Maxime Tourte

Philippe Schaeffer

Vincent Grossi

Philippe M Oger
email: philippe.oger@insa-lyon.fr

Acid Hydrolysis for the Extraction of Archaeal Core Lipids and HPLC-MS Analysis

Keywords: Archaea, Cell membrane, Core lipids, Acid hydrolysis, HPLC-MS, Extremophiles

Lipid membranes are essential cellular elements as they provide cellular integrity and selective permeability under a broad range of environmental settings upon cell growth. In particular, Archaea are commonly recognized for their tolerance to extreme conditions, which is now widely accepted to stem from the unique structure of their lipids. While enhancing the stability of the archaeal cell membrane, the exceptional properties of archaeal lipids also hinder their extraction using regular procedures initially developed for bacterial and eukaryotic lipids. The protocol described here circumvents these issues by directly hydrolyzing the polar head group(s) of archaeal lipids and extracting the resulting core lipids. Although leading to a loss of information on the nature of polar heads, this procedure allows the quantitative extraction of core lipids for most types of archaeal cells in an efficient, reproducible, and rapid manner.

the most extreme environments, be it due to temperature, pH, salinity, hydrostatic pressure, or other environmental stressors [START_REF] Komatsu | Low permeability of liposomal membranes composed of bipolar tetraether lipids from thermoacidophilic archaebacterium Sulfolobus acidocaldarius[END_REF][START_REF] Baba | Formation and characterization of planar lipid bilayer membranes from synthetic phytanyl-chained glycolipids[END_REF]. This ability to cope with multiple extreme conditions has been associated with one of their most diagnostic features, i.e., the unique structure of their membrane lipids. Indeed, instead of the typical lipids commonly found in Bacteria and Eukarya, which are built upon straight fatty-acyl chains ester-linked to a glycerol backbone in a sn-1,2 configuration, archaeal lipids contain polyisoprenoid alkyl chains that are ether-linked to a glycerol in a sn-2,3 configuration (De Rosa and Gambacorta, 1988). While polyisoprenoid alkyl chains provide greater membrane packing and impermeability compared to fatty acyl chains due to their heavy branching [START_REF] Komatsu | Low permeability of liposomal membranes composed of bipolar tetraether lipids from thermoacidophilic archaebacterium Sulfolobus acidocaldarius[END_REF], ether bonds are more chemically and thermally resistant than ester linkages and less polar [START_REF] Baba | Formation and characterization of planar lipid bilayer membranes from synthetic phytanyl-chained glycolipids[END_REF], both contributing to the enhanced stability of archaeal membranes. In addition, most Archaea are also capable of synthesizing both bilayer-forming diether lipids (one polar moiety) and membrane-spanning tetraether lipids (two polar moieties), the latter generating membrane monolayers that are even more rigid and impermeable than archaeal bilayers [START_REF] Chong | Archaebacterial bipolar tetraether lipids: physico-chemical and membrane properties[END_REF]. In contrast to their core lipids, the polar head groups of archaeal lipids remain very similar to those of Bacteria and Eukarya, with phospho-and glyco-lipids deriving from sugars (glycerol, inositol, glucose, and N-acetylhexosamine) [START_REF] Jensen | Structural characterization of ether lipids from the archaeon Sulfolobus islandicus by high-resolution shotgun lipidomics[END_REF], aminoacids (serine and ethanolamine) [START_REF] Koga | Taxonomic significance of the distribution of component parts of polar ether lipids in methanogens[END_REF], or combinations of both [START_REF] Koga | Taxonomic significance of the distribution of component parts of polar ether lipids in methanogens[END_REF]. Although substantial lipidomes, e.g., Note: Here, we used a vacuum pump at 50 Hz with a maximum displacement of 20.5 m 3 h -1 , a maximum pumping speed of 17.0 m 3 h -1 , and an ultimate vacuum of 2 × 10 -2 mbar. Although a strong suction power allows for a faster procedure, any vacuum pump able to reach an ultimate vacuum below 20 mbar can be used.

N2 drying system

Note: Here, we connected our building N2 supply (Air Products, CryoEase ® supply system) to a distributing system consisting of polycarbonate tubes (Cole-Parmer, catalog number: FV-30526-18) and splitting valves (Cole-Parmer, catalog number: FV-30600-02) ending on needles (BD, catalog number: 300700) or glass pipettes. Refer to Figure 3 for a depiction of the system.

High performance liquid chromatography (HPLC) instrument

Note: Here, we used a HP 1100 series LC system equipped with binary pumps (Agilent G1312A), a solvent degasser (Agilent G1379A), an autoinjector (Agilent G1913A), a thermostated column compartment (Agilent G1316A), and Chemstation chromatography manager software (Agilent Technologies Rev. A.09.03). However, any HPLC system that can function with a solvent gradient can be used. Note: As for every lipid extraction procedure, the growth medium may contain a significant amount of lipids unrelated to the cells to be analyzed. To avoid extracting these lipids from the culture medium, it is important to rinse the cells twice with an appropriate isotonic solution prior to freezedrying.

2. Remove all traces of potential organic contaminants by combusting all glassware, sand, celite, and glass wool at 450°C for 4 h before use. Rinse the glassware with deionized water, acetone, and DCM (in that order). Let it dry before use.

3. Silica gel is pre-extracted with DCM in a Soxhlet apparatus.

4. All reagents and solvents (except Hept) must be high grade, i.e., >99% purity, to reduce the risk of contamination.

Note: High purity solvent can also be obtained by distillation if possible.

5.

Hept is cleaned from organic contaminants by filtration over a column filled with silica gel.

6. Prepare 100 ml of 1.2 N HCl in MeOH by adding 10 ml of HCl 37% to 90 ml of MeOH.

Note: The solution can be stored for several weeks in a capped glass bottle at room temperature.

7. Prepare 100 ml of MeOH/DCM (1:1, v/v) by adding 50 ml of DCM into 50 ml of MeOH.

Note: The solution can be stored in a capped glass bottle at room temperature for several weeks. 8. Prepare 50 ml of Hept/IPA (99:1, v/v) by adding 0.5 ml of IPA to 49.5 ml of Hept.

Note: The solution can be stored for several weeks in a capped glass flask at room temperature. 9. Prepare 1,000 ml of Hept/IPA (95:5, v/v) by adding 50 ml of IPA to 950 of Hept.

Note: The solution can be stored for several weeks in a glass bottle at room temperature.

10. Prepare a 1,000-ml glass bottle filled with Hept.

Note: The solution can be stored for several weeks at room temperature.

A. Archaeal cell hydrolysis (Figure 1) 1. Transfer the archaeal cell pellet to a Pyrex TM glass tube using a small glass funnel.

2. Use 4 ml of 1.2 N HCl in MeOH to resuspend the archaeal cell pellet and rinse the glass funnel using glass pipettes and a hand pump.

Note: The volume at this step may be modified depending on the size of the cell pellet. However, do not fill the Pyrex TM tube above half of its total volume. Warning: Due to the gas generated during the heating step, hot gas and liquid may gush out from the tube when opening it.

Add 4 ml of DCM into the Pyrex TM tube (final MeOH/DCM 1:1, v/v).

Note: The volume at this step may be modified depending on the size of the cell pellet.

3. Create a filter by placing glass wool and then celite into a glass funnel. Place the glass funnel onto a 50 ml-round-bottomed flask suited for the rotary evaporator. 2020), but the nature and volume of solvents can be adjusted to fit the requirements of your chromatographic separation, i.e., nature and size of the column.

1. Start your chromatography instrument.

2. Set the temperature of the column thermostat to 30°C.

3. Empty the trash collectors and fill the vial used for rinsing (Hept/IPA 95:5, v/v).

4. Feed the chromatography manager software with the gradient used for HPLC chromatography.

Note: Here, we used the following gradient with A = Hept/IPA (95:5, v/v) and B = Hept, and a flow rate of 0.2 ml min -1 : 95% B (5 min isocratic) to 65% B in 30 min (5 min isocratic), then to 0% B in 1 min (10 min isocratic), and back to 95% B in 1 min (5 min isocratic). The total run time per sample is 57 min.

5. Double-check for stable pressure in the system to ensure proper separation. Flush your system if necessary.

Start the MS instrument.

7. Feed the MS manager software with the conditions for the MS analysis.

Note: Here, we used the following MS conditions: nebulizer pressure, 50 psi; APCI temperature, 420°C; drying temperature, 350°C; drying gas (N2) flow, 5 L min -1 ; capillary voltage, -2 kV; corona, 4 µA; and scan range m/z, 600-2,200. Note: Run a control sample every 30-40 analyses to check for appropriate separation and intensity. Reproducibility is evaluated by running selected samples in duplicate or triplicate and a reference sample. For instance, as a reference sample, we used a core lipid extract from Pyrococcus furiosus, which contains diphytanyl glycerol diethers (DGD) and multiple isomers of tetraether lipids, i.e., glycerol mono-, di-, and trialkyl glycerol tetraethers (GMGT, GDGT, and GTGT, respectively) with 0 to 4 cyclopentane rings, to assess the performance of the HPLC column. Additionally, run a standard mixture (synthetic archaeal-like lipids can be purchased from Avanti) to assess the relative response factors of the compounds detected. As a standard mixture, we used a solution of DGD/GDGT0 at 2:1 molar ratio which gave a relative response factor of DGD ca. 10 times lower than that of GDGT0 under our analytical conditions [START_REF] Tourte | Functionalized membrane domains: an ancestral feature of Archaea?[END_REF]). identification can be aided by referring to, e.g., [START_REF] Hopmans | Analysis of intact tetraether lipids in archaeal cell material and sediments by high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry[END_REF].

E. Chromatogram and mass spectrum analysis

1. Draw the total ion current (TIC) of your analysis using mass spectrum and chromatogram processing software. 2. Major compounds, i.e., the most abundant on the TIC, are tentatively identified based on their mass spectra, fragmentation patterns, retention time in LC, and comparison with published data (where possible). Use an average of several spectra of a given core lipid peak to ensure proper identification (Figure 1).

Note: Very few ions besides the protonated adduct of the intact compound are generated by our APCI procedure, and compounds are thus identified based on their protonated masses. To further ascertain the structures of identified core lipids, MS/MS can be performed.

3. Draw the extracted ion chromatogram (EIC) by extracting the masses of all compounds identified (Figure 4). For each compound, select a mass range suited to your MS system.

Note: Considering our system, we usually use a mass range of ±0.5.

  www.bio-protocol.org/e4118 Bio-protocol 11(16): e4118. DOI:10.21769/BioProtoc.4118 Please cite this article as: Tourte, M. et al. (2021). Acid Hydrolysis for the Extraction of Archaeal Core Lipids and HPLC-MS Analysis. Bio-protocol 11(16): e4118. DOI: 10.21769/BioProtoc.4118.

3 .

 3 Rotary evaporator (Büchi, models: Rotavapor RE 111; heating bath, B-491) Note: Any rotary evaporator compatible with your vacuum pump and heating bath up to 50°C can be used.

