Benzo[k]fluoranthene B(k)F	350	400	350	400
	Benz[a]pyrene	B(a)P	350	400	400	450
	Indeno[1,2,3-cd]pyrene IP	200	250	300	350
	Dibenzo[a,h]anthracene DB(ah)A	400	450	400	450
	Benzo[ghi]perylene	B(ghi)P	100	150	400	450

Table 1 for

 1 PAH abbreviation meanings.

	6

Table

Acknowledgements

This study was funded by the Lorraine Energy and Environment Carnot Institute (ICEEL) and the French Geological Survey (BRGM) (Grant numbers: 1.12Action54OTELoMACRODISP and CR213/233-BFC30033895, respectively). This work is included in the scientific program of the GISFI research consortium dedicated to the knowledge and the development on remediation technologies for degraded and polluted lands (Groupement d'Intérêt Scientifique sur les Friches Industrielles -http://www.gisfi.univ-lorraine.fr). We also thank Dr Marc Offroy for his expertise and valuable advises on multivariate analysis and Axel Bart from SRA for technical support.

Appendix A. Supplementary data


Florent Bodinier

Youssouf Sanogo

Janick Ardisson

Marie-Isabelle Lannou

Geoffroy Sorin

Low-valent dialkoxytitanium(II): a

come

Herein, we describe an unprecedented access to all-carbon or heterocyclic seven-membered ring frameworks from 1,8-ene-ynes promoted by inexpensive low-valent titanium(II) species, readily available from Ti(OiPr)4 and Grignard reagent. A broad range of cycloheptane, azepane or oxepane derivatives has been obtained (19 examples) with moderate to good yields and an excellent selectivity (up to 95/5 d.r.).

Natural products synthesis is a tremendous source of inspiration and creativity. Faced with the molecular complexity and diversity encountered in nature, a synthetic chemist has to find innovative tools to tackle these problems. [1] In this area, the construction of seven-membered ring frameworks, which are represented in numerous natural compounds or pharmaceuticals (Fig. 1), appears to be a compelling challenge. [2] Fig. 1 Natural compounds containing a seven-membered ring framework Ring expansions [START_REF] Bastos Lemos | [END_REF] or ring closing reactions mediated by a transition metal catalyst are among the classical methods employed for the access to such structures. For example, gold, [START_REF]From gold catalyst see for example[END_REF] ruthenium, [START_REF]From ruthenium catalyst see for example[END_REF] palladium, [START_REF] Peshkov | From palladium catalyst see for example[END_REF] rhodium [START_REF] Tap | From rhodium catalyst see for example[END_REF] or iridium [START_REF] Fernández | From iridium catalyst see for example[END_REF] catalysts have already proven their efficiency in the construction of sevenmembered rings. Even though these processes allow a straightforward access to such cycles, they often require an expensive catalyst, tedious reaction conditions, or sophisticated precursors involving multistep sequences. Regarding the available methods, for economic and chemoselectivity issues as well as environmental concerns, designing new routes to sevenmembered ring adducts remains a significant challenge.

