Gabriel P Garcia
email: gabrielgarcia@sfr.fr

Sylvain Sutour

Delphin Rabehaja

Loïc Tissandié

Jean-Jacques Filippi

Félix Tomi
email: felix.tomi@univ-corse.fr

Essential oil of the Malagasy grass Elionurus tristis Hack. contains several undescribed sesquiterpenoids

Keywords: Elionurus tristis Hack., Poaceae, antsorane skeleton, antsorenone, 2-epi-ziza-5en-2-ol, 7-epi-khusian-2-ol, 4, 8-di-epi-acorone, NMR, pc-GC

Essential oils (EOs) obtained from aerial parts and roots of Elionurus tristis were investigated by GC, GC-MS, pc-GC and NMR. Both aerial parts and roots EOs contained common molecules such as α-pinene, camphene, trans-α-bergamotene and calarene. Moreover, we identified several unusual sesquiterpenes and four undescribed compounds, 7-epi-khusian-2ol, 4,8-di-epi-acorone, 2-epi-ziza-5-en-2-ol and antsorenone. The last one exhibits an

Introduction

The members of the genus Elionurus (Poaceae) is mainly found in tropical and subtropical regions of America, Africa, and Australia [START_REF] Renvoize | Studies in Elionurus (Gramineae)[END_REF]. While previously the generic name has also been spelt Elyonurus, the spelling Elionurus is that now accepted by the Plant List (The Plant List, 2013). This genus contains less than 20 species, from which a very limited number have already been investigated for their essential oils and/or extracts, E.

viridulus Hack. ex Stuck. synonym of Elionurus muticus (Spreng.) Kuntze [START_REF] Chagonda | The essential oils of wild and cultivated Cymbopogon validus (Stapf) Stapf ex Burtt Davy and Elionurus muticus (Spreng.) Kunth from Zimbabwe[END_REF][START_REF] Chagonda | Constituents of the Roots Esential Oil of Cultivated Elionurus muticus (Spreng.) Kurth from Zimbabwe[END_REF][START_REF] Hefendehl | Analysis of the essential oil of Elyonurus viridulus[END_REF][START_REF] Scramin | Essential Oil of Elyonurus muticus (Sprengel) O.Kuntze (Gramineae)[END_REF], E. elegans Kunth [START_REF] Mevy | Composition and Some Biological Activities of the Essential Oils from an African Pasture Grass: Elionurus elegans Kunth[END_REF] and E. hensii K.Schum. [START_REF] Silou | Study of Essential Oil Composition of Elyonurus hensii Schum from Congo[END_REF][START_REF] Yang | Volatile Fraction Composition and Total Phenolic and Flavonoid Contents of Elionurus hensii-Antioxidant Activities of Essential Oils and Solvent Extracts[END_REF].

Elionurus tristis Hack. (Poaceae) commonly named "Antsoro" by the Malagasy is an aromatic grass endemic to the highlands of Madagascar, which has long been used by the local population as a folk medicine against colic but also as an antipyretic [START_REF] Chazan | L'Antsorenone, cétone sesquiterpénique de l'huile essentielle d'Elionurus tristis[END_REF].

Although the very first study dealing with the essential oil of E. tristis was published more than forty years ago, the scientific literature related to E. tristis remains very scarce, and the actual knowledge of its volatile constituents rather unexplored. Nonetheless, the presence of an unusual sesquiterpenic ketone named antsorenone was pointed out by Chazan in 1969 but its structure could not be elucidated [START_REF] Chazan | L'Antsorenone, cétone sesquiterpénique de l'huile essentielle d'Elionurus tristis[END_REF]. Recently another research group investigated the essential oil of E. tristis and reported a composition dominated by calarene (18.4%), neoclovene (15.8%), and nootkatone (10.4%) [START_REF] Yedomon | Elionurus tristis Essential Oil: GC-MS Analysis and Antioxidant and Antituberculosis Activities[END_REF].