6 . 1 .

 61 Prevail Cyano 3 microns column (150 mm × 2.1 mm; Grace Davison Discovery Sciences, VWR, catalog number: HICH99243) Note: When not used, our column was stored in Hept/IPA (95:5, v/v). However, the solvent mixture allows for both the storage and equilibration of the column and thus depends on the separation gradient (see Step D4). It might hence be adjusted to your requirements. 7. Mass spectrometer equipped with an ion trap and an atmospheric pressure chemical ionization (APCI) source Note: Here, we used an Ion Trap MS Esquire 3000Plus (Bruker Daltonics, GE0100-G552) and an Agilent APCI source (Agilent G1947A), but any Mass spectrometry (MS) instrument equipped with an APCI source can be used. Software Mass spectrum and chromatogram processing software. Note: Here, we used the Compass Data Analysis software (version 5.0, 2017) provided by Bruker Daltonics, but any mass spectrum and chromatogram processing software, especially the one provided by your HPLC-MS instrument manufacturer, can be used. www.bio-protocol.org/e4118 Bio-protocol 11(16): e4118. DOI:10.21769/BioProtoc.4118 Please cite this article as: Tourte, M. et al. (2021). Acid Hydrolysis for the Extraction of Archaeal Core Lipids and HPLC-MS Analysis. Bio-protocol 11(16): e4118. DOI: 10.21769/BioProtoc.4118. Archaeal biomass must be freeze-dried beforehand.

3 .

 3 Tightly close the Pyrex TM tube with a cap containing a PTFE seal.Note: Double check that the cap is closed to avoid any evaporation that could lead to the destruction of the sample.4. To avoid any cross-contamination of samples and contamination of the oven in case of leakage, put 5 cm of sand in a glass bottle, place your Pyrex TM tubes in the sand, and close the bottle with a stopper.

5 .

 5 Heat your samples at 110°C for 4 h.Note: This procedure is strong enough to remove most of the archaeal polar head groups without further degrading archaeal lipids, i.e., ether linkages remain unharmed.6. Take your samples out of the oven and let them cool down to room temperature.Note: At this step, if your tubes were not sealed tightly enough, your sample might have dried out and completely burned (black-brown color of the sample), which might result in the destruction of core lipids. As a quality control of the procedure, check that tubes still contain solvent and that cell pellets are almost completely disrupted and colorless.

Figure 1 .

 1 Figure 1. Schematic of the workflow for the direct acid methanolysis of archaeal dried biomass. Circled numbers refer to the steps in the text. Abbreviations: MeOH, methanol; PTFE, polytetrafluoroethylene.

4 .

 4 Transfer the sample onto the filter and filter it through the funnel into the flask to remove the solid particles (i.e., cell debris) from the lipid extract. 5. Rinse the Pyrex TM tube twice with 4 ml of MeOH/DCM (1:1, v/v) and filter it into the same flask as above. 6. Rinse the filter into the same flask twice with 4 ml of MeOH/DCM (1:1, v/v). www.bio-protocol.org/e4118 Bio-protocol 11(16): e4118. DOI:10.21769/BioProtoc.4118 Please cite this article as: Tourte, M. et al. (2021). Acid Hydrolysis for the Extraction of Archaeal Core Lipids and HPLC-MS Analysis. Bio-protocol 11(16): e4118. DOI: 10.21769/BioProtoc.4118.

Figure 2 .

 2 Figure 2. Schematic of the workflow for the extraction of core lipids from the hydrolyzed biomass. Circled numbers refer to the steps in the text. Abbreviations: MeOH, methanol; DCM, dichloromethane.

Figure 3 .

 3 Figure 3. Schematic of the workflow for the core lipid extract preparation for HPLC analysis. Circled numbers refer to the steps in the text. Abbreviations: MeOH, methanol; Hept, heptane; IPA, isopropanol.

8 .

 8 Double-check that all parameters properly reach the set values. 9. Place the 2 ml-vials containing your samples dissolved in 1 ml of Hept/IPA (99:1, v/v; see Step C6) onto the HPLC autosampler. 10. Fill the chromatography manager software file with your sample information and sequence. Note: The injection volume is set to 10 µl. To enhance reproducibility, adjust the concentration of your samples (Step C6) rather than change the injection volume. 11. Run your sequence.

  Note: Relatively few examples of archaeal core lipid mass spectra have been published, but www.bio-protocol.org/e4118 Bio-protocol 11(16): e4118. DOI:10.21769/BioProtoc.4118 Please cite this article as: Tourte, M. et al. (2021). Acid Hydrolysis for the Extraction of Archaeal Core Lipids and HPLC-MS Analysis. Bio-protocol 11(16): e4118. DOI: 10.21769/BioProtoc.4118.

4 .

 4 Integrate each peak on your EIC to determine the relative proportion of each core lipid and correct with the response factor of the compounds under your analytical conditions (see Step D11).

Figure 4 .

 4 Figure 4. Core lipid analysis of Thermococcus acidaminovorans. Cells were grown under optimal conditions (85°C, pH 9.0, 3.0% NaCl (w/v); Dirmeier et al., 1998). The chromatogram was drawn by extracting the following masses: 653.5, 1302.5, and 1304.5. Inserts show the mass spectrum and the resulting core structure for each colored peak: green, diphytanyl glycerol diether (DGD); blue, glycerol trialkyl glycerol tetraether with no cyclopentane ring (GTGT0); and purple, glycerol dibiphytanyl glycerol tetraether with no cyclopentane ring (GDGT0). The indicated masses correspond to protonated adducts. Correcting for the response factors

Acknowledgments

The authors would like to thank the French National Research Agency for funding the ArchaeoMembranes project (ANR-17-CE11-0012-01) and the CNRS interdisciplinary program 'Origines' for funding the ReseArch project.

Competing interests

The authors declare no conflicts of interest.


Andreia Valente

Gregory Stoclet

Fanny Bonnet

André Mortreux

Marc Visseaux

Philippe Zinck

Isoprene-styrene chain shuttling copolymerization mediated by lanthanide half-sandwich complex and lanthanidocene: a straightforward access to a new type of thermoplastic elastomers **

Keywords: Chain Shuttling, Coordinative Chain Transfer Polymerization, Block Copolymers, Copolymerization

A lanthanide half-sandwich complex and a ansa lanthanidocene have been assessed for isoprene-styrene chain shuttling copolymerization with n-butylethylmagnesium (BEM). In the presence of 1 equiv. BEM, a fully amorphous multiblock microstructure of soft and hard segments is achieved. The microstructure consists of poly(isoprene-co-styrene) blocks, with hard blocks rich in styrene and soft blocks rich in isoprene. The composition of the blocks and the resulting glass transition temperatures (Tg) can be easily modified by changing the feed and/or the relative amount of the catalysts, highlighting a new class of thermoplastic elastomers (TPEs) with tunable transition temperatures. The materials self-organize into nanostructures at the solid state.

TPEs are materials that exhibit an elastic behavior similar to that of vulcanized rubber. [START_REF] Drobny | Handbook of thermoplastic elastomers[END_REF] In contrast to thermosets elastomers, they possess thermoreversible crosslinks and can be processed as thermoplastics. TPEs are widely used to improve the properties of rigid thermoplastics, notably their impact strength. TPEs in the form of block copolymers can furthermore self-assemble in the solid state or in solution, leading to nanostructures with a variety of properties and potential functions. [START_REF] Schacher | [END_REF] Poly(styrene-co-butadiene-co-styrene) triblock copolymers have been commercialized and are used as bitumen additives and viscosity modifiers. [START_REF] Schacher | [END_REF][3] The polybutadiene block shows a glass transition temperature below room temperature, providing the elastomeric character or soft block, while polystyrene with a Tg above room temperature acts as the crosslink or hard block. Di-and triblocks copolymers are formed by anionic polymerization, [START_REF] Drobny | Handbook of thermoplastic elastomers[END_REF] while multiblock microstructures of homopolymers can be obtained by chain shuttling copolymerization (see vide infra). [4d] These blocky structures show well defined transition temperatures (Tg or melting temperature of the homopolymers) that cannot be tuned, which restricts their fields of application. An approach able to tune the transition temperatures of the blocks may widen the applications of these materials to other fields including for example shape memory polymers. The synthesis of such adjustable structures is not an easy task and requires the use of advanced synthetic methods. Chain shuttling polymerization [4] is a process based on coordinative chain transfer polymerization [5][6] that gives access to multiblock microstructures (Scheme 1). The growing macromolecular chain is allowed to "shuttle" between two catalysts presenting a difference in monomer selectivity via a chain transfer agent. Our objective in this study was to extend the potentialities of chain shuttling copolymerization to the synthesis of a new class of thermoplastic elastomers with variable transition temperatures. As the Tg of statistical copolymers can be easily varied by a change of their composition, a fully amorphous multiblock microstructure consisting of hard and soft statistical copolymers was targeted. We developed these last years lanthanide half-sandwich complexes based catalytic systems for the coordinative chain transfer polymerization and copolymerization of conjugated dienes, styrene and 1-alkenes. [6] We elaborated our strategy based on this knowledge. Borohydrido half-lanthanidocenes are known to lead to isoprene rich poly(isoprene-co-styrene) copolymers with a high 1,4trans stereoselectivity when combined to magnesium dialkyls. We envisioned the use of a sterically hindered complex as second precatalyst in order to reach styrene rich poly(isoprene-co-styrene) blocks. Lanthanum and neodymium based pre-catalysts were found to be particularly well suited for the reversible transmetalation of a growing polymeric chain with magnesium dialkyls. We report herein the first example of a chain shuttling copolymerization Mn [b] (g/mol)

DM [b] Ip [c] (%)

St [c] (%) Polyisoprene microstructure [c] Tg (°C) Reactions conducted at 50°C in toluene for a monomers / pre-catalyst(s) ratio of 2000 (e.g 1.1 ml styrene (10 -2 mol) and 1 ml isoprene (10 -2 mol) in 1.5 ml toluene for a 50/50 molar ratio of the monomers). The chain shuttling copolymerizations were conducted with a 50/50 molar ratio of the pre-catalysts. [b] Number-average molecular weight and dispersity determined by size exclusion chromatography using polystyrene standards. [c] from 1 H NMR measurements. leading to a fully amorphous multiblock microstructure of soft and hard statistical copolymers, providing a straightforward access to a new class of thermoplastic elastomers with variable transition temperatures. Isoprene and styrene were selected as monomers, the lanthane half-sandwich complex Cp*La(BH4)2(THF)2 (1) (Cp*=C5Me5) and the ansa-neodymocene [(C5H4CMe)2Nd(BH4)2]2Mg(THF)3 ( 2) as pre-catalysts (Scheme 2) and n-butylethylmagnesium as chain transfer agent. Experiments representative of the statistical copolymerization of isoprene and styrene (50/50 molar ratio) conducted in the presence of either 1 or 2 combined to 1 and 10 eq. BEM are presented in Table 1, entries 1-5. The decrease of the number-average molecular weight observed for 10 eq. BEM vs. 1 eq. shows that the transmetalation is occurring efficiently with both pre-catalysts. Statistical copolymers with a unique Tg and monomodal molecular weight distributions are obtained. 1 leads to isoprene rich statistical copolymers exhibiting a low Tg, while the copolymers obtained with 2 show higher styrene content and higher Tg. The chromatograms reveal a monomodal distribution of the molecular weight in all cases.