Titanium is a very abundant element in nature (9 th of the Earth's crust). In addition to its low cost, commercially available titanium alkoxides are non-toxic and eco-friendly. Since the beginning of the 1990s, chemistry of low-valent dialkoxytitanium(II) has known significant advances thanks to the Kulinkovich cyclopropanation. [9] It was further established that Ti(II) divalent complexes were also suitable for promoting various reductive cyclization reactions, [START_REF]For a review, see for example[END_REF] leading to five-and six-membered rings by a ligand exchange occurring at the preliminary step with -systems, for example alkynes. [START_REF]For various representative examples[END_REF] More recently, first results mentioning low-valent titanium(II) mediated seven-membered rings syntheses were published. Hence, in 2001, Sato et al. reported an intramolecular reductive alkyne-alkyne coupling leading to a fused [4-7] bicycle. [12] Micalizio and co-workers published, in 2019, an elegant transformation involving low-valent dialkoxytitanium (II) for an access to highly functionalized fused polycyclic structures. [13] In this manner, several types of polycycles, including sevenmembered rings, are thus reachable through a tandem annulative process between an alkyne and two ketones (Scheme 1). Scheme 1 Titanium(II) mediated synthesis of seven-membered rings Inspired by the seminal works of Sato on various cyclization reactions involving ene-yne systems leading to five-and sixmembered rings [START_REF]For some representative examples[END_REF] we were delighted to discover, in the course of total synthesis of thapsigargin core, that Ti(II) was able to promote the diastereoselective formation of seven-membered rings through a reductive coupling process between an alkyne and an alkene. [15] Encouraged by this preliminary result, we decided to pursue our investigations in order to determine the scope and limitations of such a transformation (Scheme 2). We started by the synthesis of the racemic cyclization precursor 1 from phthalic anhydride. After a seven steps linear sequence, the appropriate 1,8-alkene-yne system was obtained. [START_REF]For the synthesis of cyclization precursors, see S.I. 17 For complete survey on optimization reaction[END_REF] With the support of our preliminary result, [15] we first investigated the best conditions to promote this cyclization (Table 1). [17] Experimental procedure involves three steps : the first one corresponds to the deprotonation of free alcohol function (step 1), by means of an organolithium or magnesium reagent, followed by addition of a Ti(IV) source (step 2) then generation of the Ti(II) active species (step 3) using an organolithium or magnesium reagent. 13 (c) Ti(OiPr)4 iPrMgCl Toluene rt 64% (60%) (d) (a) 1 H-NMR yield was determined from 1,3,5-trimethoxybenzene as internal standard. (b) 1 equiv. of Ti(OiPr)4 and 2 equiv. of iPrMgCl were employed. (c) Respectively 6 min. and 4 min. instead of 15 min. and 10 min. were used for steps 1 and 2. (d) Isolated Yield.

First, deprotonation step (step 1) turned out to be essential. No reaction occured without deprotonating the alcohol group. Generation of sodium alcoholate was unsuitable since the alkyne was desilylated. Lithium alcoholate allows the process to occur (44%) but not in a clean manner. [17] Magnesium alcoholate from deprotonation with iPrMgCl was thus the most appropriate method. A brief screening of common Ti(IV) reagents (step 2) showed that Ti(OiPr)4 was the most suitable precursor (entry 1, 2 and 3) yielding to (±)-2 in 59 % yield (entry 1). Interestingly, using stoichiometric amount of Ti(OiPr)4 had a detrimental effect (entry 4), similarly, other reducing agents like nBuLi (entry 5) or cpentylMgBr (entry 6) proved to be inappropriate and only degradation was observed. When the reaction was conducted in ethereal solvents such as THF (entry 7) or Et2O (entry 8), instead of toluene, yields respectively dropped to 32 and 34%. We next examined the influence of the temperature on the cyclization (step 3). Running the reaction at -40 °C afforded (±)-2 as traces with degradation side-products. At -20 °C and 0 °C, yields respectively rise to 20% (entry 10) and 43 % (entry 11) still along with degradation side-products whereas increasing the temperature to 45 °C delivered (±)-2 in a clean manner (entry 12, 57%). It is worth to mention that performing the reaction at room temperature or 45 °C turned out to be crucial for a clean and complete conversion. Finally, by shortening the deprotonation time (step 1) and the Ti(OiPr)4 mixing time (step 2), the yield was improved to 64% (entry 13, 60% isolated yield and 40% on 1.5 mmol scale) while maintaining an excellent stereoselectivity in favour of the transadduct (d.r. > 95/5). It should be mentioned that the brief mixing time of alcoholate with Ti(IV) (step 2) is essential. Indeed, the direct generation of Ti(II) after addition of Ti(OiPr)4 led to a modest yield (33%) and a loss of the selectivity. [17] With the best conditions in hand, we studied the scope and limitations of the reaction on several structural derivatives of 2 (Scheme 3). First, we investigated the influence of the substitution pattern on the alkyne. Precursor 3 bearing a bulky TBS group yielded the cyclized product (4, 55%) with an excellent selectivity. Introducing an alkyl instead of a silyl group such as a methyl group ( 5) resulted in a slight decrease in yield (6, 48%). Alkyne bearing phenyl (7) or p-methoxyphenyl group ( 9) respectively afforded 8 and 10 in good yield (60% and 49%). Interestingly, these conditions were also found suitable for alkynylphosphonate [START_REF] Quntar | [END_REF] (11) even if the expected product was obtained (12) in moderate yield (31%) along with degradation side-products. Noteworthy, in all cases, an excellent selectivity toward the trans-isomer was observed (d.r. > 95/5).

