Jean-Luc Putaux
email: jean-luc.putaux@cermav.cnrs.fr

Cong-Anh-Khanh Le

Luc Choisnard

Denis Wouessidjewe

Polymorphism of crystalline complexes of V-amylose with fatty acids

Keywords: PROA: propanoic acid (C3), BUTA: butanoic acid (C4), PENA: pentanoic acid (C5), HEXA: hexanoic acid (C6), OCTA: octanoic acid (C8), DECA: decanoic acid transmission electron microscopy, ED: electron diffraction, XRD: X-ray diffraction

Polymorphism of crystalline complexes of V-amylose with fatty acids

Introduction

Amylose, a constituent of starch, is a mostly linear polymer consisting of α-D-glucose units connected by α(1→4) bonds with less than 1% α(1→6) branching linkages [START_REF] Hanashiro | Fine structure of amylose[END_REF]. The linear nature of the chain and the torsion angles around the osidic linkage promote helical molecular trajectories. Amylose can be crystallized in vitro from aqueous solutions into so-called A-and B-type allomorphs that both consist of the parallel packing of 6-fold left-handed double helices associated with a number of water molecules [START_REF] Popov | Crystal structure of A-amylose: A revisit from synchrotron microdiffraction analysis of single crystals[END_REF][START_REF] Imberty | A revisit to the three-dimensional structure of B-type starch[END_REF].

Amylose also exists as single helices when it crystallizes in the presence of a variety of organic and inorganic molecules [START_REF] Bear | Complex formation between starch and organic molecules[END_REF][START_REF] Mikus | The complexes of fatty acids with amylose[END_REF][START_REF] Bluhm | Detailed structure of the Vh-amylose-iodine complex: a linear polyiodine chain[END_REF][START_REF] Tomasik | Complexes of starch with inorganic guests[END_REF][START_REF] Tomasik | Complexes of starch with organic guests[END_REF]. The resulting inclusion compounds, known under the generic name of V-amylose, are generally crystallized from aqueous solutions [START_REF] Putseys | Amylose-inclusion complexes: formation, identity and physico-chemical properties[END_REF][START_REF] Obiro | V-amylose structural characteristics, methods of preparation, significance, and potential applications[END_REF]. In the literature, several types of V-amylose have been reported that can readily be distinguished by their X-ray diffraction (XRD) powder pattern. In addition, transmission electron microscopy (TEM) has significantly contributed to the morphological and structural characterization of V-amylose lamellar single crystals prepared from dilute solutions. Since the helical axis of amylose is perpendicular to the approximately 10 nm-thick lamellae, base-plane electron diffraction (ED) patterns were particularly helpful to identify the different crystal systems [START_REF] Yamashita | An electron microscopy study on crystals of amylose V complexes[END_REF][START_REF] Lourdin | Crystalline structure in starch[END_REF]. Depending on the guest molecule, the single helices can be made of 6, 7 or 8 glucosyl units per turn, organized into different unit cells. VH crystals prepared in the presence of ethanol [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF] or fatty acids [START_REF] Godet | Molecular modelling of the specific interactions involved in the amylose complexation by fatty acids[END_REF] are hexagonal. They contain left-handed 6-fold helices, the organization of which has been described using orthorhombic [START_REF] Mikus | The complexes of fatty acids with amylose[END_REF][START_REF] Rappenecker | Detailed refinement of the crystal structure of Vhamylose[END_REF] or hexagonal [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part X. X-Ray diffraction of amylose-fatty acid complexes[END_REF] unit cells. Six-fold helices have also been reported to occur in other types of crystals prepared with iodine [START_REF] Bluhm | Detailed structure of the Vh-amylose-iodine complex: a linear polyiodine chain[END_REF], nbutanol [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or npentanol: structural features and properties[END_REF], dimethyl sulfoxide [START_REF] Winter | Crystal and molecular structure of the amylose-DMSO complex[END_REF], ethylenediamine [START_REF] Simpson | X-Ray diffraction of ethylenediamine-amylose complex[END_REF] and glycerol [START_REF] Hulleman | Single crystals of V amylose complexed with glycerol[END_REF]. Bear [START_REF] Bear | Complex formation between starch and organic molecules[END_REF] suggested that bulkier molecules would be accommodated in a helix with a larger cross-section, i.e. with more residues per turn. Zaslow [START_REF] Zaslow | Characterization of a second helical amylose modification[END_REF] compared the volume of the pseudo-hexagonal unit cells of the partially dried complexes formed with tert-butanol and n-butanol and obtained a ratio of about 7:6 in favor of the existence of a 7-fold helix for Vtert-butanol. Yamashita and Hirai confirmed this observation by preparing crystals with branched alcohols larger than n-butanol [START_REF] Yamashita | Single crystals of amylose V complexes. II. Crystals with 71 helical configuration[END_REF]. V-amylose crystals prepared in the presence of 2-propanol have a rectangular shape and an orthorhombic unit cell [START_REF] Buléon | Single crystals of V amylose complexed with isopropanol and acetone[END_REF][START_REF] Nuessli | Crystal structure of amylose complexes with small ligands[END_REF]. Although models based on 6-and 7-fold helices have both been proposed [START_REF] Yamashita | Single crystals of amylose V complexes. II. Crystals with 71 helical configuration[END_REF][START_REF] Buléon | Single crystals of V amylose complexed with isopropanol and acetone[END_REF], using a combination of structure refinement based on conformational and packing energy analyses with classical crystalline polymer structure refinement, Nishiyama et al. have shown that the 7-fold conformation was the most probable [START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF]. Finally, when crystallized with 1-naphthol or quinoline, amylose forms square single crystals corresponding to a tetragonal organization of 8-fold helices [START_REF] Cardoso | Single crystals of V-amylose complexed with α-naphthol[END_REF].

The complexes of starch with fatty acids have been particularly studied since both types of molecules coexist and interact in some native starch granules [START_REF] Morrison | Lipids in cereal starches: A review[END_REF] and in a large variety of processed food [START_REF] Schoch | Adsorption of fatty acid by the linear component of corn starch[END_REF][START_REF] Davies | Inclusion complexes of free fatty acids with amylose[END_REF][START_REF] Cao | Facile synthesis and structural characterization of amylose-fatty acid inclusion complexes[END_REF]. Various formulations and processing methods have been tested [START_REF] Fanta | Steam jet cooking of high-amylose starch-fatty acid mixtures. An investigation of complex formation[END_REF][START_REF] Shogren | X-ray diffraction study of crystal transformations in spherulitic amylose/lipid complexes from jet-cooked starch[END_REF][START_REF] Lesmes | Effects of long chain fatty acid unsaturation on the structure and controlled release properties of amylose complexes[END_REF] to develop products with potential health benefits [START_REF] Panyoo | Amylose-lipid complex production and potential health benefits: A mini-review[END_REF]. Linear saturated fatty acids form crystalline complexes with amylose from aqueous solutions [START_REF] Godet | Molecular modelling of the specific interactions involved in the amylose complexation by fatty acids[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. Several electron diffraction (ED) and X-ray diffraction (XRD) studies have reported that the complexes corresponded to the VH-type regardless of the fatty acid chain length (C8-C18) [START_REF] Mikus | The complexes of fatty acids with amylose[END_REF][START_REF] Yamashita | An electron microscopy study on crystals of amylose V complexes[END_REF][START_REF] Lesmes | Effects of long chain fatty acid unsaturation on the structure and controlled release properties of amylose complexes[END_REF][START_REF] Godet | Crystallization of amylose-fatty acid complexes prepared with different amylose chain lengths[END_REF]. Some authors classified complexes of amylose with fatty acids into different types (I, IIa and IIb) based on thermal behaviors. However, the XRD profiles showed that they all corresponded to the VH allomorph but mostly differed by their degree of crystallinity [START_REF] Lesmes | Effects of long chain fatty acid unsaturation on the structure and controlled release properties of amylose complexes[END_REF][START_REF] Karkalas | Some factors determining the thermal properties of amylose inclusion complexes with fatty acids[END_REF][START_REF] Tufvesson | Formation of amylose-lipid complexes and effects of temperature treatment. Part 2. Fatty acids[END_REF]. Studying complexes with shortchain fatty acids, Takeo et al. [START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part X. X-Ray diffraction of amylose-fatty acid complexes[END_REF] revealed that by varying the water/propionic acid ratio, amylose could crystallize into different allomorphs that would contain 6-or 7-fold helices, organized into hexagonal or orthorhombic unit cells, respectively. After this first evidence of polymorphism of V-amylose with a given fatty acid, only a few examples have been documented.

Helbert [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF] reported that butanoic acid (C4) could also induce both 6-or 7-fold helices. Hexanoic acid (C6) was also shown to yield crystals with a VH structure [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF] as well as a second arrangement of 6-fold helices, isomorphous to that observed with complexes with n-butanol [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or npentanol: structural features and properties[END_REF][START_REF] Biais | Structural and stoichiometric studies of complexes between aroma compounds and amylose. Polymorphic transitions and quantification in amorphous and crystalline areas[END_REF].