As a part of our three decades-long investigation of EOs from Madagascar, we had the opportunity to reinvestigate the volatile constituents of E. tristis growing wild in the central regions of the island. Herein, we report the detailed analysis of the chemical composition of EOs obtained from both the aerial parts and the roots of E. tristis. This analytical study was mainly conducted by means of gas chromatographic techniques (GC, GC-MS, pc-GC) and NMR as an identification tool for the study of complex mixtures, but also for structure elucidation. Several undescribed sesquiterpenoids were isolated from E. tristis EOs and their structures were unambiguously characterized by NMR. Evident stereochemical relationships observed between these compounds led us establishing a possible biogenetic pathway for the production of the volatile constituents in the plant.

Results and discussion

Analysis of E. tristis essential oils by GC(RI), GC-MS and 13 C NMR

Essential oils of E. tristis were extracted from both the aerial parts and its roots as soon as the plant was harvested. EOs were subsequently analyzed by GC(RI) on both apolar (DB-1) and polar (DB-Wax) stationary phases using linear temperature programs for a reliable calculation of retention indices (RI). Constituents were identified upon combination of RI, MS, and 13 C NMR. The 13 C NMR based computerized methodology has been developed in our laboratory and allows the direct identification of constituents in mixtures such as essential oils or their fractions by comparison of their 13 C NMR values with those of compounds present in our in-house libraries. Each compound is identified by taking into account three parameters directly available from the software: (i) the number of carbon observed compared to what is expected, (ii) the difference between the chemical shift of each signal in the mixture and those from reference spectral data (δ), and (iii) the number of overlapped signals of carbons belonging to two components that fortuitously possess the same chemical shift [START_REF] Ouattara | The key role of 13 C NMR analysis in the identification of individual components of Polyalthia longifolia leaf oil[END_REF][START_REF] Tomi | Computer-aided identification of individual components of essential oils using carbon-13 NMR spectroscopy[END_REF].

The major constituents identified in both the aerial parts and the roots EOs were αpinene (1.7; 7.3% respectively), camphene (4.0; 13.6%), trans-α-bergamotene (7.0; 3.3%) and calarene (2.1; 12.7%) Table 1). Interestingly, we detected the presence of ten undetermined compounds (by GC(RI) and GC-MS) in appreciable amounts in EOs obtained from aerial parts (AP) and roots (R): i) compound 38 accounting for: 1.6% (AP), 2.2% (R); ii) 50: 4.0%, 6.3%; iii) 67: 1.0%, 1.3%; iv) 69: 5.1%, 1.8%; v) 71: 17.6%, 7.2%; vi) 72: 3.5%, 1.5%; vii) 75: 0.6%, 3.1%; viii) 76: 1.3%, 6.4%; ix) 77: 15.4%, 5.6%; x) 78: 3.1%, 0.2% (Fig. 1, 2 and S2).

Identification of undetermined compounds

In the process of analyzing the chemical composition of both the essential oils and their fractions, we noticed that four compounds named here 38, 71, 75, and 76 remained undetermined, providing very unsatisfactory matching with commercial or in-house MS libraries. These four compounds could however be identified by applying our in-house 13 C NMR computerized methodology. Moreover, two rather uncommon molecules which were absent from our 13 C NMR libraries were also identified after several fractionation steps, and their spectral characteristics could be reliably compared to literature data (compounds 50 and

69) (see Experimental).

Compound 76 was enriched up to 56.9% in subfraction F R 5-2, and its 13 C NMR values perfectly matched those previously reported for 1(10)-aristolen-2-one [START_REF] Rücker | Oxidized aristolane sesquiterpenes from Aristolochia debilis[END_REF] (Fig. 2 and S2, Table S1). Compound 75 was detected in several fractions and could be identified as aristolone by 13 C NMR. 75 was further purified by means of repeated column chromatography (CC) on silica gel until 82.5% purity. Its full 2D-NMR data permitted to correct wrong 13 C assignments reported by [START_REF] Su | Sesquiterpenes from Laurencia similis[END_REF], mainly concerning methyl-14 and methyl-15, and the two methylenes C-2 and C-3 (Fig. 2 and S2, Table S4). Compound 71 was purified up to 89.9% purity (in F AP 3) allowing the accurate assignment of all 1 H and 13 C signals through 2D-NMR sequences (Fig. 2 and S2, Table S3). This compound was unambiguously identified as acorenone B by its 13 C NMR values, perfectly matching those reported by [START_REF] Kutschan | Stereoisomere Acorenone. PFT-13C-NMR-Spektren und massenspektroskopische Untersuchungen[END_REF].