Chain shuttling experiments were first conducted with a 50/50 molar ratio of the monomers and a 50/50 molar ratio of the catalysts (entries 6-10, Table 1). NMR investigations of possible ligand exchange were conducted prior to polymerization, and no ligand exchange between the two complexes was observed in benzene-d6 after several hours at 50°C, before and after introduction of 10 eq. BEM. The decrease of the number-average molecular weight with increasing quantities of BEM shows that the transmetalation is occurring efficiently. The copolymer obtained using 1+2 and 1 eq. BEM exhibits an overall composition comprised between those obtained using 1 and 2 alone (39% styrene inserted in the copolymer vs. 12 and 50%, respectively) and a highly 1,4-trans microstructure of the polyisoprene part. The occurrence of chain shuttling between the two catalysts was assessed from the chromatograms obtained after 2h reactions (Figure 1). The molecular weight distribution of the copolymer obtained by chain shuttling is monomodal, and located between those obtained using 1 and 2 alone. The resulting polymer is characterized by two Tg, the lower one being ascribed to the glass transition of isoprene rich blocks and the higher one to styrene rich blocks. Such characteristics are typical of thermoplastic elastomers. Endothermic peak resulting from the melting of polyisoprene crystalline phase could not be observed, confirming the amorphous nature of the materials. The formation of the multiblock microstructure was confirmed by fractionation experiments showing that the reaction product is not a mixture of the copolymers made by 1 and 2 separately (see supporting information). Additionally, the blend of the copolymers made by 1 and 2 separately shows a phase separation (figure SI1), while the copolymers formed by chain shuttling are fully transparent (see a typical example in figure 2). Chain shuttling copolymerizations were further conducted in the presence of increasing amounts of BEM (Table 1, entries 8-10). A unique Tg was observed for these copolymers, showing that the microstructure has shifted from a thermoplastic elastomer material to a more conventional statistical copolymer. In the presence of excess BEM, the transmetalation does not allow sufficient chain growth on each of the catalysts to afford the multiblock microstructure. Keeping in mind that substantial quantities of chain shuttling agent are usually required for the formation of the multiblock microstructure via chain shuttling polymerization, it is noteworthy that we achieve this goal with only 1 eq. Chain shuttling copolymerizations experiments conducted in the presence of 1 eq. BEM with excess styrene (entries 11-12) and excess isoprene (entry 13) in the feed are presented in Table 1. Two Tg are also observed for these materials, highlighting again the formation of the multiblock microstructure. The influence of the catalyst ratio was further assessed (entries 14-16). If an excess of 2 leads to the desired multiblock microstructure, the copolymer resulting from the reaction conducted with 70% of 1 shows only one Tg. It is noteworthy that both the modification of the feed and that of the relative amount of the catalysts lead to a variation of both Tg.

The nanostructure of the copolymers was investigated by means of Small-Angle X-ray Scattering (SAXS). A scattering contrast could be observed only for samples exhibiting the multiblock microstructure (see typical examples in Figure SI2). The scattering curves could be modeled by a dispersion of dilute disks having an average thickness of 6±1nm. From a structural point of view this can be interpreted by the self-assembly of one of the blocks into lamellas. The dilute term of the model indicates furthermore that the lamellas are non-interacting or in other words that there is no nanostructuration of the lamellas at large scale. Atomic Force Microscopy (AFM) analyses were performed to pursue the investigation of the nanostructure (Figure 3). In the presence of 1 or 2 alone, when a random copolymer is formed, a uniform structure is observed (entry 4 in Figure 3). The nanostructuration of the samples resulting from chain shuttling copolymerization in the presence 1 eq. BEM can be seen Figure 3, entries 7 and 11. Assuming that bright domains are assigned to hard domains, the nanodomains correspond to a self-assembly of the rigid blocs. AFM line profile analysis (Figure SI3) shows that the top of the hard domains is flat, indicating a disk-like shape. The height of the disks around 9 nm is in good agreement with the values obtained from SAXS. Finally, the examination of a sample synthesized by chain shuttling with a higher amount of BEM amount (entry 10 in Figure 3) does not evidence the presence of disk like nanodomains. This is in agreement with the thermal properties of this material for which only one Tg was detected as expected for a random copolymer structure.

In summary, we report a synthetic approach affording a straightforward access to an unprecedented fully amorphous multiblock microstructure of soft and hard statistical copolymers. The microstructure consists of blocks of poly(isoprene-co-styrene) copolymers, with hard blocks rich in styrene and soft blocks rich in isoprene. The Tg of the blocks can be varied simply by changing the feed or the relative amount of the catalysts, and the resulting copolymers self-organize into nanostructures. Additional work is currently in progress to explore the potential applications of these new block copolymers.

Scheme 1 .

 1 Scheme 1. Chain shuttling copolymerization. Mi represents transition metals or lanthanides allowing propagation, Li ligands, and CTA the chain transfer or chain shuttling agent.

Figure 1 .

 1 Figure 1. Molecular weight distributions obtained by size exclusion chromatography after 2h reaction (entries 1, 3 and 6).

Figure 2 .

 2 Figure 2. Entry 7 cast onto a glass slide (picture)

Figure 3 .

 3 Figure 3. AFM phase contrast images of entries 4, 7, 10 and 11 (Zscale = 20°)

Table 1 .

 1 Isoprene-styrene statistical and chain shuttling copolymerizations using 1 and 2 in combination with BEM

	Entry [a]	Pre-	Ip/St	Mg	Time	Yield
		Catalysts
		1/2		Eq.	(h)	(%)

Dr G. Stoclet Unité Matériaux Et Transformations, CNRS, University Lille1, Science and Technology, 59655 Villeneuve d'Ascq, France [] This work was supported by the French Ministry of Education and Research. The authors acknowledge Aurélie Malfait for technical assistance.

Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author Chain shuttling copolymerization of isoprene and styrene using a borohydrido lanthane half-sandwich complex, a borohydrido ansa-neodymocene and a magnesium dialkyl affords direct access to new amorphous thermoplastic elastomers. The resulting multiblock microstructure of soft and hard statistical copolymeric segments self-assembles at the nanoscale.

Entry for the Table of Contents


Serge Bourbigot
email: serge.bourbigot@ensc-lille.fr

Johan Sarazin

Tsilla Bensabath

Fabienne Samyn

Maude Jimenez

Intumescent polypropylene: reaction to fire and mechanistic aspects

Keywords: Intumescence, polypropylene, kinetic analysis, heat transfer

The concept of intumescence was applied to make flame retarded polypropylene (PP). This paper examines two types of intumescence in PP based on expandable graphite (EG, physical expansion) and on modified ammonium polyphosphate (AP760, chemical expansion). Reaction to fire of PP containing EG and AP760 was first evaluated by cone calorimetry. The incorporation of intumescent additives at relatively low loading (10 wt%) in PP permits the reduction by 70% of peak of heat release rate (pHRR). The mode of action occurs via the formation of an expanded carbonaceous layer in all cases. The protective coating acts mainly as heat barrier in the case of the formulations containing AP760 or as heat dissipater with EG. The incorporation of small amount of EG in PP-AP760 modifies heat transfer in the coating creating a strong anisotropy. Upon expansion, graphite worms align normal to the surface increasing the transverse heat conductivity (lower efficiency of the heat barrier) and hence, decreasing the fire performance (decrease by only 30% of pHRR). Kinetic analysis was then performed to quantify the thermal stability of the intumescent systems. It reveals that the intumescent additives do not modify the reactional scheme of the PP thermal decomposition but they increase slightly the thermal stability of the intumescent systems. For all materials, the decomposition model follows a reactional scheme at two successive reactions. This model was determined in dynamic conditions (conditions of thermogravimetry with linear heating rates) but it is able to simulate the decomposition of the materials in isothermal conditions (excellent agreement between the simulated and experimental curves).

INTRODUCTION

Economical manufacturing methods for mass production and improvements of properties of finished products have in many applications greatly contributed to replace traditional materials like metals or wood with plastics and rubbers. In particular, polypropylene (PP) is the fastest growing commodity plastic worldwide. It has found its place in many sectors such as building, transportation (automotive, railways …), electrical engineering (electrical / household appliances, housings …) or paper industry.

The use of the organic polymer systems like PP, which are flammable, leads to greater fire risks and thus to growing importance of flame retardancy [START_REF] Troitzsch | Fires, statistics, ignition sources, and passive fire protection measures[END_REF]. Some events have shown the need to improve fire resistance. As an example, the PP fire at the BASF plant (March 1995 in Teeside, UK) which has been described as one of the largest fires ever seen in the UK during the peacetime (more than 10,000 tons of PP were consumed) [START_REF] Carty | Letter to the Editor: Polypropylene Fire at the BASF Plant[END_REF]. This clearly entails a more comprehensive appraisal of all aspects of the combustion of polymers and the ways to prevent it. The high carbon and hydrogen content of PP (like many other synthetic polymers) makes it combustible. To enable PP to participate and perform safely, flame-retardants are added in applications where regulatory or specifications require an enhanced flame retardant capability over and above the unmodified polymer. The flame retardant will not make the resulting compound non-combustible. It will however make the material more ignition resistant. During burning, the sufficiently flame retarded plastic will reduced the flame spread time over that of unmodified PP. Flame retardants exert many different modes of action as a function of the chemical nature of the polymer-flame retardant systems and of the interactions between the components. It is considered that inhibition of burning is achieved by modification of either the condensed phase or the dispersed or gaseous phase in a physical and/or chemical mode [START_REF] Bourbigot | Fire retardant polymers: Recent developments and opportunities[END_REF].