The Vfatty acids complexes have frequently been used as models to study various characteristics and properties of V-amylose: morphology and crystal structure [START_REF] Mikus | The complexes of fatty acids with amylose[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part X. X-Ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Godet | Crystallization of amylose-fatty acid complexes prepared with different amylose chain lengths[END_REF], physicochemical properties [START_REF] Karkalas | Some factors determining the thermal properties of amylose inclusion complexes with fatty acids[END_REF][START_REF] Marinopoulou | Amylose-fatty acid inclusion complexes as examined by interfacial tension measurements[END_REF][START_REF] Ozcan | The impact of thermal events on amylose-fatty acid complexes[END_REF][START_REF] Raphaelides | Thermal dissociation of amylose-fatty acid complexes[END_REF][START_REF] Raphaelides | Rheological studies of starch-fatty acid gels[END_REF][START_REF] Singh | Effect of fatty acids on the rheological properties of corn and potato starch[END_REF], enzymatic hydrolysis and digestibility [START_REF] Marinopoulou | Amylose-fatty acid inclusion complexes as examined by interfacial tension measurements[END_REF][START_REF] Gelders | Enzyme and acid resistance of amylose-lipid complexes differing in amylose chain length, lipid and complexation temperature[END_REF][START_REF] Holm | Digestibility of amylose-lipid complexes in-vitro and in-vivo[END_REF][START_REF] Marinopoulou | Morphological characteristics, oxidative stability and enzymic hydrolysis of amylosefatty acid complexes[END_REF], encapsulation and release properties [START_REF] Godet | Crystallization of amylose-fatty acid complexes prepared with different amylose chain lengths[END_REF][START_REF] Gelders | Amylose-lipid complexes as controlled lipid release agents during starch gelatinization and pasting[END_REF][START_REF] Morrison | Solvent extraction of fatty acids from amylose inclusion complexes[END_REF] as well as complexation protocols and factors affecting the crystallization [START_REF] Tufvesson | Formation of amylose-lipid complexes and effects of temperature treatment. Part 2. Fatty acids[END_REF][START_REF] Marinopoulou | Morphological characteristics, oxidative stability and enzymic hydrolysis of amylosefatty acid complexes[END_REF][START_REF] Bhatnagar | Starch-stearic acid complex development within single and twin screw extruders[END_REF].

Since some fatty acids (propanoic, butanoic and hexanoic acids) have been reported to yield different crystalline forms, it seemed likely that polymorphism would also be observed with other linear fatty acids of the homologous series, using suitable crystallization conditions. In order to shed some light on the parameters that control the polymorphism of the Vfatty acid complexes, we have investigated the crystallization of amylose in dilute aqueous solution with a series of saturated linear fatty acids of increasing length from C3 to C20. The morphology and structure of the resulting lamellar crystals were characterized by TEM, ED and XRD.

Experimental

Amylose and fatty acids

Potato amylose was purchased from Sigma-Aldrich and further purified as previously reported [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. Briefly, 30 g of amylose was dissolved in 1 L of dimethyl sulfoxide (DMSO) by stirring for 12 h at room temperature. The solution was centrifuged (2500 g, 20 min) to remove residual aggregates. Amylose was precipitated by addition of an equal volume of ethanol, kept at 4 °C for 12 h, filtered (glass filter G5) and extensively rinsed with ethanol, acetone and diethyl ether, successively, using a Büchner device hooked to a water suction pump. Amylose was then dried for one week in an exhaust hood at room temperature. A series of fatty acids was purchased from Sigma-Aldrich: propanoic acid (referred to as PROA -C3), butanoic acid (BUTA -C4), pentanoic acid (PENA -C5), hexanoic acid (HEXA -C6), octanoic acid (OCTA -C8), decanoic acid (DECA -C10), dodecanoic acid (DODA -C12), tetradecanoic acid (TEDA -C14), octadecanoic acid (OCDA -C18) and icosanoic acid (ICOA -C20).

Crystallization protocols

Aqueous amylose dispersions (0.1 wt%) were submitted to nitrogen bubbling for 20 min and autoclaved in an oil bath at 160 °C for 30 min. In other tests, amylose solutions were prepared by dissolving amylose in DMSO at 100 °C, then diluting with boiling water to the desired concentration (0.1 wt% amylose). Two and 10 vol% DMSO were tested. The concentration of fatty acids varied depending on their chain length and physical state: 5-50 vol% for C3 and C4 (water-miscible liquids), 2.5-5.0 vol% for C5 (water-immiscible liquid), and saturation concentration for C6 to C20 (poorly water-soluble liquids or solids). Preheated liquid fatty acids were mixed with amylose solutions at 90 °C, while solid fatty acids were added either before or after the dissolution of amylose. The mixtures were then kept at a predetermined temperature (between 40 and 90 °C, depending on the fatty acid) for one week or slowly cooled down to room temperature in a Dewar bottle initially filled with boiling water. In the case of watermiscible fatty acids, the resulting crystals were stored in their mother liquor. For waterimmiscible fatty acids, the excess of liquid floating on top of the dispersions was removed by pipetting. The excess of insoluble solid fatty acids was removed by washing the wet solid fraction of the decanted dispersion three times with diethyl ether.

X-ray diffraction (XRD)

The crystal suspensions were centrifuged and the excess solvent was removed. The pellets were deposited on a 7-µm nylon bolting cloth, equilibrated over a salt-saturated aqueous solution with 95% relative humidity (RH). Fragments of the resulting films were inserted into 1.0 mm (outer diameter) X-ray glass capillaries which were immediately flame-sealed. The specimens were X-rayed in vacuum by a Ni-filtered CuKα radiation (λ = 0.15418 nm) using a Philips PW3830 generator operating at 30 kV and 20 mA, equipped with a Wahrus camera. Twodimensional diagrams were recorded on Fujifilm image plates over 2-h exposures and read with a Fujifilm BAS 1800-II bioimaging analyzer. The 2D diffraction diagrams were rotationally averaged to obtain diffraction profiles that were normalized between 5 and 30° (2θ). The XRD data were calibrated using the diffraction pattern of calcite (CaCO3) powder and the cell parameters were calculated using the CelRef module (version 3) of the LMGP package [START_REF] Laugier | LMGP-Suite: Suite of programs for the interpretation of X-ray experiments[END_REF].

Transmission electron microscopy (TEM) and electron diffraction (ED)

Drops of dilute crystal suspensions were deposited on glow-discharged carbon-coated grids and allowed to dry. The specimens were observed under low dose illumination with a FEI-Philips CM200 'Cryo' microscope operating at 200 kV. For electron diffraction, the TEM grids were mounted on a Gatan 626 specimen holder and fast-frozen into liquid nitrogen just after air drying and prior to being introduced in the microscope. The holder was then cooled down with liquid nitrogen and the crystals were observed under low dose illumination at low temperature (-177 °C). Base-plane electron diffraction patterns were recorded from selected areas of about 1 µm 2 .

Images and diffraction patterns were recorded with a TVIPS TemCam F216 camera. The diffraction patterns were calibrated using a gold-coated carbon film as standard.

Results

In the following, we will refer to three distinct allomorphs of V-amylose, using a notation based on that proposed by Helbert [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. The compact hexagonal packing of 6-fold helices obtained with linear alcohols [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF] and fatty acids [START_REF] Godet | Molecular modelling of the specific interactions involved in the amylose complexation by fatty acids[END_REF], often called "VH", will be referred to as "V6I" whereas the structure proposed by Helbert and Chanzy [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or npentanol: structural features and properties[END_REF] to describe Vn-butanol or Vn-pentanol crystals will be called "V6II". While Zaslow [START_REF] Zaslow | Characterization of a second helical amylose modification[END_REF] had modeled the unit cell of complexes prepared with tertbutanol using 7-fold helices, Buléon et al. [START_REF] Buléon | Single crystals of V amylose complexed with isopropanol and acetone[END_REF] later proposed an alternate model based on 6-fold helices to describe the structure of similar crystals prepared with 2-propanol or acetone, hence the "V6III" denomination that can be found in some articles [START_REF] Putseys | Amylose-inclusion complexes: formation, identity and physico-chemical properties[END_REF][START_REF] Biais | Structural and stoichiometric studies of complexes between aroma compounds and amylose. Polymorphic transitions and quantification in amorphous and crystalline areas[END_REF][START_REF] Bail | Structural investigation of amylose complexes with small ligands: Helical conformation, crystalline structure and thermostability[END_REF]. However, Nishiyama et al. [START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF] have recently demonstrated that the constituting helices were more likely 7-fold.

Consequently, we will use the notation "V7" in the following sections.

Morphology and structure of individual allomorphs

Depending on the crystallization conditions (solvent, temperature, etc.), three basic types of lamellar crystals were distinguished, individually or in mixtures, according to their crystallization pattern, morphology and crystal structure, although for each type, some minor variations in shape were observed. S1). The XRD powder patterns recorded from crystals equilibrated at 95% RH were all identical as well (Figure 1f, Supplementary Data Figure S7) and could be indexed along a hexagonal unit cell. The parameters calculated for the whole series of fatty acids are given in Supplementary Data Table S2. The average parameters of the hexagonal unit cell are a = b = 1.37 ± 0.01 nm and c = 0.81 ± 0.01 nm, and a = 1.37 ± 0.01, b = a√3 = 2.38 ± 0.01 nm and c = 0.81 ± 0.01 nm for the orthorhombic unit cell. The reflection indices are provided for one example (VPENA) in Supplementary Data Table S3. All these hexagonal crystals thus belong to the V6I family [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF].