Scheme 3 Cyclization reaction onto ene-yne containing phenyl framework.

Compound 13, containing a tertiary methyl-vinyl-alcohol, structurally close to the cyclization precursor of thapsigargin core firstly engaged with low-valent dialkoxytitanium(II), [13] provided the corresponding seven-membered ring adduct 14 in Please do not adjust margins Please do not adjust margins modest yield (43%) with a total diastereoselectivity in favour of the trans-isomer. Finally, we surveyed the substitution of the alkene moiety. Compound 15 bearing internal methyl substituent onto the double bond, was reluctant to cyclize under our conditions, even in a prolonged reaction time or by heating at 50 °C. From an inseparable cis/trans mixture (36/64) of the precursor 16, only 20% of the desired product 17 was obtained as well as the alkyne reduction product (~14% with side products). These results could be explained by a steric hindrance between the alkene moiety and the titanacycloproprene intermediate, which precludes the ring closure. Concerning compound 16, we hypothesized that only the cis isomer was able to cyclize explaining the poor yield. Indeed, Sato et al. have already evidenced that such cyclization was highly sensitive to the geometry of the double bond. [14c] Finally, the conditions developed herein allowed the trapping of electrophiles like acetone (18, 27%). However, the yield was disappointingly low, which is probably due to a competitive deprotonation in α-position to the ketone moiety by the intermediate titanacyclopentene since a large amount of compound 2 was recovered at the end of the reaction.

All the cyclization reactions gathered in Scheme 2 led to excellent selectivity in favour of the trans-isomer. We presumed that the diastereoselectivity observed would arise from the free allylic alcohol. [START_REF]For assumed mechanism and selectivity, see S.I. 20 For some representative examples[END_REF] Indeed, its directing effect in the cyclization of enynol and allenynols systems has already been underlined. [20] Moreover, the need to keep a short mixing time with two equivalents of Ti(IV) could witness the real implication of the alcohol toward the selectivity observed through an interaction with one equivalent of titanium reagent. In order to assess this assumption regarding seven-membered ring adducts, we ran two control reactions with the vinylic alcohol protected as a silylated ether (19) or a methyl ether (20) (Scheme 4).

The cyclization reaction conducted onto TBS ether provided poor selectivity (ratio: 37/63) and yield (12% and 21% as determined by 1 H NMR) along with reduction of the alkyne moiety (20, 20% as determined by 1 H NMR) and numerous others inseparable undefined side products. In a similar manner, an important drop in yield and selectivity was observed when the reaction was performed onto methyl-ether 21. However, we were pleased to isolate the 8-membered ring adduct (22, 38%) as the major product, which would probably result from a SN2' mechanism. As a conclusion, the presence of a free alcohol in the allylic position seems to play a crucial role onto both selectivity and yield. Hence, less side reaction products were observed when substrates comporting a free allylic alcohol were involved compared to their homologues bearing a protected alcohol, free hydroxy group would then help to prevent side reactions. For these reasons, we pursued our scope investigation from allylic alcohol-type compounds.

We first regarded all-carbon chain containing ene-yne systems like 24, 26 and 28 (Scheme 5).

Scheme 5 Synthesis of cycloheptane derivatives

The conditions turned out to be suitable for 24, 26 and 28 and good yields were obtained respectively for 25 (53% and 48% on 1 mmol scale), 27 (41%) and 29 (54%) as well as excellent diastereoselectivities (up to 95/5). However, it is important to mention that the time for step 2 was shortened in order to circumscribe side reactions between Ti(IV) and the ketal moiety. This reaction can also be extended to N-or O-containing substrates in order to access to azepane (Scheme 6) or oxepane derivatives (Scheme 7).