The motivation of this paper is to examine the flame retardancy of PP using the concept of intumescence (mode of action in the condensed phase). When heated beyond a critical temperature, an intumescent material begins to swell and then to expand. The result of this process is a foamed cellular charred layer on the surface, which protects the underlying material from the action of the heat flux or the flame. Visually, the swelling and the expansion looks like 'black waves' swollen at the surface of the material and the final char exhibits hemispheric shape with a roughed or smooth surface. The concept of intumescence enables to make flame retarded polymeric materials (including PP-based materials) exhibiting high performance [START_REF] Alongi | Intumescence: Tradition versus novelty. A comprehensive review[END_REF]. Typically, the ingredients of intumescence are mainly composed of an inorganic acid or a material yielding acidic species upon heating (e.g. phosphate), of a char former (e.g. pentaerythritol) and of a component that decomposes at the right temperature and at the right time to enable the expansion of the system (e.g. melamine). Those types of ingredients were incorporated in PP in the 80s and 90s and gave high limiting oxygen index (LOI) and low heat release rate (HRR) [START_REF] Camino | Study of the mechanism of intumescence in fire retardant polymers: Part II-Mechanism of action in polypropylene-ammonium polyphosphate-pentaerythritol mixtures[END_REF][START_REF] Morice | Heat transfer study of polypropylene-based intumescent systems during combustion[END_REF]. More sophisticated flame-retardants combining the ingredients' triplet in all in one molecule and made from organic syntheses were designed but they provided similar performance as the conventional formulation [START_REF] Alongi | Intumescence: Tradition versus novelty. A comprehensive review[END_REF][START_REF] Fontaine | Neutralized flame retardant phosphorus agent: Facile synthesis, reaction to fire in PP and synergy with zinc borate[END_REF][START_REF] Zhu | Synthesis of a novel hyperbranched and phosphorus-containing charringfoaming agent and its application in polypropylene[END_REF][START_REF] Xu | Synthesis and characterization of a novel organic-inorganic hybrid char-forming agent and its flame-retardant application in polypropylene composites[END_REF]. Incorporation of additional filler in an intumescent PP can also involve synergistic effects. Performance is enhanced dramatically adding small amount of an additional compound [START_REF] Fontaine | Neutralized flame retardant phosphorus agent: Facile synthesis, reaction to fire in PP and synergy with zinc borate[END_REF][START_REF] Han | Synergistic flame retardant effect of BiFeO3 in intumescent flame-retardant polypropylene composites[END_REF][START_REF] Dong | Synergistic Effects of Novel Intumescent Flame Retardant Polypropylene Composites[END_REF][START_REF] Nie | Morphology influence of nanoporous nickel phosphate on intumescent flame retardant polypropylene composites[END_REF]. Numerous synergists (micro-and nanofillers) have been used in conventional "three-based ingredients" intumescent formulations. It includes the boron compounds (zinc borates, B2O3, borophosphate, borosiloxane), phosphorus compounds (phosphazene, ZrPO4), silicon compounds (silica, silicone, silicalite), aluminosilicates (mordenite, zeolite, montmorillonite), rare earth oxides (La2O3, Nd2O3), metal oxides (MnO2, ZnO, Ni2O3, Bi2O3, TiO2, ZrO2, Fe2O3) and others (carbon nanotubes, silsesquioxanes, layered double hydroxides, Cu, Pt, talc, sepiolite, zinc and nickel salts). The presence of this additional filler can modify the chemical (reactivity of the filler versus the ingredients of the intumescent system) [START_REF] Bourbigot | PA-6 clay nanocomposite hybrid as char forming agent in intumescent formulations[END_REF] and physical (expansion, char strength and thermophysical properties) [START_REF] Bourbigot | Polyhedral oligomeric silsesquioxane as flame retardant for thermoplastic polyurethane[END_REF] behavior of the intumescent char when undergoing flame or heat flux leading to enhanced performance. This short survey on intumescent PP shows that the method to develop the intumescence phenomenon is mainly based on series of chemical reactions occurring timely. Another way to make intumescence in a polymeric matrix is the physical expansion. In other words, the rapid sublimation of a molecule (and/or the decomposing products) creates the expansion of the top degraded layer of the polymer to make an intumescent coating. This mechanism occurs when using expandable graphite (EG). EG is a synthesized intercalation compound of graphite that expands or exfoliates when heated. A wide variety of chemical species can be used to intercalate graphite materials (e.g. sulfate, nitrate, various organic acids …) [START_REF] Focke | Characterization of commercial expandable graphite fire retardants[END_REF]. It was applied in PP in combination with conventional intumescent ingredients [START_REF] Zheng | Synergistic effect of expandable graphite and intumescent flame retardants on the flame retardancy and thermal stability of polypropylene[END_REF][START_REF] Qi | Efficient organic-inorganic intumescent interfacial flame retardants to prepare flame retarded polypropylene with excellent performance[END_REF]. The mechanism involved was investigated in some aspects (chemistry of the system) but it remains poorly understood. It is the purpose of this work to revisit the mode of action of physical intumescence in PP and to make comparisons with chemical intumescence. This paper examines two types of intumescence in PP based on EG (physical expansion) and on modified ammonium polyphosphate (AP760, chemical expansion). Some examples of using ammonium polyphosphate and EG can be found elsewhere [START_REF] Duquesne | A comparative study of the mechanism of action of ammonium polyphosphate and expandable graphite in polyurethane[END_REF][START_REF] Lu | Flame retardancy and mechanical properties of ethylene-vinyl acetate rubber with expandable graphite/ammonium polyphosphate/dipentaerythritol system[END_REF][START_REF] Guo | Effects of expandable graphite and modified ammonium polyphosphate on the flame-retardant and mechanical properties of wood flour-polypropylene composites[END_REF] but not in PP. Reaction to fire of PP containing EG and AP760 is first evaluated by cone calorimetry and the efficiency of the intumescent barrier is measured quantitatively at the same time. Kinetic analysis is then performed to quantify the thermal stability of the intumescent systems.

EXPERIMENTAL

Materials and processing

Commercial grade of PP was used in this work: PP (PPH9060) was supplied by Total petrochemicals (Feluy, Belgium). PPH9060 has a melt flow rate (MFR) for a load of 2.16 g at 230°C of 25 g/10 min.

Expandable graphite (EG) is the commercial grade ES350F5 from Graphitwerk Kropfmühl (Germany) with an average particle size of 300 µm. Sulfate was used in this grade as intercalation compound to make graphite bisulfate. Modified ammonium polyphosphate (AP760) is the commercial grade of Clariant (Germany) with the brand name Exolit AP760. It is an intrinsic intumescent system containing 20 wt% phosphorus and 14 wt% nitrogen acting in synergy. It is mainly based on ammonium polyphosphate as acid source and tris(hydroxyethyl) isocyanurate (THEIC) as char former.

Processing

The strategy was to blend PP with flame-retardants (FRs) in a twin-screw extruder. The total loading of FRs in PP was 10 wt% varying the ratio AP760/EG (wt/wt) at 10:0; 5:5; 9:1 and 0:10. This loading was selected because it provides an acceptable performance according to cone calorimetry.

Compounding was performed using HAAKE Rheomix OS PTW 16 twin-screw extruder. The extruder is a co-rotating intermeshing twin screw with a barrel length of 400 mm and screw diameter of 16 mm (L/D = 25) with 10 zones. PP and FRs were incorporated using two gravimetric side feeders into the extruder. Polymer flow rate is fixed to extrude about 500 g/h with a screw speed of 300 rpm. The

Cone calorimetry

FTT (Fire Testing Technology) Mass Loss Calorimeter (MLC) was used to carry out measurements on samples following the procedure defined in ASTM E 906. The equipment is identical to that used in oxygen consumption cone calorimetry (ASTM E-1354-90), except that a thermopile in the chimney is used to obtain heat release rate (HRR) rather than employing the oxygen consumption principle. Our procedure involved exposing specimens measuring 100 mm x 100 mm x 3 mm in horizontal orientation. External heat flux of 35 kW/m² was used to perform the experiments. This flux corresponds to common heat flux in mild fire scenario. MLC was used to determine heat release rate (HRR) and total heat release (THR). When measured at 35 kW/m², HRR is reproducible to within 10%. The cone data reported in this paper are the average of three replicated experiments.

In addition to those measurements, a thermocouple (K-type thermocouple of 0.5 mm diameter) was embedded on the backside of the materials in horizontal position. It measured temperature as a function of time during a regular cone experiment. In this experimental set-up, it is necessary to assume that additional conductive effects due to the thermocouple are negligible.

X-ray tomography

The expanded intumescent specimens obtained after cone calorimetry were placed in X-ray μCT (computed microtomograpy) for the analysis of the inner morphology without structural damage.

The resulting recordings over 360• were reconstructed to obtain the computerized 3D CT volumes.

The tomography was performed using the microtomography setup at ISIS4D X-ray CT platform (equipped with UltraTom from RX Solutions). The set-up consisted of two X-ray tubes (micro and nano focus), a sliding and rotating stage holder, a flat panel detector (1920x1496 px-127 μm/px-0.2 to 60 frame/s), a linear detector (2560 px-200 μm/px-0.2 to 60 frame/s), a CCD camera (4000x2624 px-11.8 μm/px-up to 3.4 frame/s) and an image intensifier. Samples were placed on styrofoam holders and then mounted on the rotating stage to minimize the signal noise due to the holder.

Samples were rotated by 360• with an angular step of 0.25•. The subsequent tomography volumes were reconstructed from 1440 projection images produced by classical attenuation contrast technique and visualized using X-Act (from RX Solutions).

Kinetic analysis

ThermoGravimetric Analysis (TGA) was carried out using a Netzsch TG 209 F1 Libra at five heating rates (1, 2, 5, 10, 20 and 50°C/min) from 30°C to 800 °C in nitrogen flow (50 cm 3 /min). Samples of exactly 10 mg (0.3 mg) were put in open alumina pans. Typically, two replicates were run for each sample, and the average was reported. Both the onset (5% mass fraction loss) and peak mass loss rate have an uncertainty of 0.9°C (2). We corrected for each heating rate the buoyancy force (calibration with empty pan). Kinetic analysis and modeling of the degradation of the samples was made using an advanced thermokinetic software package developed by Netzsch Company [START_REF] Opfermann | Kinetic analysis using multivariate non-linear regression. I. Basic concepts[END_REF]. For kinetic analysis, it is assumed that the material decomposes according to the Eq. ( 1):

Asolid → Bsolid or liquid + Cgas (1)
The rate expression d/dt, where  is the degree of conversion, is assumed to be defined by Eq. ( 2):

d/dt = k(T).f() ( 2
)
where k is the kinetic constant, k = A.exp(-E/RT) according to the Arrhenius law, A is the frequency factor, E is the activation energy and f() is the so-called "reaction model". All reactions are assumed to be irreversible. In the case of decomposition and since the evolved gases were continuously removed by the fluid flow in the TGA chamber, this is a reasonable assumption. It is also assumed that the overall reaction (Eq. ( 1)) is the sum of individual reaction steps (formal or true step) with constant activation energy, as generally accepted in chemistry. The model can then include competitive, independent and successive reactions. The equations were solved with multivariate kinetic analysis (determination of the parameter via an hybrid regularized Gauss-Newton method or Marquardt method).