In addition, two types of rectangular crystals were observed. The first one consisted of elongated particles made of several lamellae stacked on their base plane, most likely via epitaxial growth (Figure 2, Supplementary Data Figure S3). 60° and 90° twinned assemblies were frequently observed (Figure 2b-d). This type of crystals was obtained with all fatty acids of the series except PROA. The XRD patterns recorded from hydrated powders clearly corresponded to the orthorhombic V6II allomorph, with average cell parameters a = 2.64 ± 0.01 nm, b = 2.70 ± 0.01 nm and c = 0.81 ± 0.00 nm (Figure 2f, Supplementary Data Tables S2 andS3, and Figure S7). However, base-plane ED patterns corresponding to the V6II structure were particularly elusive as it was difficult to prevent a partial drying during the preparation of TEM specimens.

When the structure was preserved, the two-dimensional orthorhombic lattice of the base-plane ED pattern was unambiguously identified as that of the V6II allomorph, with parameters a = 2.65 ± 0.01 nm and b = 2.68 ± 0.01 nm. (Figure 2e, Supplementary Data Figure S4 and Table S1).

The crystals are thus isomorphous to those prepared in the presence of n-butanol or n-pentanol [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or npentanol: structural features and properties[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] St | Chain folding in amylose crystals[END_REF][START_REF] Booy | Electron diffraction study of single crystals of amylose complexed with n-butanol[END_REF]. However, for consistency with the other V-amylose allomorphs and following the convention proposed by Donnay [START_REF] Donnay | Rules for the conventional orientation of crystals[END_REF], i.e. a < b, we have switched the a and b values with respect to those given in the previous studies. The ED pattern of partially dried specimens was an intermediate between those of V6I and V6II (Supplementary Data Figure S4), in agreement with the observation of Helbert and Chanzy [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or npentanol: structural features and properties[END_REF] that V6II converts into V6I upon air-drying.

Moreover, a characteristic feature of the V6II crystals was the longitudinal cracks that occurred upon drying. As described by Helbert and Chanzy [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or npentanol: structural features and properties[END_REF], these cracks appear upon drying due to the V6II to V6I transition that involves a reorganization of the amylose helices within the lattice.

The second type of rectangular lamellar crystals generally grew in the form of more or less dense flower-like aggregates (Figure 3, Supplementary Data Figure S5) and were obtained with all fatty acids. Although this type of complex was easily identified from this typical crystal organization, base-plane ED patterns could only be recorded when the single lamellae were sufficiently individualized at the periphery of the aggregates or from broken lamellae (Figure 3e, Supplementary Data Figure S6). The ED patterns were indexed in a two-dimensional orthorhombic unit cell with parameters a = 2.82 ± 0.03 nm and b = 2.94 ± 0.07 nm (Supplementary Data Table S1), in good agreement with those calculated from XRD powder patterns (a = 2.80 ± 0.01 nm, b = 2.97 ± 0.01 nm and c = 0.80 ± 0.01 nm) (Figure 3f, Supplementary Data Tables S2 andS3, and Figure S7). These crystals are isomorphous to those prepared in the presence of acetone, various alcohols such as 2-propanol [START_REF] Buléon | Single crystals of V amylose complexed with isopropanol and acetone[END_REF], geraniol, fenchone, menthone [START_REF] Nuessli | Crystal structure of amylose complexes with small ligands[END_REF], thymol or terpineol [START_REF] Putaux | Helical conformation in crystalline inclusion complexes of V-amylose: A historical perspective[END_REF] and thus correspond to the V7 allomorph [START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF].

Mixtures of crystal types

Since the three types of morphologies described above have distinct features, the coexistence of V-amylose allomorphs could be unambiguously identified from TEM images (Figure 4) and XRD diagrams (Figure 5). Table 1 summarizes the allomorphic types observed for complexes formed with each fatty acid. A general observation is that all complexing agents could induce more than one crystalline structure. Specifically, PROA (C3) formed V6I and V7 while all longer-chain fatty acids (C4-C20) promoted the formation of V6I, V6II and V7. Tables 234detail the results obtained with different solvent compositions, fatty acid concentrations and crystallization temperatures. The relative amount of allomorphs was evaluated from the peak intensities in the XRD profile. TEM images were used to identify different crystal forms, even when one of both forms was in minor amount and the corresponding XRD patterns showed only one allomorph. Selected XRD profiles for different mixtures of allomorphs are shown in Figure 5. Depending on the crystallization conditions, the product contained only one type of crystal or coexisting allomorphs. The two allomorphs of VPROA complexes, V6I and V7, always formed simultaneously but the relative amounts could be modified by varying the crystallization temperature. Typically, V6I was favored at higher temperatures. Upon crystallization in water in the presence of 40 vol% of PROA at 40 °C, V7 was the major form. Keeping similar crystallization mixtures at 50-75 °C resulted in V6I as the main allomorph (Table 2, Supplementary Data Figure S8). However, the result was different when the solvent composition and concentration of PROA were changed. By increasing PROA to 50 vol% or upon crystallization in the presence of 10 vol% DMSO, V6I was the major allomorph at all tested temperatures (Tables 2 and4, Supplementary Data Figure S8).

Similar results were obtained for VBUTA complexes. Higher BUTA concentrations and the presence of DMSO favored the formation of V6I (Tables 2 and4, Supplementary Data Figure S9). Besides, at 40 °C and low concentrations of BUTA (5-10 vol%), amylose crystallized into a mixture of V6II and V7. Despite the fact that V6II crystals were recognized in TEM images (Figure 4e), the corresponding reflections were not visible in the XRD profile (Supplementary Data Figure S9), suggesting that the amount of V6II was lower than 5%.

For longer fatty acids (C5-C20), V6II crystals were also formed at lower temperatures (40-50 °C) than V6I complexes (Tables 234, Supplementary Data Figures S10 andS11). It must be noted that a successful preparation of V6II with C10-C20 fatty acids required an incubation of the amylose-lipid mixture at 100-110 °C for 24 h before cooling down to the crystallization temperature (40-50 °C). Without this incubation period, amorphous and B-type amylose formed as well. Besides, V6II was generally not favored with 10 vol% DMSO. On the other hand, with C5-C20 fatty acids, V7 crystals only formed in the presence of the appropriate amount of DMSO (2 vol%) (Table 3). 10 vol% DMSO favored V6I instead. In addition, for the complexation with PENA (C5), the formation of V7 was preferred to that of V6II at lower fatty acid concentrations and higher temperatures (Table 3, Supplementary Data Figure S10). For C6-C20, the conditions in which V7 was the unique or the main crystallized form were not determined. V7 crystals always formed in minor amount concomitantly with V6I. In these cases, the crystals were always present attached to those of V6I, possibly implying a twinning mechanism (Figure 4c,d). A similar pattern was observed for the coexistence of V6I and V6II (Figure 4a,b) whereas coexisting V6II and V7 crystals seemed to have grown independently (Figure 4e,f).

Discussion

Our data clearly show that highly crystalline complexes of V-amylose could be prepared with a series of saturated linear fatty acids (C3 to C20) at temperatures as low as 40 °C, which agrees with the conclusions of Marinopoulou et al. [START_REF] Marinopoulou | Structural characterization and thermal properties of amylose-fatty acid complexes prepared at different temperatures[END_REF] but contradicts other reports that mention that a crystallization at a temperature lower than 60 °C only yields amorphous complexes [START_REF] Karkalas | Some factors determining the thermal properties of amylose inclusion complexes with fatty acids[END_REF][START_REF] Biliaderis | Crystallization behavior of amylose-V complexes: Structure-property relationships[END_REF].

However, a successful crystallization requires to mix the fatty acids and amylose solutions at a high temperature (90-100 °C) and, for the V6II allomorph prepared with C10-C20 ligands, to maintain the mixture at this temperature for 24 h before cooling down to the crystallization temperature. According to Whittam et al. [START_REF] Whittam | Aqueous dissolution of crystalline and amorphous amylose-alcohol complexes[END_REF], the formation of crystalline V-amylose requires two steps: i) the association of amylose and complexing agent into helical complexes, followed by ii) the formation of crystal by association of the helices under suitable conditions. The interaction of fatty acids with amylose in solution at a temperature higher than the crystallization temperature is thus critical to promote the formation of V-amylose single helices.

A salient and unexpected result of our study is that the polymorphism of V-amylose was observed for all tested fatty acids rather than only for PROA, BUTA and HEXA as reported in previous articles (Table 1) [START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part X. X-Ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Biais | Structural and stoichiometric studies of complexes between aroma compounds and amylose. Polymorphic transitions and quantification in amorphous and crystalline areas[END_REF]. The observed allomorphs differ by the 6-or 7-fold helical conformation of amylose, packing of helices and location of the guest fatty acids in the unit cells.

In V6I crystals, the helices are packed into a compact hexagonal unit cell and, therefore, the guest molecules can only be located inside the helices [START_REF] Godet | Crystallization of amylose-fatty acid complexes prepared with different amylose chain lengths[END_REF]. In contrast, V6II and V7 are less compact structures where the guest and water molecules can be located inside and between the helices (Figure 6) [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or npentanol: structural features and properties[END_REF][START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF].