RIs and MS spectrum of compound 38 were nearly identical to those of cyclosativene.

However, its 13 C NMR data showed significant differences with those of cyclosativene (for instance, C-2 was deshielded by +3.74 ppm and in contrast, C-6, C-8 and C-11 were shielded by -5.39, -4.26 and -3.51 ppm), mostly related to the presence of specific steric γ-effects, indicative of its stereoisomer, cyclocopacamphene, previously described by Garcia et al.

(2017) (Fig. 2 and S2, Table S1).

Similarly, compound 50 showed similar RIs and MS data to those of prezizaene. However, its 13 C NMR chemical shifts differed from those of prezizaene, due to the presence of specific steric γ-effects (C-5 was deshielded by +4.82 ppm whereas, C-10 and C-12 were respectively shielded by -11.86 and -5.63 ppm). This led us to identify 50 as 2-epi-prezizaene which was previously described by [START_REF] Weyerstahl | 1,7-Cyclogermacra-1(10),4dien-15-al, a sesquiterpene with a novel skeleton, and other sesquiterpenes from Haitian vetiver oil[END_REF] (Fig. 2 and S2, Table S1).

Compound [START_REF] Weyerstahl | 1,7-Cyclogermacra-1(10),4dien-15-al, a sesquiterpene with a novel skeleton, and other sesquiterpenes from Haitian vetiver oil[END_REF] as constituent of vetiver oil. The full 2D-NMR eventually allowed us to correct the wrong assignment made by [START_REF] Weyerstahl | 1,7-Cyclogermacra-1(10),4dien-15-al, a sesquiterpene with a novel skeleton, and other sesquiterpenes from Haitian vetiver oil[END_REF] for methyl groups 13 and 15 (Fig. 2 and S2, Table S2). were then isolated from F AP 4 and F AP 5-3 using pc-GC with respectively 95.8% and 82.4% purity (see Experimental).

Identification

7-epi-Khusian-2-ol (72)

The combination of MS spectra and NMR data led to the molecular formula C 15 H 26 O. [START_REF] Weyerstahl | 1,7-Cyclogermacra-1(10),4dien-15-al, a sesquiterpene with a novel skeleton, and other sesquiterpenes from Haitian vetiver oil[END_REF]. These differences could be explained by a different relative configuration at C-7. Indeed, in the case of khusian-2-ol, the steric γ effect is observed between C-1 and C-13, whereas it appears between C-5 and C-13 for compound 72. The C-7 stereochemistry of 72 was confirmed by the NOESY correlation between H-5 and H-13 and the lack of the one between H-1 et H-13.

Finally, 72 was identified as 7-epi-khusian-2-ol. Total assignment of its 1 H and 13 C NMR spectra was achieved by means of 2D-NMR (Fig. 2, 3 and S2, Table 2). and 1-epi-acorone (commonly named cryptoacorone) except for some carbons [START_REF] Garneau | Aromas from Quebec. l. Composition of the essential oil of the rhizomes of Acorus calamus L[END_REF][START_REF] Nawamaki | Sesquiterpenoids from Acorus calamus as germination inhibitors[END_REF]. Thus, we focused our attention toward an epimer of acorone which 1 H and 13 C assignments were established through the accurate examination of HSQC, HMBC and COSY spectra. Concerning the relative configuration, the NOESY cross peak between hydrogens 3b and 6b indicated the stereochemistry of C-5. The NOESY correlation between H-3a and H-15 and the one between H-6a and H-13 showed that the two substituents of the cyclopentane were both oriented in a syn-periplanar manner. These previous observations allowed to deduce the relative configuration of C-5, C-1 and C-4. The NOESY cross peak of H-9b and H-10b with H-15 allowed to establish the inverted chair conformation of the cyclohexane. Finally, for minimal steric hindrance the methyl-14 was in the equatorial position. Consequently, compound 78 was identified as 4,8-di-epi-acorone (Fig.

4,8-di

2, 3 and S2, Table 3).