RESULTS AND DISCUSSION

Reaction to fire

EG and AP760 and some of their combinations (AP760/EG at 5:5 (wt/wt) and 9:1 (wt/wt)) were incorporated in PP at 10 wt% total loading. The four formulations were evaluated by cone calorimetry at an external heat flux of 35 kW/m² and compared to neat PP (Fig. 1). 50 MJ/m²) and by 20% for PP-AP760/EG (5:5) (82 MJ/m² vs. 60 MJ/m²). The formation of an expanded charred layer is observed at the surface of the materials evidencing an intumescent phenomenon (Fig. 2). The char formed from PP-AP760 is fully expanded and its surface is flat (Fig. 2 -a) but on the opposite, the two formulations PP-EG and PP-AP760/EG (5:5) exhibit an expanded 'hairy' char during the experiment (Fig. 2 -b andc). The intercalation compounds contained in EG decompose rapidly into gaseous products, which blast off the graphite flakes. Those flakes make then worms forming an entangled network at the surface of the material. This network acts as a protective layer (PP-EG, Fig. 2 -b). The combination of EG and AP760 in the formulation PP-AP760/EG (5:5) exhibits a reduced expansion compared to PP-EG. The char is also formed of entangled worms of graphite, which can be clearly distinguished at the surface (Fig. 2 -c). The relative limited expansion is probably due to the embedded graphite worms in the char increasing its viscosity. The formulation containing 1 wt% EG exhibits a pHRR only decreased by 30% and a THR decreased by 10% (82 MJ/m² vs. 70 MJ/m²). An intumescent phenomenon is also observed but its efficiency is not as high as the other intumescent PPs. The low quantity of EG in the formulation does not permit the formation of an entangled network and only graphite worms coming out of the charred surface can be observed (Fig. 2 -d). It is noteworthy the graphite worms rise up straight upon heating: they do not flatten and remain in line on the expanding char (Fig. 3).

Overall, the intumescent char formed from PP-AP760 exhibits high expansion and a smooth surface up to 150s and then their temperature rise exhibits a similar value as that of PP-EG (0.15°C/s). They reach a steady state at about 700s with similar final temperatures (520°C at 1100s). Note PP-EG does not show any steady state. It is assigned to smoldering occurring on the graphite worms (Fig. 2 -b).

The higher performance of PP-EG compared to PP-AP760 and PP-AP760/EG(5:5) can be explained by the faster expansion of its intumescent coating thanks to the fast sublimation of the inserted molecules. The protective layer is therefore formed at shorter time as evidenced by the slope break at 190s and then the entangled network of graphite worms can limit the heat transfer to the bottom layer. PP-AP760/EG(9:1) exhibits an unexpected behavior because its backside temperature increases extremely rapidly to 650°C (temperature rise of 5.2°C/s). Its backside temperature reaches a pseudo steady state at 670°C for 350s (50s<t<400s) and then decreases to 545°C. This temperature drop (125°C) at 400s is unclear and it is not due to the flameout of the materials since it occurs at 660s. The temperature on the backside of PP-AP760/EG(9:1) strongly decreases after 400s while it is not observed with the other samples. To explain this, the structure of the chars was examined by X-ray tomography. The main advantage of tomography is to observe the internal structure of the samples without damaging it. The intumescent samples are brittle and it is difficult to cut them off without any modification. The cross-sections of the intumescent residues show two types of internal structures (Fig. 5). The residues of PP-AP760 and PP-AP760/EG(9:1) exhibit an expanded hollow structure with thin walls (Fig. 5 (a) and (d)). Some graphite worms can be distinguished for PP-AP760/EG(9:1) to the wall (Fig. 5 (d)). Note also that a part of the sample is expanded.

The residues of PP-EG and PP-AP760/EG(5:5) are also expanded but they are filled by graphite worms showing an entangled network as mentioned above (Fig. 5 (b) and (c)). The comparison of the 4 structures does not permit to explain the temperature drop occurring for PP-AP760/EG(9:1) but they confirm the comments made about the mode of protection, namely the formation of an entangled network when using EG and of an expanded charred coating when using AP760. Hence, additional experiments were performed for preparing residues stopped at characteristic times of the temperature/time curve, namely 130s (highest temperature), 460s (decreasing temperature) and 900s (end of the experiment). Because of ease, the so-prepared samples were cut off to get a rapid view of their cross-sections (Fig. 6). At 130s, the sample is relatively compact and few expanded (Fig.

(a)

). The graphite worms are embedded in the char and are staked because of the cooling of the sample (the worms were aligned and normal to the surface during the experiment). The heat conduction inside the solid should be high. At t = 460 s, the temperature on the backside of the sample is decreasing. Concurrently, the char is expanding creating a hollow charred coating (Fig. 6 (b)). At this time, the heat transfer is reducing and then the temperature drops down. The final residue is also a hollow coating (Fig. 6 (c)). Those observations give an explanation why the temperature decreases after a certain time: the incorporation of EG delays the expansion of the intumescent coating and hence limits the performance of the intumescent char. The results suggest the involvement different mechanisms. PP-A760 forms an char at the surface, which reduces the heat transfer from the flame to the material. The incorporation of small amount of EG in a conventional intumescent system (PP-AP760) strongly affects its performance. Visual observation shows graphite worms grows up at the surface but there is no entanglement of the worms (Fig. 3). The worms exhibit an alignment normal to the surface and hence, they might enhance heat conductivity (transverse heat conductivity) through the material as

shown by temperature measurement (see the cartoon of Fig. 7). In the other cases, EG can form an entangled network, which flattens because of the expansion. Hence, in-plane heat conductivity is enhanced (parallel to the surface) and the worms play the role of heat dissipater (this effect is limited when EG is combined with AP760). Even if the cone heater irradiates the whole surface, it is reasonable to assume heat dissipation occurs on side surfaces of the samples. Those two types of behaviour leads to huge difference in terms of performance and it evidences the anisotropic character of EG when used as FR.

Kinetic analysis

The previous section has shown the fire performance of PP was strongly enhanced incorporating AP760 and EG. The goal of this section is to examine whether the thermal stability of the intumescent PPs could be modified. Fig. 8 shows TG curves of the intumescent PPs compared to neat PP. All materials exhibit a single apparent step of degradation but only the intumescent PPs have residues at high temperature (about 4.5 wt.-% for PP containing AP760 and 7.5 wt.-% for PP-EG). It is easily explained by the charring of the samples due to the intumescent ingredients. It is noteworthy PP-EG gives higher char yield than the others probably because of the high thermal stability of graphite. The onset temperature of decomposition of PP materials containing AP760 is slightly lower than that of neat PP (365°C vs. 390°C) while it is the contrary for PP-EG: its onset temperature of decomposition is at 415°C. The curves of AP760 containing PPs are superimposed and no difference can be distinguished. At T > 430°C, the thermal stability of those materials become higher than that of neat PP while it is always the case for PP-EG. The comparison of the thermal stabilities of the PP-based materials only brings information on the temperature range where the material decomposes but it does not give any physical parameter. The approach was then to perform kinetic analysis on the thermal decomposition of the neat PP and of the 4 intumescent formulations to get kinetic parameters, namely the triplet including the frequency factor (A), the activation energy (E) and the reaction order (n). Before starting any fitting procedure, it is necessary to define a model (combination of reactions) and to preset starting values for the kinetic parameters. A convenient approach is to use model-free analysis as a preliminary step of the kinetic analysis. A model-free analysis provides the plot of the activation energy versus the conversion degree. For the 5 materials, it reveals that the plot of activation energy exhibits a pseudoplateau at conversion degree within the range 0.2 -0.9 between 180 and 270 kJ/mol and a more complicated shape at low and high conversion degrees (the 5 materials exhibits similar curves and Fig. 9 shows a typical plot calculated from Friedman analysis [START_REF] Friedman | Kinetics of thermal degradation of char-forming plastics from thermogravimetry. Application to a phenolic plastic[END_REF]). This indicates that the decomposition does not take place as a one-step reaction but as multi-step reactions. TG curve of neat PP (Fig. 8) showed an apparent single step of decomposition but the Friedman analysis suggested a multi-step of decomposition. The simulation of the curves including only one single step does not give satisfactory results. They were done using reaction models including n-th order function, Avrami-Erofeev function and auto-catalyzed n-th order function but the beginning of the decomposition could not be captured by the model (Fig. 10 (a)). The combination of two successive reactions considering an Avrami-Erofeev function followed by an auto-catalyzed order function gives an excellent fit (Fig. 10 (b)). The number of steps and the resulting calculated kinetic parameters (Table 1) are consistent with the conclusions of the Friedman analysis. The thermal decomposition of PP is a typical radical chain mechanism where initiation, H-abstraction, -scission and radical recombination reactions are the relevant classes [START_REF] Lattimer | Direct analysis of polypropylene compounds by thermal desorption and pyrolysis-mass spectrometry[END_REF]. The first reaction described by an Avrami-Erofeev function (small contribution of about 8% on the whole decomposition) is attributed to chain initiation reactions, which produce radicals and require lower activation energy. The second reaction is attributed to -scission reactions, which are known to be the main decomposition reactions of PP. The autocatalysis is justified by the formation of allyl radicals and allyl H atoms during the decomposition of PP [START_REF] Ranzi | Kinetic modeling of polyethylene and polypropylene thermal degradation[END_REF], which can enhance the decomposition of PP. Our reactional scheme makes then sense with the decomposition pathway of PP. Intumescence occurs mainly in the condensed phase and it involves numerous chemical reactions.

So, kinetic analysis of the reaction in the physical-chemical sense is not possible. That is why we have considered that the overall process of the intumescence could be described by multi-step processes.

For the 4 intumescent PPs, TG curves were simulated using the same reactional scheme as the neat PP suggesting the decomposition of the intumescent PPs is mainly governed by the thermal decomposition of the neat PP. The fits are presented using derivative curves to clearly highlight the effect of the assumed reactional scheme (DTG) (Fig. 11). All the fits are excellent and they can capture all the maxima of the DTG curves. It can be then assumed our reactional scheme is appropriate to describe the thermal decomposition of the intumescent materials. The calculated kinetic parameters are shown in Table 1. The activation energies of the intumescent PPs are slightly higher than that of the neat PP indicating some enhancement of the thermal stability of the intumescent PPs. A goal of kinetic analysis is also to simulate the thermal behavior/decomposition of a material. If our assumptions are accurate, we should be able to simulate the decomposition of PP and intumescent PPs in isothermal conditions far from the dynamic conditions used to make our modeling. Thus, to verify the accuracy of our model, the decomposition PP-based materials was simulated in pyrolytic conditions such described as follows:

-Isothermal at 40°C for 10 min (stabilization of the temperature),

-Ramp from 40 to 425°C at 50°C/min, -Isothermal at 425°C for 120 min. Step 1: n-dim. Avrami-Erofeev

Step 2: n-th order with autocatalysis by C 50.0 K/min 20.0 K/min 10.0 K/min 5.0 K/min 2.0 K/min

As it is observed in Fig. 12 in the case of neat PP, the simulated curve fits very well the experimental curve. It means that our approach can be used to simulate the thermal behavior and the pyrolytic decomposition of neat PP. It also validates our model in two successive steps. Note the two-step decomposing model for PP was also recently evidenced by Thiry-Muller using an extended approach of activated complex applied to kinetic analysis [START_REF] Thiry-Muller | Modélisation de la décomposition thermique des solides[END_REF]. The same comparisons were repeated for the intumescent materials (Fig. 13). In all cases, the simulation can capture the decomposition of the materials in isothermal conditions. It supports therefore the selection of the reactional scheme determined for those materials and it gives a series of models for simulating the decomposition of intumescent materials in various conditions.