We have shown that all tested fatty acids could induce the formation of both 6-and 7-fold helices whereas earlier works reported that the amylose conformation depended on the chain length of complexing agent [START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part X. X-Ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds. Part VI. X-ray diffraction of amylose-n-aliphatic ketone complexes[END_REF]. In particular, the occurrence of V7 with long-chain fatty acids C6-C20 has never been reported before (Table 1). If the cross-sectional diameter of the linear fatty acids (ca. 0.30 nm) is considered, V6 structures are expected rather than V7. V7 is usually obtained with bulkier complexing agents such as branched alcohols or monoterpenes whose cross-sectional diameter is 0.45-0.60 nm [START_REF] Yamashita | An electron microscopy study on crystals of amylose V complexes[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part X. X-Ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes. II. Crystals with 71 helical configuration[END_REF][START_REF] Nuessli | Crystal structure of amylose complexes with small ligands[END_REF][START_REF] Biais | Structural and stoichiometric studies of complexes between aroma compounds and amylose. Polymorphic transitions and quantification in amorphous and crystalline areas[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds. Part VI. X-ray diffraction of amylose-n-aliphatic ketone complexes[END_REF]. In this context, Shogren et al. [START_REF] Shogren | X-ray diffraction study of crystal transformations in spherulitic amylose/lipid complexes from jet-cooked starch[END_REF] proposed that the formation of 7-fold helices with fatty acids was due to the complexation of amylose with fatty acid "dimers", the association of which would be promoted by hydrophobic interactions between the alkyl moieties [START_REF] Mukerjee | Dimerization of long-chain fatty acids in aqueous solutions and the hydrophobic properties of the acids[END_REF]. However, our results showed that V7 was more favorable with water-miscible fatty acids (C3 and C4) than with the poorly soluble ones. Moreover, several complexes formed at high temperature (90 °C) at which the existence of fatty acid "dimers" in solution was unlikely. Therefore, the 7-fold helical conformation is more likely controlled by a specific interaction between fatty acids and amylose rather than by the molecular dimension of the guest molecule. As proposed by Takeo et al. [START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part X. X-Ray diffraction of amylose-fatty acid complexes[END_REF], the inclusion of the polar head (i.e. the carboxyl group) of the fatty acid in the amylose helix could be responsible for its enlargement to the V7 conformation.

In contrast, straight-chain molecules bearing a different functional group such as aliphatic nalcohols and aldehydes have been reported to only induce the formation of 6-fold helices [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Conde-Petit | Structural features of starch-flavor complexation in food model systems[END_REF].

Besides, previous numerical simulations and NMR data concluded that the linear alkyl moiety was included inside the 6-fold helix while the polar head remained outside [START_REF] Godet | Molecular modelling of the specific interactions involved in the amylose complexation by fatty acids[END_REF][START_REF] Kawada | Solid state NMR and X-ray studies on amylose complexes with small organic molecules[END_REF][START_REF] Lebail | Mobility of lipid in complexes of amylose-fatty acids by deuterium and 13 C solid state NMR[END_REF][START_REF] Snape | Solid state 13 C NMR investigation of lipid ligands in V-amylose inclusion complexes[END_REF]. In addition, the hypothesis that the inclusion of carboxyl group controls the helical conformation somehow agrees with the fact that V7 is more preferred with shorter fatty acids (C3-C5). In the case of C6-C20, V7 crystals always formed in minor amount concomitantly with V6I. It is possible that with a longer chain length, the influence of the linear aliphatic chain gets predominant over that of the carboxyl group in determining the size of helix and thus would induce mainly 6-fold helical complexes [START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part X. X-Ray diffraction of amylose-fatty acid complexes[END_REF]. Expanding from the modeling work of Godet et al. [START_REF] Godet | Molecular modelling of the specific interactions involved in the amylose complexation by fatty acids[END_REF], further simulations must be carried out to elucidate the role of the carboxyl group and its interaction with amylose in the determination of its helical conformation. Moreover, the crystallization of amylose with other homologous series of aliphatic linear guests with different functional groups (straight-chain alcohols, aldehydes, esters, ketones, etc.) should be systematically investigated to see whether the formation of the V7 allomorph can be induced.

The present study showed that a systematic screening of a variety of crystallization conditions was necessary to explore the polymorphism of Vfatty acid complexes. Our results revealed that with a given fatty acid, different allomorphs could crystallize, alone or in mixtures, depending on the conditions (Tables 234). A similar behavior has previously been reported for many polymorphic systems, as reviewed by Bernstein et al. [START_REF] Bernstein | Concomitant polymorphs[END_REF]. The phenomenon has been explained by the fact that so-called occurrence domains, defined by sets of conditions at which specific allomorphs crystallize, could be independent or overlap. When the occurrence domain is unique, only one form is obtained, which is usually the most thermodynamically stable. In contrast, in regions where the domains overlap, two or more forms would crystallize under identical conditions. These concomitant allomorphs are energetically close and their relative amount is mainly governed by kinetic stability [START_REF] Bernstein | Concomitant polymorphs[END_REF][START_REF] Bučar | Disappearing polymorphs revisited[END_REF].

Since we have only characterized the morphology and structure of the final products, the precise chronology of crystallization events was lost. The possibility that the amylose chains form nuclei and crystallize under conditions that depend on their length (so-called fractionated crystallization) or on the temperature (during cooling) cannot be ruled out. One allomorph would crystallize after the other and the allomorph formed first may even act as a seed for nucleation and epitaxial growth of the second one. As illustrated by Figures 4a and4b, rectangular V6II and hexagonal V6I crystals are often associated, exhibiting specific orientation relationships that were not precisely analyzed in the present study. However, since the images are projections of the crystal assemblies, one cannot clearly identify the crystallization sequence of each allomorph.

In the case of coexisting V6I and V7 crystals, the latter ones are mostly observed as outgrowths of the former (Figure 4d), suggesting that they formed the last. V6II and V7 crystals did not appear to be associated but rather independent from one another (Figures 4e and4f).

In our study, three parameters were shown to play important roles to regulate the complexforming ability and molecular packing, namely the fatty acid concentration, the temperature, and the solvent composition. It appears that the compact V6I structure was favored at higher concentrations of fatty acid and DMSO (10 vol%), and a higher temperature (60-90 °C). However, milder conditions of these parameters result in the formation of the less dense structure that contains inter-helical cavities (V6II and V7 -Figure 6b,c). Similar observations on the effect of complexing agent concentration were reported earlier for PROA and BUTA as well as 2-propanol and acetone [START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part X. X-Ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds. Part VI. X-ray diffraction of amylose-n-aliphatic ketone complexes[END_REF]. A common feature of these molecules is that they are water-miscible.

To ensure the crystallization of V-amylose, they must be used at a higher concentration (5-50 vol%) than that used for insoluble complexing agents, i.e. long-chain fatty acids (< 0.1 vol%).

In addition to promoting the interaction with amylose, high concentrations of complexing agents may induce a significant dehydration on the polymer and thus favor the formation of the more compact V6I structure [START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part X. X-Ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. Moreover, previous works have shown that V6II and V7 crystals were stable in an aqueous environment whereas drying or washing with methanol resulted in the conversion into the more compact V6I structure [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or npentanol: structural features and properties[END_REF][START_REF] Buléon | Single crystals of V amylose complexed with isopropanol and acetone[END_REF]. In the case of BUTA, V6II was more sensitive to dehydration than V7, the former appearing only with a BUTA concentration ≤ 10 vol% while the later was formed at higher concentration. This also explains why V6II could not be obtained with PROA for which at least 30 vol% was required to form the complexes.

The impact of DMSO on the formation of specific allomorphs has been clearly highlighted.

This molecule is a good solvent of amylose but also of fatty acids which increases the concentration of weakly soluble fatty acids and therefore, their availability for complexation.

Helbert [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF] also observed different allomorphs for complexes prepared with n-butanol that exhibited a V6II structure when crystallized in water and V6I in DMSO:water mixtures. It must also be noted that DMSO can form crystalline complexes with amylose [START_REF] French | X-ray diffraction of oriented amylose fibers. I. Amylose dimethyl sulfoxide complex[END_REF][START_REF] Germino | Amylose V complexes from dimethyl sulfoxide solutions[END_REF]. While Simpson et al. [START_REF] Simpson | A V7 conformation of dimethyl sulfoxideamylose complex[END_REF] mentioned the occurrence of two allomorphs based on 6-and 7-fold amylose helices, Winter and Sarko [START_REF] Winter | Crystal and molecular structure of the amylose-DMSO complex[END_REF] described the VDMSO unit cell as pseudo-tetragonal and containing antiparallel 6-fold helices, with DMSO molecules located inside and between helices. However, this complex was only formed under anhydrous conditions by casting amylose / DMSO solutions and letting them to dry in a vacuum oven. Nevertheless, this shows that in specific conditions, DMSO no longer acts as a solvent of amylose but rather like a complexing agent that specifically interacts with amylose chains to form helical inclusion compounds.

The most remarkable effect of DMSO was its crucial role in the formation of V7. Our results showed that the complexes with C5-C20 fatty acids exhibited a V7 structure only if DMSO was present. A significant role of DMSO on the conformation of amylose has been suggested by other authors [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Germino | Amylose V complexes from dimethyl sulfoxide solutions[END_REF][START_REF] Simpson | A V7 conformation of dimethyl sulfoxideamylose complex[END_REF][START_REF] Jane | 13C-NMR study of the conformation of helical complexes of amylodextrin and of amylose in solution[END_REF][START_REF] Cheetham | Amylose conformational transitions in binary DMSO/water mixtures[END_REF]. DMSO may induce specific conformations for both amylose and fatty acids, although, so far, the nature of the interactions remains unknown and no evidence were produced regarding the presence of DMSO in the crystal structure of Vfatty acids complexes.