2.3.2. Structure Elucidation of 2-epi-Ziza-5-en-2-ol (67) and Antsorenone (77)

Compounds 67 and 77 eluted respectively at RIs 1582/2103 and 1749/2433 on both apolar and polar columns. These values did not match any constituents of our databases. In order to identify these two molecules, the AP EO was subjected to several chromatography techniques (see Experimental).

2-epi-Ziza-5-en-2-ol (67)

Aerial parts EO containing 1.0% of 67 was chromatographied to yield F AP 4 (containing 3.7% of 67), which was finally subjected to pc-GC to isolate 67 with 85.0% purity (GC). The C-13, C-14 and C-15) were very similar to those of 2-epi-isozizaene and 12-norisoziza-5-ene [START_REF] Filippi | Norsesquiterpenes as markers of overheating in Indonesian vetiver oil[END_REF][START_REF] Lin | Genome Mining in Streptomyces coelicolor: Molecular Cloning and Characterization of a New Sesquiterpene Synthase[END_REF]. Nevertheless, significant differences were observed for the rest of the signals mostly related to the α, β, and γ electronic effects induced by the alcohol function located on C-2. Thus, compound 67 was identified as 2-epi-ziza-5-en-2-ol (Fig. 2,3 and S2, Table 4).

Antsorenone (77)

Compound 77 was enriched up to 75.7% in F AP 5-2 by mean of repeated CC of E.

tristis AP EO, and was finally purified up to 90.1% by pc-GC. The molecular formula of compound 77 was deduced according to MS and NMR data as C 15 H 22 O.

Additionally, its 13 C NMR spectrum clearly showed the presence of a carbonyl function displaying interesting values (δ C 209.2 (C-3)), and a double bond (δ C 182.3 (C-1) and 130.9

(C-2)), due to the mesomeric effect of the α,β-unsaturated ketone, which are characteristic of an α,β-unsaturated ketone in a cyclopentane ring [START_REF] Pretsch | Structure Determination of Organic Compounds[END_REF]. nigritene (Champagnat et al., 2006;[START_REF] Filippi | Norsesquiterpenes as markers of overheating in Indonesian vetiver oil[END_REF]. However, in contrast with that previous study, this ketone constitutes the first occurrence of a complete sesquiterpene framework (15 carbons) for this skeleton. In agreement with its previous partial description by [START_REF] Chazan | L'Antsorenone, cétone sesquiterpénique de l'huile essentielle d'Elionurus tristis[END_REF], this ketone was named after the local name of the plant, Anstorenone [START_REF] Chazan | L'Antsorenone, cétone sesquiterpénique de l'huile essentielle d'Elionurus tristis[END_REF].

We thus propose the name Antsorane for this undescribed sesquiterpene family. The relative stereochemistry of this ketone could be easily deduced from the NOESY spectrum since all three aliphatic methyl groups displayed clear spatial correlations with specific hydrogens in the molecule. Thus, methyl-14 was correlated to H-9a, while methyl-15 was correlated to both H-6 and H-11a, suggesting thus the -orientation of methyl-13. Finally, the spatial correlation observed between H-10a and methyl-13 gave the ultimate clue to ensure the relative configuration of the entire structure (Fig. 2, 3 and S2, Table 5).

Putative Biogenetic Relationships

While the biogenetic relationships between the acorane, helifolane, prezizaane, and zizaane skeletons are well known [START_REF] Hong | Consequences of Conformational Preorganization in Sesquiterpene Biosynthesis: Theoretical Studies on the Formation of the Bisabolene, Curcumene, Acoradiene, Zizaene, Cedrene, Duprezianene, and Sesquithuriferol Sesquiterpenes[END_REF], the identification of compounds

Conclusion

Despite being a common plant used in folk medicine in Madagascar, Elionurus tristis, locally known as Antsoro, has rarely been investigated from a chemical standpoint. The aim of this study was to establish the detailed chemical compositions of its essentials oils extracted from both its aerial parts (AP) and its roots (R). The combined analysis of the two EOs by GC(RI), GC-MS and NMR allowed to respectively determine 93.8% and 97.2% of their entire chemical compositions in which 75 and 56 components were identified. This work also permitted to reveal the presence of several uncommon compounds such as cyclocopacamphene ( 38), 2-epi-prezizaene (50), 2-epi-prezizaan-7-ol (69), acorenone B (71), aristolone (75) and 1(10)-aristolen-2-one (76). Finally, four undescribed compounds could be entirely characterized: 2-epi-ziza-5-en-2-ol (67), 7-epi-khusian-2-ol (72), 4,8-di-epi-acorone (78), and antsorenone (77); this last one exhibits an undescribed natural sesquiterpene. Thanks to clear stereochemical relationships between the isolated constituents, our findings allowed us to decipher a probably active formation pathway of sesquiterpenes in Elionurus tristis, leading to the newly established antosrane skeleton.