CONCLUSION

The incorporation of intumescent additives at relatively low loading (10 wt%) in PP permits the reduction by 70% of pHRR. The mode of action occurs via the formation of an expanded carbonaceous layer in all cases. The protective coating acts mainly as heat barrier in the case of the formulations containing AP760 or as heat dissipater with EG. The incorporation of small amount of EG in PP-AP760 modifies heat transfer in the coating creating a strong anisotropy. Upon expansion graphite worms align normal to the surface increasing the transverse heat conductivity (lower efficiency of the heat barrier) and hence, decreasing the fire performance (decrease by only 30% of pHRR). Kinetic analysis done in dynamic conditions on the intumescent PPs permits to determine the kinetic parameters of the decomposition using a reactional scheme at two successive reactions. This model is able to simulate the decomposition of all the materials in isothermal conditions and hence, it can be considered as validated. On the other hand, the kinetic analysis reveals that the intumescent additives do not modify the reactional scheme of the PP thermal decomposition but they increase slightly the thermal stability of the intumescent systems. Further simulations show it could happen only when the heating rate is not too high. Otherwise, the decomposition of the intumescent is the same as that of neat PP.

  temperature profile of the extruder from feeder to die was set at 200/ 200/ 200/ 200/ 200/ 170/ 185/ 180/ 200/ 200°C.

Fig. 1 .

 1 Fig. 1. HRR curves as a function of time of intumescent PP (external heat flux = 35 kW/m²)

  while the chars containing EG exhibit lower expansion and 'hairy' surface. It evidences therefore two distinct mechanisms: (i) the physical expansion of graphite flakes forming a protective entangled network and (ii) the formation of an intumescent char by a series of chemical reactions. Those two types of protective coating provide the same level of protection according to the cone scenario (except for the combination AP760/EG (9:1)).

Fig. 2 .

 2 Fig. 2. Intumescent coatings formed from intumescent-based PP during the cone calorimetry experiment and after the ignition

Fig. 3 .

 3 Fig. 3. Charring of the formulation PP-AP760/EG (9:1) before the ignition

Fig. 4 .

 4 Fig. 4. Backside temperature as a function of time of intumescent PPs during cone calorimetry experiment (external heat flux = 35 kW/m²);

Fig. 6 .

 6 Fig. 5. X-ray tomography images of cross-section of intumescent coatings obtained after cone calorimetry

Fig. 7 .

 7 Fig. 7. Schematic description of the effects of graphite worms in PP-AP760/EG (9:1) on heat transfer through the charred network.

Fig. 8 .

 8 Fig. 8. TG curves of intumescent PPs compared to neat PP (heating rate = 10 K/min; nitrogen flow)

Fig. 9 .

 9 Fig. 9. Typical Friedman plot showing activation energies as a function of conversion degrees (plot for PP-AP760/EG (5:5))

Fig. 10 .

 10 Fig. 10. Experimental (dots) and simulated (line) TG curves at different heating rates of neat PP fitted with (a) one decomposition step (n-th order function) and (b) with 2 decomposition steps

Fig. 11 .

 11 Fig. 11. Experimental (dots) and simulated (line) TG curves at different heating rates of (a) PP-AP760; (b) PP-EG; (c) PP-AP760/EG(5:5) and (d) PP-AP760/EG(9:1).

Fig. 12 .

 12 Fig. 12. Experimental (dots) and simulated (line) decomposition curves of neat at 425°C for 120 min (N2 flow).

Fig. 13 .

 13 Fig. 13. Experimental (dots) and simulated (line) decomposition curves in isothermal conditions (T = 425°C) of (a) PP-AP760; (b) PP-EG; (c) PP-AP760/EG(5:5) and (d) PP-AP760/EG(9:1).

Fig. 4 Fig. 14 .

 414 Fig.4showed temperature profiles measured on the backside of the PP-based materials. Two extreme profiles can be distinguished: (i) that of PP-EG exhibiting moderate temperature rise (lowest heating rate of 9°C/min) and (ii) that of PP-AP760/EG(9:1) exhibiting high temperature rise (highest heating rate of 310°C/min). The purpose is then to simulate the decomposition curves of all PP-based materials undergoing the two identified extreme temperature profiles. The simulated curves and the temperature profiles (the curves of temperature profiles were built with linear segments extracted from time/temperature curves of Fig.4) are shown on Fig.14. The first profile (low heating rates, Fig.14 (a)) gives curves exhibiting the same behavior as TG curves already commented for Fig.8. The second profile (high heating rates, Fig.14 (b)) shows all PP-based materials decompose in one steep step and all the curves are superimposed (except the char yield, which is the highest at 5 wt.-% for the formulation PP-EG). The decomposition is extremely fast and no further stabilization is provided by the fillers in such conditions. This simulation evidences the incorporated additives do not limit the decomposition rate of PP at high heating rate. It means the intumescent protective coating must be formed rapidly to lower the temperature rise inside the materials and hence, it creates a relatively low heating rate in the materials (creation of large heat gradient in the intumescent coating) and the decomposition of PP can be slowed down. It is consistent with our previous results showing large heat gradient inside the intumescing material and slow temperature rise in the deeper layers of the material[START_REF] Gérard | Reaction to fire of an intumescent epoxy resin: Protection mechanisms and synergy[END_REF].

Table 1 .

 1 Computed kinetic parameters of the decomposition of neat PP and of the 4 intumescent PPs

	Reaction N°	log(A) (A: s -1 )	E (kJ/mol)	Reaction order or Dimension
			PP
	1	11.9  0.3	182  5	0.87  0.07
	2	15.3  0.3	245  3	0.91  0.11
			PP-AP760
	1	13.5  0.3	204  4	0.53  0.02
	2	16.9  0.1	270  2	0.90  0.05
			PP-EG
	1	20.4  1.9	260  15	0.20  0.06
	2	17.2  0.1	276  2	1.10  0.05
			PP-AP760/EG(5:5)
	1	14.9  0.6	219  9	0.58  0.02
	2	16.1  0.1	259  2	0.90  0.03
			PP-AP760/EG(9:1)
	1	13.3  0.4	205  5	0.53  0.02
	2	16.5  0.1	264  2	0.77  0.05

ACKNOWLEDGEMENT

This work has received funding from the European Research Council (ERC) under the European Union's H2020 -the Framework programme for Research and Innovation (2014-2020) / ERC Grant Agreement n°670747 -ERC 2014 AdG/FireBar-Concept.


Guillaume Force

Anna Perfetto

Dr Robert J Mayer

Dr Ilaria Ciofini
email: ilaria.ciofini@chimie-paristech.fr

Dr David Leboeuf
email: dleboeuf@unistra.fr

Dr A Perfetto

Macrolactonization Reactions Driven by a Pentafluorobenzoyl Group

Keywords: macrolactones, hydroxyacids, macrodiolides, mixed anhydride, macrocyclization

Macrolactones constitute a privileged class of natural and synthetic products with a broad range of applications in the fine chemicals and pharmaceutical industry. Despite all the progress made towards their synthesis, notably from seco-acids, a macrolactonization promoter system that is effective, selective, flexible, readily available, and, insofar as possible, compatible with manifold functional groups is still lacking. Herein, we describe a strategy that relies on the formation of a mixed anhydride incorporating a pentafluorophenyl group which, due to its high electronic activation enables a convenient access to macrolactones, macrodiolides and esters with a broad versatility. Kinetic studies and DFT computations were performed to rationalize the reactivity of the pentafluorophenyl group in macrolactonization reactions.

Introduction

Macrolactones are encountered in a broad range of fine chemicals and natural products, notably marine macrolides, pharmaceuticals, cosmetics and agrochemicals. [1] They are one of the foremost pillars of the flavor and fragrance industry with compounds such as Exaltolide ® (Firmenich) and Musc T ® (Takasago) that are prevalent in our everyday lives. [1c] In light of their industrial relevance and the challenges associated with their synthesis, the design of efficient strategies to access macrolactones has sparked many efforts in the synthetic community and the interest in this field is not likely to wane. [2] Though the methods devised for macrolactonizations are numerous, they remain substrate-dependent and the quest for a user-friendly system applicable to a vast array of substrates is as compelling as ever. One of the most common approaches involves the macrocyclization of seco-acids (Scheme 1). In the seventies, a groundbreaking discovery by Yamaguchi revealed that the stoichiometric activation of the carboxylic acid via the formation of a mixed anhydride from 2,4,6trichlorobenzoylchloride (TCBC), could be used as a driving force to promote macrocyclizations. [3] This approach was further improved by Shiina with the development of the 2-methyl-6nitrobenzoic anhydride (MNBA) reagent and related compounds. [4] Other classic examples based on the same concept include Corey-Nicolaou, [5] Steglich-Keck-Boden [6] and Mukaiyama macrolactonizations. [7] However, most of these methods are fraught with distinctive drawbacks that can limit their appeal. To cite but a few examples, they can require highly diluted reactions or a slow addition procedure to prevent oligomerizations. Moreover, their use is often accompanied by the formation of several side-products, which can make the purification of the targeted product cumbersome and be potentially toxic, without forgetting to mention their costeffectiveness as they must be used in stoichiometric amounts. To answer these various challenges, synthetic chemists have mainly focused on three alternative strategies: (i) the development of catalytic systems featuring transition metals, Brønsted and Lewis acids in order to limit the formation of side products, [2i,8] (ii) the use of pre-organized substrates relying in general on non-covalent interactions to facilitate and control the macrocyclization process, [2g] and (iii) ring expansion strategies that usually do not require highly diluted reaction conditions. [2j,2m,9] However, these variants have their own limitations, such as a narrow substrate scope or the requirement for additional synthetic steps to introduce the reactive functional groups. In this context, we aimed at combining the best aspects of the various approaches to achieve a general method for macrolactonizations. Based on their respective efficacy, we reasoned that the formation of a mixed anhydride featuring pentafluorobenzoyl chloride as an activating agent might allow a convenient and optimal access to macrolactones via a supramolecular template. This type of electron-poor receptor can be suitable for numerous non-covalent Scheme 1. Approaches towards the macrolactonization of seco-acids interactions such H-bonding (with respect to the carbonyl moiety), - interactions via a charge-transfer complex and -lone pair (lp) interactions (with respect to the pentafluorophenyl moiety). [10] The latter was particularly of interest as we hypothesized that such interaction could bring both reactive sites in close proximity to favor the intramolecular macrolactonization over oligomerization processes. [11][START_REF] Gallivan | For selected computational studies regarding lone pair- interactions[END_REF][13] Surprisingly, such interactions have hardly been used in synthesis. To the best of our knowledge, the only major application of those interactions was reported by Collins' group to aid the pre-organization in ring-closing metathesis. [14][START_REF] Schmidt | Even if non-covalent interactions were never explicitly mentioned to explain the reactivity, it is noteworthy that related compounds (pentafluorophenylesters) have been used for macrolactamizations[END_REF] In the same vein, we assumed that our approach could be applied to less reactive and underexplored phenol derivatives via - interactions. In addition, due to the strong nucleofugality of the pentafluorobenzoate group, [16] we anticipated that, beside the possibility for supramolecular interactions, we should get a superior activation of the reacting carbonyl group for the cyclization. Lastly, pentafluorobenzoyl chloride is also inexpensive, [START_REF]To draw a comparison, the cost of pentafluorobenzoyl chloride is 100 euros ~ 100 g (1 mmol ~ 0.25 euros) while the one of TCBC is 280 euros ~ 100 g (1 mmol ~ 0.68 euros) and the[END_REF] and the highly water-soluble by-product (pentafluorobenzoic acid) can be easily removed by aqueous treatment, counterbalancing the fact that a stoichiometric amount of pentafluorobenzoyl chloride had to be used. Herein, we thus describe the development of a simple and efficient protocol to access not only macrolactones but also macrodiolides and esters from aliphatic alcohols and phenols.