Conclusion

The crystallization of amylose with a series of homologous saturated straight-chain fatty acids confirmed the polymorphism of Vfatty acid complexes hinted at by previous authors. The helical conformation of amylose was independent of the fatty acid chain length. By adjusting the crystallization conditions, each fatty acid of the series (C3 to C20) induced both 6-and 7-fold helices that packed into different unit cells. Propanoic acid yielded V6I or V7 while the fatty acids having 4 to 20 carbons induced the formation of crystals with the V6I, V6II or V7 structure.

The results highlight the important role of fatty acid concentration, temperature and solvent composition on the crystal structure of the complexes. Depending on these parameters, a unique allomorph or mixtures of several were obtained. Some trends could be identified: i) a higher concentration of fatty acid and DMSO and a higher temperature promoted the formation of the compact V6I structure; ii) V6II and V7 preferably crystallized at a lower concentration of fatty acids and DMSO and at lower temperatures; iii) with C5-C20 fatty acids, V7 was only obtained in the presence of DMSO. Further studies on the stoichiometry, location of the guest fatty acid molecules and their interaction with amylose are in progress. Conducting similar studies with homologous series of other molecules, such as linear alcohols, aldehydes, ketones or esters should be undertaken since the amylose polymorphism has not been systematically investigated with these compounds.

Tables and figures

Table 1. Summary of the allomorphs observed for the crystalline complexes of V-amylose with linear fatty acids as a function of the number of carbon atoms in the fatty chain and comparison with previous works. * The crystal type of VICOA has not previously been reported.

Table 2. Allomorphic type of Vfatty acid crystals formed in water at different fatty acid concentrations (Cfatty acid) and temperatures.

The amylose solutions and fatty acids were mixed at 90 °C, then at a given crystallization temperature, or allowed to slowly cool down to room temperature in a Dewar container. For mixtures, the major and minor allomorphs are indicated. For mixtures (d-f), the major and minor allomorphs are indicated. those proposed for VH [START_REF] Rappenecker | Detailed refinement of the crystal structure of Vhamylose[END_REF], Vn-butanol [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or npentanol: structural features and properties[END_REF] and V2-propanol [START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF]. In these base-plane projections along the helical c-axis, only the amylose helices have been drawn. Hydrogen atoms have been removed for clarity. The conformation of the hydroxymethyl group has been fixed as gg in the three cases for simplicity. The blue ovals mark the inter-helical cavities. The unit cells have been superimposed on the models.

  The first type exhibited a characteristic hexagonal morphology and occurred as individual lamellae or, more frequently, stacks of lamellae involving spiral dislocation growth. Examples of such crystals are shown in Figure 1a-d but hexagonal crystals were obtained with the whole series of fatty acids (Supplementary Data Figure S1). The base-plane ED patterns recorded on frozen-hydrated crystals were all hexagonal (Figure 1e, Supplementary Data Figure S2) and were indexed on the basis of a hexagonal unit cell with parameters a = b = 1.36 ± 0.02 nm or an orthorhombic unit cell with a = 1.36 ± 0.02 nm and b = a√3 = 2.36 ± 0.03 nm (Supplementary Data Table

a

  Takeo et al. [16], b Helbert[START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF], c Biais et al.[START_REF] Biais | Structural and stoichiometric studies of complexes between aroma compounds and amylose. Polymorphic transitions and quantification in amorphous and crystalline areas[END_REF]

Figure 1 .

 1 Figure 1. The V6I structure: a-d) TEM images of crystals of V-amylose crystallized with OCTA (a), DODA (b) and TEDA (c,d); e) base-plane ED recorded at low temperature from frozenhydrated VTEDA crystals; f) powder XRD diagram recorded from VTEDA crystals equilibrated at 95% RH.

Figure 2 .

 2 Figure 2. The V6II structure: a-d) TEM images of crystals of V-amylose crystallized with PENA (a,b), DECA (c) and HEDA (d); e) base-plane ED recorded at low temperature from frozen-hydrated VPENA crystals; f) powder XRD diagram recorded from VPENA crystals equilibrated at 95% RH.

Figure 3 .

 3 Figure 3. The V7 structure: a-d) TEM images of crystals of V-amylose crystallized with PROA (a), BUTA (b), PENA (c) and DECA (d); e) base-plane ED recorded at low temperature from frozen-hydrated VPENA crystals; f) powder XRD diagram recorded from VPENA crystals equilibrated at 95% RH.

Figure 4 .

 4 Figure 4. Examples of coexisting crystals with different morphologies and allomorphic types prepared with a given fatty acid: a,b) V6I and V6II prepared in water with HEXA (C6) at 50 °C and TEDA (C14) at 60 °C, respectively, both fatty acids at saturation; c,d) V6I and V7 prepared in the presence of 10 vol% DMSO with 50 vol% BUTA (C4) at 60 °C and OCDA (C18) at 90 °C, respectively; e,f) V6II and V7 prepared with 5 vol% BUTA (C4) in water and 3 vol% PENA (C5) at 40 °C in the presence of 2 vol% DMSO, respectively (TEM images).

Figure 5 .

 5 Figure 5. XRD profiles of V-amylose crystallized from aqueous solutions in the presence of a series of linear fatty acids. Unique allomorphs: a) V6I prepared with TEDA (C14); b) V6II prepared with HEDA (C16); c) V7 prepared with PENA (C5). Coexisting allomorphs: d) V6II and V7 prepared with 3 vol% PENA (C5) at 40 °C in the presence of 2 vol% DMSO; e) V6I and V7 prepared in the presence of 2 vol% DMSO with OCDA (C18) at 90 °C; f) V6I and V6II prepared in water with TEDA (C14) at 60 °C. The crystals have been equilibrated at 95% RH.

Figure 6 .

 6 Figure 6. Molecular models of the three allomorphs of V-amylose complexed with fatty acids considered in this study: a) V6I (orthorhombic unit cell), b) V6II and c) V7. The models are

aFigure S1 .

 S1 Figure S1. Morphology of Vfatty acid crystals with a V6I structure (TEM images).

Figure S2 .Figure S3 .

 S2S3 Figure S2. Base-plane electron diffraction patterns of frozen-hydrated Vfatty acid crystals with a V6I

Figure S4 .

 S4 Figure S4. Base-plane electron diffraction patterns of frozen-hydrated Vfatty acid crystals with a V6II

Figure S5 .

 S5 Figure S5. Morphology of lamellar Vfatty acid crystals with a V7 structure (TEM images).

Figure S6 .Figure S7 .

 S6S7 Figure S6. Base-plane electron diffraction patterns of frozen-hydrated Vfatty acid crystals with a V7

Figure S8 .

 S8 Figure S8. XRD profiles of VPROA (C3) complexes prepared at different temperatures in the presence

Figure S9 .

 S9 Figure S9. XRD profiles of VBUTA (C4) complexes prepared with different concentrations of BUTA in

Figure S10 .

 S10 Figure S10. XRD profiles of VPENA (C5) prepared in pure water and in a 1:9 or 1:49 DMSO:water

Figure S11 .

 S11 Figure S11. XRD profiles of VHEXA (C6), VOCTA (C8) and VICOA (C20) prepared at different

Table 3 .

 3 Allomorphic type of Vfatty acid crystals formed in the presence of 2 vol% DMSO at different fatty acid concentrations (Cfatty acid) and temperatures. The amylose solutions and fatty acids were mixed at 90 °C, then kept at a given crystallization temperature. For mixtures, the major and minor allomorphs are indicated.

				Crystallization temperature (°C)
	Fatty acid	Cfatty acid
			40	60	75	90
	PROA (C3)	n. t.	n. t.	n. t.	n. t.	n. t.
	BUTA (C4)	n. t.	n. t.	n. t.	n. t.	n. t.
	PENA (C5)	2.5 vol%	V7	V7	-	-
	"	3 vol%	V6II	V7	-	-
	"	4-5 vol%	V6II	V7	-	-
	HEXA (C6)	*	V6II	V6I > V7	V6I > V7	-
	OCTA (C8)	n. t.	n. t.	n. t.	n. t.	n. t.
	DECA, DODA, TEDA,
	HEDA, OCDA, ICOA
	(C10-C20)

Table 4 .

 4 Allomorphic type of Vfatty acid crystals formed in the presence of 10 vol% DMSO at different fatty acid concentrations (Cfatty acid) and temperatures. The amylose solutions and fatty acids were mixed at 90 °C, then kept at a given crystallization temperature, or allowed to slowly cool down to room temperature in a Dewar container. For mixtures, the major and minor allomorphs are indicated.