Experimental Part

Plant material, isolation of essential oils

The essential oils were obtained by water distillation (for 4 hours) using a Clevengertype apparatus from aerial parts (AP; 235.5 g) and roots (R; 481.5 g) of Elionurus tristis harvested at Lazaina Avaradrano (central region of Madagascar) on the 1 st June 2015 (dry season). Both samples were obtained as light-yellow colored oils in 0.33% (771.3 mg) and 0.21% (998.3 mg) yields, respectively. A voucher specimen of E. tristis has been deposited at the Malagasy Institute of Applied Research, (Antananarivo, Madagascar), under the accession number LHE015.

Oils fractionation

Aerial Parts oil: the essential oil obtained from the aerial parts of Elionurus tristis (771.3 mg) was separated into five fractions F AP 1-F AP 5 by silica gel column chromatography (60-200 μm, 60 Å, 49.6 g) using an increasing gradient of pentane/Et 2 O 100/0 (F AP 1=159.9 mg), 95/5 (F AP 2=86.4 mg), 90/10 (F AP 3=157.8 mg), 80/20 (F AP 4=151.1 mg) and 0/100 (F AP 5=212.6 mg).

Fraction F AP 1 contained 38 and 50 with the following contents: 4.7 and 13.0%.

Fraction F AP 3 contained 71 with 89.9% purity.

Fraction F AP 4 containing compounds 67 (3.7%), 69 (18.8%) and 72 (16.2%) and was directly used in pc-GC to purify the target compounds: 67 (85.0%), 69 (94.6%), 72 (95.8%). These three compounds were subject to NMR analysis for structure elucidation.

F AP 5 was further fractionated by CC with a finer silica gel (35-70 μm, 60 Å, 22.4 g) using pentane/acetone (93/7) to yield three subfractions: F AP 5-1, 15.1 mg, F AP 5-2, 120.7 mg, and F AP 5-3, 71.9 mg. The last two subfractions respectively contained 77 (75.7%) and 78 (54.8%).

Compounds 77 and 78 were finally isolated by pc-GC with respective purities of 90.1% and 82.4% (Fig. S1).

Roots oil: the roots EO (998.3 mg) was subjected to the same procedure as above to provide five main fractions: F R 1, 347.4 mg, F R 2, 66.3 mg, F R 3, 151.7 mg, F R 4, 226.2 mg, and F R 5, 129.7 mg. The last one was chromatographied a second time to provide F R 5-1 (45.1 mg), F R 5-2 (78.7 mg) and F R 5-3 (4.1 mg). The first two subfractions respectively contained 75 (82.5%) and 76 (56.9%).

Isomerization of Antsorenone (77)

Reactions were realized in a 50 mL round bottom flask equipped with a reflux condenser. Every assay was performed by taking approximately 15 mg (5.2•10 -2 mmol of 77)

of F AP 5-2 which were mixed with different acids in excess (see below) and heated to reflux for 6 hours under magnetic stirring as follows: (i) 1 mL of pure formic acid (26.0 mmol), (ii)

1 mL of pure trifluoroacetic acid (12.9 mmol), (iii) 1 g of pure pTsOH.H 2 O (5.2 mmol) suspended in 5 mL of MeOH, (iv) a mixture of 1 g of pure pTsOH.H 2 O (5.2 mmol) and 1 mL of BF 3 .Et 2 O (7.8 mmol) was suspended in 7 mL of MeOH. After every assay, the acid was evaporated under reduced pressure. The residue was thus recovered by adding 3 mL of anhydrous Et 2 O, then washed by addition of NaHCO 3 until pH 7-8. The organic layer was separated and dried over MgSO 4 . The mixtures obtained for each of the four assays were directly analyzed by GC(RI) and GC-MS.