Results and Discussion

Optimization studies for the macrolactonization of seco-acids. In our initial studies, we investigated the reactivity of 1a to provide access to Exaltolide ® (2a) by screening a variety of benzoyl chlorides and benzoic anhydrides in toluene (Table 1). For practical reasons, we set the reaction time at 24 h to ease the comparison between the various sources of activating agent. To test our initial assumption, we employed pentafluorobenzoyl chloride 1'a as an activating agent and triethylamine as a base, which delivered the targeted product in 88% along with a high selectivity with respect to the macrodiolide 3a (ratio 16:1). More importantly, NMR spectra of the crude product did not show any traces of the pentafluorobenzoic acid after an aqueous treatment with saturated sodium bicarbonate solution (see the Supporting Information for details). As anticipated, reducing the number of fluorine atoms on the benzoyl chloride reagent, which was expected to weaken the interaction between the alcohol and the arene, led to a corresponding decrease in selectivity (1'b-1'f). Interestingly, 3,5-bis(trifluoromethyl)benzoyl chloride 1'g gave the same selectivity as pentafluorobenzoyl chloride 1'a with a slightly lower yield (83%). However, the resulting benzoic acid by-product proved not to be soluble in water, which led us to discard this option. On the other hand, in the presence of electron-withdrawing groups other than fluorine, notably with Yamaguchi's reagent 1'l and Shiina's reagent 1'm, yields of less than 35% were observed along with decomposition of the substrate (TCBC) and formation of higher order oligomers (MNBA), emphasizing the critical role of fluorine for the reactivity. Pentafluorobenzoic anhydride 1'n gave identical results with pentafluorobenzoyl chloride 1'a. Reaction monitoring by 1 H NMR indicated that the reaction reached full conversion within 2 h. Moreover, the transformation could be achieved at room temperature (48 h), but at the expense of a lower selectivity (12:1 2a:3a ratio). Of note, the macrolactonization did not require the use a distilled solvent as neither the selectivity nor the yield was affected in these conditions. However, the macrolactonizations using 1'a requires performing the reaction in diluted solution (1.5 mM in substrate) to avoid a significant decrease in yield because of the formation of oligomers. We also performed a systematic survey of bases and solvents but the initially investigated combination of triethylamine and toluene proved to be superior among those tested (see the Supporting Information for details). Predictably, the utilization of coordinating solvents (THF and MeCN), which might compete with the alcohol for the electron-Table 1. Optimization of the macrolactonization of 1a. a a The yields given are the isolated yields for 2a. The ratios were determined by 1 H NMR using para-anisaldehyde as an external standard. b Reaction conducted with toluene not distilled. c Reaction conducted at room temperature for 48 h. NR = no reaction.

poor host receptor, tends to diminish the selectivity (10:1 and 12:1, respectively).

Scope of macrolactones and macrodiolides. With these optimized conditions in hand, we explored the scope of the reaction using simple all-carbon linkers (2a-2h, Table 2). The macrolactonization was particularly effective for large ring sizes (from 17-to 29membered rings 2b-2e, yields up to 98%), without observing the formation of the corresponding macrodiolides. On the other hand, the proportion of macrodiolide increased with smaller ring sizes (2f-2h). While the yields remained high for 13-and 15-membered rings (72 and 81% yields, respectively), the ratio between macrolactone and macrodiolide attained 1:1 with 10hydroxydecanoic acid 1h, providing 2h in 31% yield. Further, the reaction was compatible with various functional groups, including triazole, strained 1,3-butadiyne, alkene and diol (protected or not) moieties, affording the target macrolactones in yields ranging from 65% to 89%. The method could also be applied to secondary aliphatic alcohols and carboxylic acids (2n-2t) and even challenging 8-membered rings such as 2q were obtained in 42% yield. [START_REF] Shiina | [END_REF] In addition, the framework of resorcylic lactones (2n) [19] and cyclodepsipeptide 2t were generated in 76 and 74% yields. Lastly, tertiary aliphatic alcohols were also tolerated, albeit in a lower yield (2t, 35%). The devised strategy is not limited to macrolactonization of aliphatic hydroxyacids, but was also extended to substrates incorporating phenols, [START_REF] Liu | For rare examples of macrolactonizations with the formation of a mixed anhydride, using a phenol as a partner[END_REF] delivering the target products, notably arduous meta-and para-cyclophanes in high yields (82 to 85%), compounds that have many applications in materials science and supramolecular chemistry. [21] Nevertheless, the reaction with phenols proved to be slower as the reactions required 48 h to reach full conversion. Finally, we turned our attention to the preparation of macrodiolides (compounds 6-11), which are widespread scaffolds in medicinal chemistry. [22] Their synthesis is known to be more challenging than the precedent case since two chemical events (esterification then macrolactonization) are involved when starting from seco-acids or diacids. [23] We were pleased to find that 16-to 34-membered macrodiolides could be rapidly afforded in high yields (up to 89%). Moreover, Musk T ® and Zenolide ® (10 and 11), two compounds with major industrial applications, were generated in a single step within 48 h in 74% and 68% yields.

Comparison with prior art. The major strengths of our approach are (i) its compatibility with a large variety of primary, secondary, tertiary aliphatic alcohols, diols, and phenols, (ii) an inexpensive activating reagent (pentafluorobenzoyl chloride), and (iii) the easily removable side-products through a simple aqueous workup. The key question is where our method stands compared to previous reports from the literature featuring seco-acids, including the methods involving the formation of a mixed anhydride (e.g., the use of MNBA) and the catalytic approaches. In the case of MNBA, we compared our data on identical substrates (see Table 1). [4c] With respect to traditional substrates, we noticed that both methods were similarly efficient. However, in the case of more challenging reactions, such as the formation of macrodiolides and macrolactones incorporating a phenol moiety, our method formed products in significantly higher yields than the previous method, e.g., 84% vs <10% yield of 2w and 81% vs 30% yield of 8, respectively. Another important factor is that pentafluorobenzoyl chloride is significantly cheaper than MNBA, [START_REF]To draw a comparison, the cost of pentafluorobenzoyl chloride is 100 euros ~ 100 g (1 mmol ~ 0.25 euros) while the one of TCBC is 280 euros ~ 100 g (1 mmol ~ 0.68 euros) and the[END_REF] without requiring any slow addition of the reagents during the reaction set-up. More recently, the group of Collins reported a method to access macrolactones and macrodiolides (13-to 26-membered macrolactones and macrodiolides in yields ranging from 55 to 98%) by employing Hf(OTf)4 as a catalyst. However, those reactions conditions were not compatible with secondary and tertiary aliphatic alcohols and were less effective with ring sizes smaller than 13-membered rings. In turn, Zhao and co-workers depicted the use of an ynamide (N-ethynyl-N,4dimethylbenzenesulfonamide) as a reacting agent with the carboxylic acid to deliver an enamide that undergoes a cyclization with the alcohol in the presence of p-toluenesulfonic acid. This strategy afforded 7-to 19-membered macrolactones in yields ranging from 52 to 96% over 2 steps. However, not only do the reaction requires the use of an expensive ynamide, but stoichiometric amounts of N-methyl-N-tosylacetamide were formed, which tends to make the purification of the products troublesome. Of note, the possibility of using substrates bearing phenols was not reported in any of those studies. Regarding the preparation of compound 10 (Musk T ® ), apart from industrial processes involving the thermal cleavage of oligoesters, the only macrodilactonization described led to this compound in only 20% yield via the use of Brønsted and Lewis acids. [24] Scope of esters. We also investigated intermolecular esterifications (Table 3). Once more, this procedure showcased a broad functional group tolerance at a higher concentration when compared to macrolactonizations (0.1 M). The reaction proved to be compatible with primary as well as secondary and tertiary alcohols producing high yields (up to 98%). In a similar manner, primary, secondary and tertiary carboxylic acids exhibited the same efficiency. Moreover, we succeeded to accomplish the mono-acetylation of 1,4-butanediol (14ae) in 71% yield. Even a poorly nucleophilic alcohol, trifluoroethanol, delivered the corresponding ester 14ad in 72% yield. Reactions of less reactive aryl alcohols such as phenol and naphthol provided the ester products in 80% and 72%, respectively, in 48 h. Of note, substrates bearing acid-sensitive functional groups (TBS, Boc and acetal) were well-tolerated. Finally, compounds of interest such as amino alcohols (14ag), amino acids (14ba), carbohydrates (14ah), steroids (14ak) and fatty acids (14em) underwent esterifications with yields ranging from 54% to 91%.

Having identified the pentafluorobenzoyl group as an excellent activating agent for macrolactonization processes, we next turned to study the origins of its reactivity and selectivity. In particular, we wondered whether the cyclization occurred via supramolecular interactions as initially hypothesized. To shed light on the mode of operation of our system, we conducted experimental studies, which were further corroborated by Density Functional Theory (DFT) computations.

Kinetic studies. First, we studied the kinetics of the reaction of 1a with 1'a in the presence of triethylamine by 1 H and 19 F NMR spectroscopy in toluene-d8 at 23 °C (the proposed mechanism is shown in Figure 1A). From the first spectrum recorded, disappearance of the α-CH2 group of 1a was observed and a new downfield-shifted resonance appeared which we attributed to the formation of the mixed anhydride I-1a. While I-1a could not be isolated due to its slow cyclization to 2a, evidence for its identity was found by comparison of the 1 H and 19 F NMR spectra with those of the mixed anhydride obtained by the analogous reaction of hexanoic acid with 1'a (see the Supporting Information). Over the course of 65 h, disappearance of the 1 H resonance of the CH2-OH group of I-1a was accompanied by an increase of the signal intensity of the CH2-O-C(O) moiety of macrolactone 2a (Figure 1B). During the first 5 h of the reaction, 19 F NMR shows the disappearance of the resonances of remaining 1'a and initially the Table 3. Reaction scope for intermolecular esterifications. a The yield was determined by 1 H NMR using para-anisaldehyde as an external standard since the silyl group was cleaved on silica gel to give 14ae. b Reaction conducted at 80 °C. c Phenol (2 equiv). d Alcohol 13l (0.8 equiv). e Alcohol 13a (1.5 equiv).

formation of pentafluorobenzoic anhydride. Only after complete consumption of remaining 1'a/Et3N, appearance of the 19 F resonances of pentafluorobenzoic acid were observed (Figure 1C).