					Crystallization temperature (°C)
	Fatty acid	Cfatty
			40	50	60	75	90	Slow cooling
	PROA (C3)	30 vol%	-	-	-	-	-	-
	"	40-50 vol%	V6I > V7	V6I > V7	V6I > V7	V6I > V7	-	V6I > V7
	BUTA (C4)	5-10 vol%	V7 > V6II, B	-	-	-	-	V7 > V6II, B
	"	20-30 vol%	V7 > V6I	V7 > V6I	V7 > V6I	V7 > V6I	-	V7 > V6I
	"	40-50 vol%	V6I > V7	V6I > V7	V6I > V7	V6I > V7	-	V6I > V7
	PENA (C5)	2.5 vol%	V7 > V6II	V7 > V6I, V6II	-	-	-	n. t.
	"	5 vol%	V6II > V7	V6II > V6I, V7	V6I > V7	-	-	V6II > V6I
	HEXA (C6)	*	V6I > V6II	V6I > V6II	V6I > V7	V6I > V7	-	V6I > V6II
	OCTA (C8)	*	V6I > V6II	V6I	V6I	V6I > V7	-	V6I > V6II
	DECA (C10), DODA (C12)	*	V6I	V6I	V6I	V6I > V7	V6I > V7	V6I > V7
	TEDA, HEDA, OCDA,
	ICOA (C14-C20)

Table S2 .

 S2 Unit cell parameters for V6I (hexagonal), V6II and V7 (orthorhombic) allomorphs calculated from XRD powder patterns from V-amylose crystals prepared with a series of fatty acids (C3-C20).

		V6I			V6II			V7
	Fatty acid
		a = b (nm) c (nm) a (nm) b (nm) c (nm) a (nm) b (nm) c (nm)
	PROA	1.37	0.82	-	-	-	2.80	2.97	-
	BUTA	1.36	0.80	-	-	-	2.79	2.96	0.80
	PENA	1.38	0.80	2.63	2.69	0.82	2.81	2.98	-
	HEXA	1.37	0.81	2.65	2.71	0.81	-	-	-
	OCTA	1.36	0.81	2.65	2.72	0.81	-	-	-
	DECA	1.38	0.81	2.63	2.69	0.81	-	-	-
	DODA	1.36	0.81	2.64	2.70	0.81	-	-	-
	TEDA	1.38	0.81	2.64	2.70	0.81	-	-	-
	HEDA	1.37	0.82	2.63	2.69	0.81	-	-	-
	OCDA	1.38	0.81	2.65	2.71	0.81	-	-	-
	ICOA	1.37	0.81	2.64	2.70	0.81	-	-	-
	Average	1.37	0.81	2.64	2.70	0.81	2.80	2.97	0.80
	SD	0.01	0.01	0.01	0.01	0.00	0.01	0.01	-
	-: data not available
	SD: standard deviation

Table S3 .

 S3 Miller indices, diffraction angles and unit cell parameters for three allomorphs of VPENA calculated from X-ray diffraction data. The crystals equilibrated at 95% RH.

		V6I (a)			V6II (b)			V7 (b)
	h k l	2θobs (°)	2θcal (°)	h k l	2θobs (°)	2θcal (°)	h k l	2θobs (°)	2θcal (°)
	0 1 0	7.40	7.38	2 1 0	7.45	7.49	1 1 0	4.38	4.32
	1 1 0	12.84	12.80	2 2 0	9.46	9.41	2 0 0	6.36	6.29
	1 1 1	16.88	16.98	3 2 0	12.11	12.06	2 1 0	6.99	6.96
	0 2 1	18.69	18.54	2 0 1	12.78	12.76	2 2 0	8.72	8.65
	1 2 0	19.65	19.61	1 4 0	13.62	13.60	1 3 0	9.46	9.45
	0 3 0	22.28	22.27	4 3 0	16.67	16.73	3 1 0	9.91	9.90
	0 3 1	24.88	24.95	1 4 1	17.42	17.42	2 3 0	10.93	10.91
	1 3 0	26.83	26.84	5 2 0	18.16	18.11	1 4 0	12.28	12.29
				1 6 0	20.15	20.11	4 1 0	12.95	12.95
				2 6 0	20.97	20.95	1 5 0	15.15	15.20
				5 4 0	21.43	21.46	5 2 0	16.88	16.87
				3 6 0	22.28	22.29	3 5 0	17.63	17.64
				4 6 0	24.02	24.04	5 3 0	18.11	18.14
				3 6 1	24.79	24.84	6 1 0	19.16	19.19
				5 6 0	26.13	26.13	6 2 0	19.84	19.88
							5 5 0	21.73	21.74
							4 7 0	24.46	24.46
							7 4 0	25.18	25.21
							6 6 0	26.27	26.16
							2 9 0	27.67	27.69

Acknowledgement

We thank LabEx Arcane (grant agreement #ANR-11-LABX-0003-01) for funding the PhD thesis of C.A.K.L, the NanoBio-ICMG platform (UAR 2607, Grenoble) for granting access to the Electron Microscopy facility, Christine Lancelon-Pin (CERMAV) for her help with sample preparation, the Glyco@Alps program (Investissements d'Avenir grant agreement #ANR-15-IDEX-02), and Henri Chanzy for stimulating discussions and critical reading of the manuscript.

Supplementary Data

Table S1. Base-plane unit cell parameters of V6I, V6II and V7 allomorphs determined from ED patterns of single crystals prepared with each fatty acid of the tested series (C3-C20).

Fatty acid


Dr Wenyao Zhang

J Esfflsb Arrio,C Hristel Gervais

Andraz ˇkocjan

Aiping Yu

Xin Wang
email: wangx@njust.edu.cn

Prof J M Barrio

Shalom

Prof X Wang

Prof A Yu

Prof C Gervais

Dr A Kocjan

Synthesis of Carbon-Nitrogen-Phosphorous Materials with an Unprecedented High Amount of Phosphorous toward an Efficient Fire-Retardant Material

Phosphorus incorporation into carbon can greatly modify its chemical, electronic,a nd thermal stability properties.T odate this has been limited to lowlevels of phosphorus. Now as imple,l arge-scale synthesis of carbon-nitrogenphosphorus (CNP) materials is reported with tunable elemental composition, leading to excellent thermal stability to oxidation and fire-retardant properties.T he synthesis consists of using monomers that are liquid at high temperatures as the reaction precursors.T he molten-state stage leads to good monomer miscibility and enhanced reactivity at high temperatures and formation of CNP materials with up to 32 wt % phosphorus incorporation. The CNP composition and fireretardant properties can be tuned by modifying the starting monomers ratio and the final calcination temperature.T he CNP materials demonstrate great resistance to oxidation and excellent fire-retardant properties,w ith up to 90 %o ft he materials preserved upon heating to 800 8 8Ci na ir.

Carbon materials are widely used for arange of applications such as batteries,s upercapacitors,f uel cells,h eterogeneous catalysis,s ensors,w ater treatment, among others. [1] Insertion of heteroatoms with ad ifferent electronegativity into the carbon network can dramatically alter its electronic, [2] magnetic, [3] optical, [4] and electrochemical properties [5] along with its chemical reactivity. [6] Fore xample,s everal studies have shown that the introduction of nitrogen and phosphorus as electron donors into ac arbon structure alters its electronic states, [7] (electro)catalytic properties [8] and its chemical stability to both oxidation and exposition to high temperatures. [9] More specifically,d oping ac arbon matrix with phosphorus significantly enhances its thermal stability [10] and its electrical conductivity. [11] Thec ovalent radius of nitrogen permits its facile incorporation into carbon materials;s tructures with up to 70 % nitrogen are easily synthesized. [12] In contrast, the large covalent radius of phosphorus strongly impedes its insertion into the carbon matrix. During the traditional solid-state synthesis of carbon materials,the carbon and nitrogen atoms can build auniform network which is energetically favorable, whereas the phosphorus is mainly bonded to oxygen and carbon atoms on the surface. [7b, 13] Furthermore,i nm aterials based on carbon and nitrogen, the phosphorus is seldom bonded to nitrogen atoms.T herefore,uptonow,only carbon materials doped with low amounts of phosphorus have been prepared and the presence of PÀNb onds within the carbon structure has rarely been reported. [7b, 14] Its presence within ac arbon network can strongly enhance its chemical and thermal stability, [15] as well as its electronic and catalytic properties. [16] Polyphosphazenes are polymers with aP À N backbone,w hich are usually prepared from poly(dichlorophosphazene). Thes ynthesis of the latter usually requires ahighly inert environment and involves the use of catalysts to avoid crosslinking. [17] Herein, we report the simple and large-scale synthesis of carbon-nitrogen-phosphorus materials with al arge amount of heteroatoms incorporation and up to 32 wt %p hosphorus in the final materials.The new materials were obtained by the calcination of mixtures of two monomers,namely hexachlorophosphazene (abbreviated as PNCl) and 2,4-diamino-6phenyl-1,3,5-triazine (DPT), which form au niform liquid solution at high temperatures (250-400 8 8C). Thel iquid state facilitates the ring-opening of PNCl to poly(dichlorophosphazene), followed by their reaction with amines groups in DPT.Subsequently,upon condensation at high temperatures, ar ange of new carbon-nitrogen-phosphorus materials are formed;their properties can be tuned by changing the initial monomers composition and the final reaction temperature.

Thes ynthetic route,i ntermediates,a nd final materials structure were thoroughly studied by aw ide range of techniques.T he optimal nanocomposites demonstrate very high stability to oxidation and act as efficient fire-retardant materials.