General Experimental Procedures

Optical rotations were measured on a JASCO P-2000 polarimeter. UV and ECD spectra were recorded on a JASCO J-1500 spectrometer. IR spectra was recorded on a

ThermoFisher SCIENTIFIC Nicolet iS5 FTIR spectrometer.

Gas Chromatography

Gas Chromatography-High Resolution Mass Spectrometry

High-resolution EI-mass spectra were recorded using an Agilent 7200 GC-QTOF system (Agilent, Santa Clara, CA, USA), equipped with an Agilent J&W, DB-1 capillary column (30 m x 0.25 mm; 0.25 μm film thick). The mass spectrometer was operated at 70 eV with an acquisition rate of 2 GHz over a 35-450 m/z range, affording a resolution of ~8000. with the following oven temperature programming: 140 to 250 °C at 10 °C/min. A Graphpack effluent splitter was connected to the column outlet, and was additionally mounted with 0.1 mm and 0.32 mm deactivated fused-silica capillary restrictors (1 m each) to provide an FID/PFC ratio of ~1/9. The transfer line and the PFC were maintained at 230 °C. The injected volume was 1 μL in splitless mode. The system was operated in constant pressure mode at 35 kPa. Compounds were trapped at room temperature in Gerstel U-type glass tubes by programming cutting times into the operating software allowing accurate automated operation. The isolation of any unknown compound in amounts sufficient for NMR analysis required 150-400 GC runs, and to avoid any contaminations, each product was collected directly in a NMR tube.

Nuclear Magnetic Resonance

NMR spectra for compounds: (i) 67, 69, 72, 78, were recorded in C 6 D 6 ; (ii) 77 was recorded in CDCl 3 ; at 298 K on a Bruker Avance DRX 500 spectrometer operating at 500.13

MHz for 1 H and 125.75 MHz for 13 C. In order to increase sensitivity, 13 C NMR spectra such as broadband-13 C, DEPT 135 and DEPT 90 were run with a direct probe head (5 mm PADUL 13 C-1 H Z-GRD). 1D-and 2D-NMR spectra such as 1 H, COSY, NOESY, HSQC, HMBC were run with an inverse probe head (5 mm PHTXI 1 H-13 C/ 15 N ZGRD). Spectrum calibration was performed by using the C 6 D 6 signal (7.16 ppm for 1 H NMR, 128.06 ppm for 13 C NMR) or CDCl 3 signal (7.26 ppm for 1 H NMR, 77.16 ppm for 13 C NMR) as internal reference.

Chemical shifts (δ) are expressed in parts per million (ppm) and coupling constants (J) in hertz. All NMR experiments were carried out using pulse sequences supplied by the spectrometer manufacturer (Bruker Topspin TM ) and processed via Mestrelab MestreNOVA software (Version 6.0.2-5475).

Other spectra (including 38, 50, 71, 75, 76) were recorded on a Bruker AVANCE 400 (100.623 MHz for 13 C) equipped with a 5-mm probe (DUL 13 C-

Identification and quantification of individual components

Identification of individual components was based: a) on a comparison of their GC retention indice (RI) values on both polar and apolar stationary phases, with the literature [START_REF] König | Terpenoids and Related Constituents of Essential Oils Terpenoids[END_REF]. b) on computer search using digital libraries of mass spectral data and comparison with published data [START_REF] Adams | Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry[END_REF][START_REF] Mclafferty | Wiley Registry of Mass Spectral Data[END_REF]. c) on comparison of the signals in the 13 C NMR spectra of the mixtures with those of reference spectra compiled in the laboratory spectral library, with the help of a laboratory-made software [START_REF] Ouattara | The key role of 13 C NMR analysis in the identification of individual components of Polyalthia longifolia leaf oil[END_REF][START_REF] Tomi | Computer-aided identification of individual components of essential oils using carbon-13 NMR spectroscopy[END_REF].