As shown in Figure 1B/C, both the increase of the concentration of 2a (Figure 1A, black points) and the disappearance of I-1a were found to be first-order processes which could be analyzed by least-squares fitting with the single exponential A[1exp(-kobs t)] + C and A*exp(-kobs t)] + C, respectively (for all individual fits, see the Supporting Information). Analogously, the relative concentrations of I-1a and pentafluorobenzoic acid as obtained from 19 F NMR spectroscopy were analyzed. For all increase/decrease processes, the analogous first-order rate constant kobs = (1.52 ± 0.14)  10 -5 s -1 was observed, which corresponds to a half-life of t1/2 ≈ 13 h (Figure 1B/C). Such firstorder kinetics are in line with the expected rate-determining intramolecular cyclization between the hydroxy group and the anhydride intermediate I-1a (Figure 1A), followed by elimination of pentafluorobenzoic acid to yield 2a.

To verify this interpretation and to exclude a bi-molecular reaction being involved in the rate-determining step, the rate constants were additionally determined at twice lower concentration of all reactants. Identical rate constants as determined for the higherconcentrated reaction were observed for the formation of 2a and disappearance of I-1a with kobs = (1.45 ± 0.06)  10 -5 s -1 (k from 1 H data), thereby supporting our mechanistic interpretation. The mono-molecular nature of the rate-determining step is further F NMR (471 MHz) NMR spectroscopy for the reaction of 1a (1.6 mM) with 1'a (1.8 mM) in d8-toluene at 23 °C. The dashed line corresponds to the fitted kinetic curve used for the determination of the rate constants k1. Note: The first data-point for I-1a was not included in the fitting, as the reaction does not immediately dissolve. The concentrations in (B) were determined from the integral ratios relative to tetrachloroethane used as internal standard while for (C) the normalized integral concentrations are depicted.

supported by measurement of the rate constants of the reaction (1.4 mM in 1a) in the presence of excess Et3N (2.5 equiv) which only resulted in an insignificantly different value of kobs = (1.62 ± 0.06)  10 -5 s -1 (k from 1 H data).

Slightly faster than intramolecular cyclization is bimolecular cyclization to dimer 3a, which can also be described by a monoexponential increase function. During the reaction, however, decreasing concentrations of I-1a disfavor the bimolecular reaction, and finally after 65 h, a ratio of 2a:3a of 1:12 was observed which is in line with the observed ratio on preparative scale (Table 1). At twice lower concentration of all reactants the bimolecular reaction becomes less favored. Accordingly, a smaller rate constant for dimer-formation is observed as well as a higher selectivity for the cyclization product with 2a:3a now being 22:1.

To test, if the reaction is promoted by additional hydrogenbonding from ammonium ions (Et3NH + ) formed as a by-product, the reaction was conducted in the presence of additional 0.5 equiv triethylammonium pentafluorobenzoate (Figure S7). In that case, a slightly reduced rate constant was observed compared to the standard conditions (kobs = (1.33 ± 0.07)  10 -5 s -1 (k from 1 H data)).

DFT studies. To gain further insights in the role of potential noncovalent interactions in the macrolactonization mechanism, DFT calculations (see the Supporting Information for computational details) were performed on different supramolecular template molecules (1α-1δ in Figure 2) obtained from 11hydroxyundecanoic acid complexed with different activating agents. Namely, we considered pentafluorobenzoyl chloride (1'a), p-fluorobenzoyl chloride (1'f), p-nitrobenzoyl chloride (1'i) and benzoyl chloride (1'm) to test the effect of the presence, the nature and strength of electron-withdrawing group(s). For each of these systems several stable conformers were obtained whose energies and 3D structures are collected in the Supporting Information. In the case of 1α (Figure 3), the most stable conformers are the ones stabilized either by a H-bond interaction (B and C) or by a -lp interaction (A). [11-12] Among these conformers, A is the conformer presenting most likely the best preorganization to access conformer F, which has the correct orientation to effectively react to form the final macrolactone. On the other hand, in the case of 1α, the non-reactive conformers (D and E) are more than 3.5 kcal/mol higher in energy with respect to the most stable conformer B. Of note, calculations using either a global or a double hybrid functional (PBE0 [25] and PBE0-DH, [26] respectively) returned the same trends, indicating that the theoretical outcomes are not an artifact of the DFT approach chosen. H bond interactions are efficient in stabilizing the conformers: The H bond interaction between the alcoholic group with the oxygen of the anhydride residue (with a distance of 2.00 Å) stabilizes the conformer B, while the interaction of the same alcoholic proton with the fluorine atoms of the benzoyl group stabilizes conformer C. This interaction is indeed weaker by 1 kcal/mol and the corresponding distance is slightly larger (in average 2.82 Å). Interestingly, in the case of the other supramolecular templates (1β-1δ), for which a decrease in reactivity and selectivity has been experimentally observed, such -lp interaction could not be located. For these systems, a nonreactive conformer A is thermodynamically accessible presenting the reaction sites in opposite positions (Figures S8-S10). To verify the presence of non-covalent interactions involved in this reaction, we also plotted noncovalent interaction (NCI) surfaces computed for all six conformers of 1α complex (Figure S11). [27] The NCI plots show the H-bond interactions that stabilizes the conformers B and C but, more importantly, NCI plots recovers also the -lp interaction between the lone pair of the oxygen and the fluorinated ring in the case of conformers F and A that are the keys for the cyclization.

Based on the kinetic studies, we then studied the one-step reaction path leading to the final macrodiolide 16 starting from 1α-1δ to validate the macrolactonization mechanism. The computed Gibbs energy diagrams for 1α-1δ with respect to the most stable conformer are depicted in Figure 4. The corresponding reaction pathways computed for all templates are reported in the Supporting Information together with all corresponding optimized structures (Figures S12-S15). A simultaneously proton transfer and a cyclization leads to the formation of the final product 16: the proton transfer takes place from the alcoholic residue towards the carboxylic oxygen, thereby leading to the ring closure with the formation of a new C-O bond. Analysis of the atomic (Mulliken) charges computed for the initial reagent (15-1α) reveals that the complexes 1β-1δ, the computed activation energies are between 1.3 and 1.6 kcal/mol higher than for complex TS-1α, in line with the experimental observation of lower conversion for these systems (Table 1). This can be rationalized by the reacting carbonyl groups of 1β-1δ being less electrophilic as showed by the calculated partial charges on the carbon (for 1β: δ-= -0.04 |e-|, for 1β: δ+= 0.19 |e-| and for 1δ: δ-= -0.04 |e-|). Consequently, the computational analysis supports our initial hypothesis that the pentafluorophenyl group is a superior promoter for macrolactonization as it increases the electrophilicity of the relevant carbonyl group and has the potential to pre-organize the substrates through lp- interactions. Those results also explain why, in the methods developed by Yamaguchi and Shiina, 4dimethylaminopyridine (DMAP) is typically required as an acyl transfer reagent to enable the macrolactonization, which is not necessary with our method. [3,4]

Conclusion

In summary, we have discovered an efficient and user-friendly protocol to achieve macrolactonizations, generating products that have the potential to make an impact in important applied fields such as medicinal chemistry and the fragrance industry. This transformation, which featured the formation of a mixed anhydride based on the utilization of an inexpensive activating agent (pentafluorobenzoyl chloride), displayed a remarkable efficacy to access a wide range of ring size, while overcoming most of the common limitations associated with such processes. For instance, this unique method could be equally applied to intramolecular macrolactonizations of seco-acids or to the intermolecular formation of macrodiolides from diacids and diols and even esters. Kinetic studies showed that after rapid formation of the mixed anhydride from pentafluorobenzoyl chloride (1'a) and the substrate, mono-molecular cyclization is the rate-determining step of the reaction. The potential formation of non-covalent interactions is underpinned by DFT computations, which indicated that, in substrates featuring the pentafluorophenyl moiety, both strong-hydrogen bonding as well as -lp interactions are present. Further applications of the pentafluorophenyl moiety are presently investigated to take advantage of the unique reactivity of this structural motif.

  Figure 1. (A) Proposed reaction mechanism. (B) Kinetics followed by 1 H (500 MHz) and (C) 19F NMR (471 MHz) NMR spectroscopy for the reaction of 1a (1.6 mM) with 1'a (1.8 mM) in d8-toluene at 23 °C. The dashed line corresponds to the fitted kinetic curve used for the determination of the rate constants k1. Note: The first data-point for I-1a was not included in the fitting, as the reaction does not immediately dissolve. The concentrations in (B) were determined from the integral ratios relative to tetrachloroethane used as internal standard while for (C) the normalized integral concentrations are depicted.

Figure 2 .

 2 Figure 2. Structural formulae of supramolecular template molecules 1α-1δ.

Figure 3 .

 3 Figure 3. Molecular structures of all optimized conformers of 1α complex together with their relative energy values computed using a global hybrid (PBE0) and a double hybrid (PBE0-DH) functional. carbonyl carbon presents a partial positive charge (δ+= 0.32 |e-|), so that a nucleophilic attack can be envisaged from the oxygen (δ-= -0.53 |e-|). Moreover, the H-bond favors the formation of a six-membered ring structure. Thus, the corresponding transition state (TS-1α) lying 19.8 kcal/mol above 15-1α clearly corresponds to a concerted ring closure and proton transfer, as indicated by the shortening of C-O (1.81 Å) bond and the simultaneous elongation of the O-H (1.02 Å) bond. The

Figure 4 .

 4 Figure 4. Computed energy diagrams for 1-1. The zero-energy value has been assigned to the reagent for each case.

Table 2 .

 2 Reaction scope for the formation of macrolactones and macrodiolides. a Ratio macrolactone/macrodiolide 8:1 (separable by flash column chromatography). Ratio macrolactone/macrodiolide 8:1 (separable by flash column chromatography). c Ratio macrolactone/macrodiolide 1:1 (separable by flash column chromatography). d Reaction conducted at 50 °C. d Reaction conducted at 80 °C. f Reaction conducted for 48 h.

b

Acknowledgements

G.F. and D.L. gratefully thank the ANR (ANR-16-CE07-0022 funding for GF), the CNRS and the Université Paris-Saclay for the support of this work. A.P. and I.C. thank the European Research Council (ERC) for funding under the European Union's Horizon 2020 research and innovation program (grant agreement No 648558, STRIGES CoG grant). We thank Dr. Pawel Dydio and Pr. Joseph Moran for useful discussions regarding the manuscript. We also thank Dr. Marie Vayer and Shaofei Zhang for their help during the revisions of the manuscript.

Entry for the Table of Contents

Old made new again: The onset of macrolactonizations can be initiated using the inexpensive pentafluorobenzoyl chloride as an activating agent to deliver a large variety of macrolactones, macrodiolides and esters with high efficiency through a robust protocol. Notably, this process could be easily extended to less reactive phenol derivatives.

Institute and/or researcher Twitter usernames: @djpleboeuf; @group_ctm