Thec arbon-nitrogen-phosphorus materials were synthesized through ar eaction involving am olten-state step and afinal solid-state condensation as shown in Figure 1a.Briefly, DPT and PNCl powders in various molar ratios (6:1,3:1,1:1, is covalently bonded to the PN chain. Additionally,t he spectrum for D 6 PN 1 -550 showed two shoulder peaks,a ttributed to P2p 3/2 and P2p 1/2 ,a tl ower binding energies,w hich correspond to the ternary Patoms from PN chain end-groups. This suggests that at high DPT amount mainly PhÀCN x is formed and the polymerization with PNCl is inhibited. Consequently,i nt he final structure,t he result of PNCl condensation mostly consists of PN oligomers.T he detailed reaction paths and the effect of monomers ratio are discussed below.

C1sa nd N1sX PS measurements show the evolution of CN heterocycles (C and Na toms in CÀN=Ca nd N-(C) 3 species) at high amounts of DPT in the starting materials reflecting the PhÀCN x formation. Thes ubstitution of PN structure with Ph À CN x was further supported by X-ray diffraction (XRD) measurements, [START_REF]CCDC 224695[END_REF] Fourier transform infrared (FTIR) spectroscopy,a nd elemental analysis (EA) data. TheX RD patterns (Supporting Information, Figure S1a) reveal that increasing the relative amount of DPT results in the formation of more organized layered structure,a s witnessed by the typical stacking peak of carbon nitride gradually appearing and shifting to higher angles,a ta round 27.58 8.T he FTIR spectrum of D 1 PN 6 -550 (Supporting Information, Figure S1b) featured strong and broad absorption bands at 1180 cm À1 and 874 cm À1 ,corresponding to stretching P=N [18] and PÀNv ibrations, [19] respectively.F or materials prepared with al arger relative amount of DPT,t he typical peaks corresponding to i) the breathing vibration mode of tris-triazine subunits at 810 cm À1 and ii)vibrations of aromatic C À Nh eterocycles at 1200-1600 cm À1 were detected. EA measurements (Supporting Information, Table S2, Table S4) show that the starting monomers ratio is directly related to the relative amounts of elements in the final materials:t he relative increase in DPT resulted in more C, Nand less P, Cl in the final materials and vice versa. TheC /N atomic ratio approached that for Ph À CN x when using higher DPT/PNCl ratio.

All the above-mentioned measurements suggest that the synthesis involves first the ring opening into linear À[PNCl] n À, followed by its substitution with DPT monomers.Afterward, the attached DPT monomers are further condensed into Ph À CN x .T he alteration of the products by the change of the monomers ratio indicates that there is acompetition between the DPT condensation and its reaction with the linear PN polymer.

In the next set of experiments,t he effect of the final condensation temperature was explored. Thethermal properties that are presented later in this manuscript indicate that the D 1 PN 3 sample is the most stable to oxidation at high temperature,s ow ef ocused on further studying materials of this composition at different calcination temperatures.T he C1shigh-resolution XPS spectrum of the D 1 PN 3 -450 sample confirms the creation of PhÀCN x phase (Supporting Information, Figure S2) and C À Na nd C = Nb onds were detected. These carbon components show an egligible change upon calcination at 450-650 8 8C, suggesting that the basic structure of D 1 PN 3 was undamaged with increasing temperature. Further increase of the calcination temperature to 750 8 8C leads to small decomposition of the structure.At850 8 8C, most 1:3and 1:6) were ground into ahomogeneous solid mixture. Afterward, the mixture was transferred into aceramic crucible and heated to 260 8 8Cfor 2h,followed by its calcination under nitrogen atmosphere for another 2h at adesignated target temperature (450, 550, 650, 750, 850 8 8C). Theobtained composites were labeled as D x PN y -T (x:y denotes the molar ratio of DPT to PNCl, Tdenotes the target temperature of the reaction). Thereaction yields are given in the Supporting Information, Table S1. Themolten-state step allows to shape the final structure as exemplified in Figure 1a. Thecylinder shape can be prepared by preforming the reaction in aglass-tube. The confined reaction vessel together with polyphosphazene structure lead to the formation of CNP monolith.

X-ray photoelectron spectroscopy (XPS) measurements show the typical evolution of the chemical states of P, C, and Nfor all samples. The P2pspectrum reveals four phosphorus environments (Figure 1b). Twodominant species, attributed to the terminal and bridging Penvironments from the backbone of À [PNR] n À polymer chains, appeared in all samples. Importantly,the increase of DPT in the precursor mixture led to asmall shift of P2ptolower binding energies. This shift is due to the electron-donating substituents, from the branched phenyl-modified carbon nitride (PhÀCN x )that of the functional groups disappeared and mainly sp 2 carbon was detected, accounting for the collapse of the polymer structure.Asreported in heteroatom-doped carbon materials, the dopant atoms incorporate into the p-conjugated system by delocalizing their p-electrons, [20] this process occurs at 700 8 8Co rh igher temperatures owing to the formation of thermodynamically stable hybridized carbon. This carbonization process can also be evidenced by the additional new peaks in P2pa nd N1sX PS spectra comparing D 1 PN 3 -850 samples with the materials that were calcinated at lower temperatures.Apart from XPS,EAand ICP data (Supporting Information, Table S3) were used to demonstrate the variation of the elements ratio at D 1 PN 3 with varying calcination temperatures.T he EA data shows that up to 750 8 8C, the structure remained almost intact and more than 30 %Pwas incorporated within the structure.T he increase in temperature to 850 8 8Cresulted in the loss of most Pand only 6% was detected.

To reveal the reaction mechanism of DPT/PNCl we focused on the elucidation of the synthesis path for the D 1 PN 3 sample.Amelting point apparatus (MPA), solid-state NMR, scanning electron microscope (SEM), and also thermogravimetric analysis (TGA) and differential thermogravimetry (DTG) were employed to study the growth mechanism, intermediate and final materials.F igure 2a displays photographs of DPT-PNCl mixture (molar ratio of 1:3) in acapillary tube heated at various temperatures in the MPA. Tw ochanges can be observed from the MPAimages;first, the solid mixture turned into aq uicksand-like mixture at 115 8 8Co wing to the melting of PNCl. Afterward, slightly before 260 8 8Ct he DPT was melted and at ransparent homogeneous molten solution was observed. In accordance with the MPAd ata, TGA and DTG curves (Supporting Information, Figure S3) show that as ignificant weight loss occurs from 115-200 8 8Cd ue to the monomers phase change,f ollowed by their condensation up to 600 8 8C.

To confirm the critical role of the molten phase,w e performed aseries of modifications to the reported synthesis.

In our experiments,c alcination of PNCl alone led to its full decomposition and no material remained after the same synthetic procedure.T he importance of choosing two monomers that form ahomogeneous molten state is demonstrated by replacing DPT with other nitrogen-rich organic compounds,n amely dicyandiamide,m elamine,a nd urea, which are the common precursors to materials based on carbon and nitrogen:n one of these monomers exhibit ac lear molten phase.W henm ixed with PNCl and calcinated at high temperatures,t hey did not form am olten phase:a sar esult, the PNCl completely evaporated and only carbon-nitrogen materials with an egligible amount of Pw ere formed with non-uniform distribution (Supporting Information, Figures S4, S5).

Solid-state NMR measurements at various reaction temperatures were acquired to elucidate the reaction path as well the intermediates and final materials structure. 13 CCPM AS spectrum of DPT molecule shows two broad signals centered at 130 and 170 ppm, corresponding to carbon within the phenyl and triazine units,r espectively (Figure 3a). Am ore precise assignment was obtained from the calculation of 13 C chemical shift values on the crystalline structure of DPT. [21] Thefull chemical shift values assignments are summarized in the Supporting Information, Table S5. Little change is observed in the carbon environments upon the reaction with PNCl at 260 8 8C(1:3molar ratio of DPT:PNCl) as illustrated by abroadening of the 13 Csignals without significant shifts (Figure 3a.). After heating to 550 8 8C, the 18.8 and 17.6 ppm (Figure 3b)t hat can be attributed to the two phosphorous inequivalent sites (1:2 ratio) in the crystalline structure. [24] After reaction with DPT at 260 8 8C, the spectrum was drastically changed and broad signals ranging from 10 to À40 ppm appeared. Thep rominent changes are probably due to the formation of PN 3 Cl and PN 4 environments owing to the replacement of Cl group with the NH 2 groups.T ofurther elucidate the Pchemical environment, we applied structural modeling (Figure 3c)t oc alculate the possible 31 Pc hemical shift values (Supporting Information, Table S6) on simple systems with representative phosphorus first neighbors.T he absence of signals between 20 and 50 ppm, where peaks for [PCl 2 N] 3 rings would be expected, confirmed the opening of [PCl 2 N] 3 rings.

Thef ormation of P À Nb onds leads to phosphorous environments which match our experimental data. Indeed, the calculated chemical shift values for P(NP) x (NH) 4Àx and P(NP) 2 (NH)Cl cover the whole experimental observed range. This suggests alarge distribution of Penvironments at 550 8 8C. Am ore precise assignment would necessitate am uch more sophisticated modeling,b ut it can be noticed that after calcination at 850 8 8C(Supporting Information, Figure S6) the signal becomes narrower and centered around À3ppm. This suggests the DPN lost most of its Pand turns into carbon, in ag ood agreement with XPS and EA results.