Scheme 6 Synthesis of azepane derivatives

We were pleased to observe good conversion and selectivity from 30 offering the azepane derivatives 31 in moderate yield (51%). Intriguingly, replacing TMS group by a phenyl (32) or pmethoxyphenyl group (34) onto the alkyne led to a drop in yield (33, 42% and 35, 14%) and selectivity (33, 60/40 and 35, 69/31).

Although the yield was modest (27%), it was also possible to trap the titanabicycle intermediate by a ketone [START_REF]For assumed mechanism and selectivity, see S.I. 20 For some representative examples[END_REF] . Thus, the expected product 36, was obtained from 30, with an excellent diastereoselectivity in favour of the trans-isomer.

We finally carried out the cyclization reaction on compounds 37 and 39 and we were delighted to respectively obtain the corresponding oxepane derivatives 38 (43%) and 40 (28%) (Scheme 7).

Scheme 7 Synthesis of oxepane derivatives and assumed elimination mechanism

Astonishingly, conducting the reaction on 37 over a prolonged time (20 min. instead of 10 min.) decreased the yield to 19%. At the purification step, diol 42 was isolated with a modest yield (16%) [START_REF]For assumed mechanism and selectivity, see S.I. 20 For some representative examples[END_REF] . The presence of this product could explain the disappointing yield in cyclization adduct 18. Indeed, if the elimination of a leaving group at the α-position of a titanacyclopropene is well known, [21] the critical drop of the yield over a longer reaction time allows to presume that the This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins expected cyclization process occurs first and the titanabicycle 41 intermediate undergoes an irreversible competitive elimination process leading to diol 42. [22] Thenceforth, a rapid quench of the intermediate, after 10 min as described, is required in order to limit this undesired pathway.

Conclusion

In summary, we have extended the intramolecular low-valent dialkoxytitanium(II) mediated cyclization reaction of ene-yne systems to seven-membered ring compounds with moderate to good yields. In most cases, a very good selectivity in favour of the trans-isomer was obtained. The conditions developed herein are mild, easy to set-up, relying on the use of inexpensive Ti(OiPr)4 and the cyclic adducts are obtained after a short time (~ 10 min) as a single diastereoisomer. Thanks to the conditions established, we were able to reach various (hetero)cycles i.e. cycloheptane as well as azepane or oxepane frameworks in a single step. We also highlighted the crucial role of the free allylic alcohol regarding the selectivity and the yield of the cyclization reaction. Thanks to the titanabicyclic intermediate, a complete control of the exocyclic double bond geometry is also to mention in all cases. Finally, we demonstrated the possibility to access 8-membered rings in moderate yield through a SN2' mechanism, which could open the way to the synthesis of larger cycles. Thus, the conditions need to be optimized and are currently undergoing in our laboratory.

a 1 H

 1 NMR conversion calculated from 1,3,5-trimethoxybenzene as internal standard. b Isolated yield. Scheme 4 Influence of the protecting group on allylic alcohol

Table 1

 1 Optimization of reaction parameters

	Entr y	Catalyst	Reductant	Solvent	Temp.	Yield (a)
	1	Ti(OiPr)4	iPrMgCl	Toluene	rt	59%
	2	TiCl2Cp2	iPrMgCl	Toluene	rt	0%
	3	TiCl(OiPr)3	iPrMgCl	Toluene	rt	33%
	4 (b)	Ti(OiPr)4	iPrMgCl	Toluene	rt	0%
	5	Ti(OiPr)4	nBuLi	Toluene	rt	0%
	6	Ti(OiPr)4	cpentylMgB r	Toluene	rt	0%
	7	Ti(OiPr)4	iPrMgCl	Et2O	rt	32%
	8	Ti(OiPr)4	iPrMgCl	THF	rt	34%
	9	Ti(OiPr)4	iPrMgCl	Toluene	-40°C	0%
	10	Ti(OiPr)4	iPrMgCl	Toluene	-20°C	20%
	11	Ti(OiPr)4	iPrMgCl	Toluene	0°C	43%
	12	Ti(OiPr)4	iPrMgCl	Toluene	45°C	57%

Conflicts of interest

There are no conflicts to declare.

Notes and references