Having all the data at hand we suggest the following growth mechanism. Theh eating of PNCl monomer to high temperatures and its interaction with DPT molecules can facilitate and initiate the opening of the cyclic structure to ac hain linear polymer.F TIR measurements suggest the formation of as upramolecular assembly that is built from hydrogen bonds interaction between the two monomers at 260 8 8C( Figure 2b;S upporting Information, Figure S7). The antisymmetric stretching vibration of PN ring was shifted from 783 to 806 cm À1 and the stretching vibration of the N À H group in DPT fully disappeared. It is worth noting that the ring-opening reactions usually require the utilization of initiators/catalysts. [25] Here,t he hydrogen bonds network leads to the opening of the ring without any additional catalysts.S ubsequently,t he chlorine atoms linked to phosphorus are replaced by the active amine groups in DPT as depicted in Figure 2b.At350 8 8C, the unreacted amine groups in DPT start to polymerize and establish ascaffold based on carbon and nitrogen. In this case,anordered carbon-nitrogen structure that substitutes À[PNR] n À polymer chains with aw ell-organized, dense,f lat, and smooth morphology (Supporting Information, Figures S8,S9) is obtained from ap olymerization in the molten phase at high temperature.T he molten phase allows the two monomers to react similarly to the traditional organic chemistry approach owing to the molecules miscibility and reactivity.T herefore,a ss hown before,itissimple to tune the final elements ratio by adapting the initial monomers ratio.A th igher calcination temperatures,the polymerization is followed by afull carbonization of the structure to DPN.O ur results indicate that the materials compose from PÀNÀCb onds.

TGA in air was conducted to evaluate the stability of the new materials to oxidation and their suitability as fire-retardant materials.F irst, all the DPNs-550, with DPT:PNCl ratio ranging from 6:1t o1 :6, were analyzed to probe the effect of the elements ratio on the thermal stability (Figure 4a). TheT 10 (the temperature at 10 %m aterial loss) strongly depends on the starting and final elements ratio and dramatically increases with higher PNCl amounts (inset in Figure 4a and the Supporting Information, Table S7). The best T 10 ,747 8 8C, was obtained for the D 1 PN 3 -550 sample. At higher PNCl ratio, a small decrease of T 10 to 731 8 8Cwas obtained. Noticeably,the T 10 of the carbon-nitrogen scaffold from DPT-550 shows alower T 10 value of 574 8 8Cand the material was fully decomposed at about 700 8 8C. Thegreater stability of the DPN materials can be attributed to the strong PÀNlinear backbones within the structure which are known to be stable to oxidation.

We opted to further optimize the best sample (D 1 PN 3 )by exploring final condensation temperatures from 450 to 850 8 8C (Figure 4b;Supporting Information, Table S8). Theincrease of the condensation temperature from 450 to 650 8 8Cresults in an increase in T 10 ,uptoanimpressive 795 8 8Cfor D 1 PN 3 -650. Further increase in the condensation temperature of the synthesis to 750 8 8Cleads to aslight decrease in T 10 .The materials synthesized at 850 8 8Cshow asimilar behavior to the pristine carbon-nitrogen scaffold due to the massive loss of PN units within the structure as shown before. Therefore, based on these results, weconclude that D 1 PN 3 -650 exhibits the best stability to oxidation and flame-retardant properties.

Thef ire-retardant behavior of D 1 PN 3 -650 was further evaluated by burning it for 30 and 60 min under the hot flame of an alcohol lamp (Figure 4c). Compared with the recent state-of-the-art fire-retardant carbon-based materials, [10b] our material displays an excellent stability enhancement and only as light mass loss was obtained (Supporting Information, Figures S10,S11). Images of burned D 1 PN 3 -650 after soaking it in ethanol are shown in the Supporting Information, Figure S12. Importantly,t he sample almost remained intact and the flame did not self-propagate.T he FTIR, XPS,a nd SEM results (Supporting Information, Figures S13-S15) illustrate that the -[PNR] n -p olymeric chains remain even after 30 min burning, despite some PÀOspecies inevitably appearing. XPS data (Supporting Information, Figure S14) suggests that the carbon was barely oxidized. We proposed that the Pis mainly oxidized, protecting the carbon from burning. Recently it was reported that heteroatoms insertion into carbon matrix may lead to the formation of noble carbons with very high resistance to oxidation. [7d] Thec hemical stability of D 1 PN 3 -650 was investigated by immersing it into strong acid and base.The material demonstrated high stability under these harsh condition as shown in Figure S16.

UV/Vis and impedance spectroscopy measurements (Supporting Information, Figure S17) disclose that D 1 PN 3 -650 is an-type semiconductor with aband gap of 1.45 eV (Supporting Information, Figure S17). Thee lectric conductivity of D 1 PN 3 -650 is slightly improved compared to DPT-550 (Supporting Information, Figure S18).

Thet hermal properties of the D 1 PN 3 -650 were then evaluated and the corresponding values for effective thermal conductivity (l eff ), diffusivity (k)a nd specific heat were 0.157(AE 0.009) WmK À1 ,0 .31(AE 0.05) mm 2 s À1 ,a nd 0.52(AE 0.05) MJ m À3 K À1 .I ts hould be mentioned here that both the l eff and k are possibly slightly overestimated due to the experimental set-up,w here the carbonaceous foam was partially crushed. Thel ow thermal diffusivity can be compared to some polymeric materials,s ilica or adobe bricks, corroborates well with the observed flame-retardant stability enhancement preventing the excessive propagation of the heat front through the material as displayed in Figure 4c. Analysis of mercury porosimetry shows that the D 1 PN 3 -650 sample possesses ar ather small pore volume (0.144 cm 3 g À1 ) and al ow mesoporosity (only as mall fraction of mesopores around 3.5 nm in size), which results the value of l eff that is not as low as anticipated (! 0.100 WmK À1 ).

In conclusion, we demonstrate au nique and straightforward approach involving am olten-state step to synthesize CNP materials with unprecedentedly high amount of phosphorus,u pt o3 2wt%,a nd controllable elemental composition and chemical properties.T od os o, we used two monomers that melt at high temperatures and establish am ixture that is ah omogenous liquid up to 400 8 8C. The molten-state stage leads to excellent monomers miscibility at high temperatures,a llowing their reaction as in solution and prevents PNCl evaporation. Detailed structural analysis of the molten-state intermediates and the final carbon-nitrogen-phosphorus materials with various monomers ratio and calcination temperatures reveals that the reaction occurs by the opening of hexachlorophosphazene rings,followed by the replacement of the chlorine atoms by amine groups of the DPT monomer at the molten-state stage.W ed emonstrated that the chemical composition and the thermal stability to oxidation at high temperature can be tuned by the starting monomers ratio and the final calcination temperature.C onsequently,t he materials exhibit great resistance to oxidation and for the best composite up to 90 %o ft he materials are preserved at 800 8 8Cu nder air. We believe that the moltenstate approach opens the opportunity to overcome some of the traditional solid-state limitations in the synthesis of new materials with controllable and tunable elemental composition as well as variable chemical and electronic properties toward their utilization in fire-retardant materials and electrochemical devices.

Figure 1 .

 1 Figure 1. a) Illustration of the process for preparing DPN samples involving amolten-state step. b) High-resolution P2p, C1s, N1s XPS spectra of i) D 6 PN 1 -550, ii)D 3 PN 1 -550, iii) D 1 PN 1 -550, iv) D 1 PN 3 -550, v) D 1 PN 6 -550.

Figure 2 .

 2 Figure 2. a) Melting of the DPT-PNCl precursor seen in the melting point apparatusa tcontrolled temperatures. b) Proposed reaction path of CNP materials.

Figure 3 .

 3 Figure 3. a) 13 CCPMAS NMR spectra of i) DPT and reaction products with PNCl (molar ratio of DPT:PNCl of 1:3) at ii) 260 8 8Cand iii)550 8 8C. b) 31 PMAS NMR spectra of i) PNCl and reaction products with DPT (molar ratio of DPT:PNCl of 1:3) at ii)260 8 8C, and iii) 550 8 8C. c) Representation of the models I, II, III, and IV obtained through geometry optimization.

Figure 4 .

 4 Figure 4. TGA curves of DPN samples synthesized in a) various raw materials ratio, and b) various calcination temperature measured in air with aheating rate of 10 8 8Cmin À1 .The insets are the T 10 temperatures vs. samples, i) DPT-550, ii) D 6 PN 1 -550, iii) D 3 PN 1 -550, iv) D 1 PN 1 -550, v) D 1 PN 3 -550, vi)D 1 PN 6 -550, vii) D 1 PN 3 -450, viii) D 1 PN 3 -650, ix) D 1 PN 3 -750, x) D 1 PN 3 -850. c) Photographs of the D 1 PN 3 -650 exposed to aflame (alcohol lamp) after various exposition times.

Csignals remain in the same chemical shift range and only asignificant broadening of the signals was observed, which is probably due to the carbonization of the structure, suggesting the formation of graphitic carbon nitride[22] and aromatic carbon domains.[23]

Acknowledgements M.S.t hanks the financial support of the Israel National Research Center for Electrochemical Propulsion (INREP), NSF of China (No.5 1572125) and PA PD of Jiangsu. NMR spectroscopic calculations were performed using HPC resources from GENCI-IDRIS (Grant 097535). TheFrench RØgion Ile de France-SESAME program is acknowledged for financial support (700 MHz spectrometer).

Conflict of interest

Theauthors declare no conflict of interest.

Keywords: carbon-nitrogen-phosphorus materials • fire-retardant materials •molten-state reactions • tunable elemental composition