Spectral data

  The 13 C NMR spectrum displayed 15 signals whose multiplicities were deduced by DEPT 135 and 90 as 3 C, 3 CH, 5 CH 2 and 4 CH 3 . The only characteristic 13 C NMR value was a methine at 70.5 ppm. Taken altogether, MS and NMR data indicated that 72 is a tricyclic sesquiterpene alcohol. Here again, 72 exhibited 13 C NMR values very similar to those of khusian-2-ol except for C-1, C-5 and C-13 (Δ(δ) > 3 ppm)

  -epi-Acorone (78) MS and NMR data (DEPT 135 and 90) for 78 indicated a C 15 H 24 O 2 molecular formula and multiplicities of 13 C NMR signals as 3 C, 4 CH, 4 CH 2 , and 4 CH 3 . The two signals at 209.7 ppm and 215.5 ppm indicated the presence of two ketone groups. The set of 13 C NMR values of 78 were similar to those of acorone, 1,8-di-epi-acorone (also named epi-acorone),

  difference of 500 points between the polar and apolar RI values, and the molecular ion peak in MS (m/z 220) suggested 67 was a sesquiterpene alcohol with a C 15 H 24 O molecular formula. By considering the degree of unsaturation and the carbon signals' multiplicities, compound 67 corresponded to an unsaturated tricyclic sesquiterpene alcohol. A DEPT experiment permitted to identify 5 C, 1 CH, 5 CH 2 , and 4 CH 3 . Moreover, we could deduce from the 13 C spectrum the presence of a tetrasubstituted double bond at 141.6 ppm/129.2 ppm and a tertiary alcohol at 80.3 ppm. Due to the similarities between our EOs with those of vetiver, and the presence of some prezizaane derivatives into E. tristis samples, we oriented our research towards a prezizaane/zizaane skeleton-based alcohol. Six out fifteen 13 C NMR signals (C-7, C-8, C-9,

  This observation was confirmed by the long-range correlations between H-4a/4b and carbons C-1, C-2, C-3, and C-5. Moreover, MS and NMR data suggested that 77 was a tricyclic sesquiterpene ketone. Interestingly, C-12 resonance was shielded with a 13 C NMR value of 7.6 ppm (H-12 at 1.64 ppm) and was assigned to the methyl in β position to the ketone thanks to the HMBC correlations observed between H-12 and C-2/C-3.The examination of other long-range 1 H-13 C correlations in the HMBC spectrum allowed to build two additional patterns: -CH(CH 3 )-C(CH 3 ) 2 -and -CH 2 -CH-CH 2 -CH 2 -. The final tricyclic skeleton was first deduced from the connections of C-1/C-11 and C-5/C-10, and secondly of C-5/C-6 and C-7/C-8 giving a basic skeleton comparable to that previously observed for norsesquiterpene constituent of Indonesian vetiver oils, nigritene and 6-epi-

  and 78 helped to establish a complete formation pathway of these molecules in E. tristis. Most unexpectedly, the specific configuration observed at C f , C g and C j in the acorane skeleton (cyclopentane ring) appeared to remain untouched in all the succeeding sesquiterpenic skeletons. Hence, compounds 72 (helifolane), 50 and 69 (prezizaane), and 67 (isozizaane) were all displaying the same -orientation in position g as opposed, for instance, to what is naturally observed in vetiver oil constituents where methyl-g (or its functionalized version) is most of the time -orientated. Moreover, the higher steric hindrance generated by the -orientation of methyl-g in the isozizaane skeleton, as seen for compound 67, might have well triggered the Wagner-Meerwein transposition of the C d -C e bridge into position j, finally producing the newly identified antsorane skeleton.Under acidic conditions, several attempts to transform 77 into zizaen-3-ones following a reverse Wagner-Merweein rearrangement failed. Several Brönsted and Lewis acids were tested but none of them succeeded in inducing the transposition. The high stability of 77 was extremely interesting, and it supported the hypothesis of the antsorane skeleton being the final step of the biogenetic pathway. Finally, it is worth mentioning that compound 77 was also detected in a solvent extract of E. tristis (made at room temperature with n-hexane). (Fig.4).

  GC analyses were performed on a Clarus 500 PerkinElmer (PerkinElmer, Courtaboeuf, France) system equipped with a FID and two fused-silica capillary columns DB-1 (50m  0.22 mm, film thickness 0.25 µm) and DB-WAX (50m  0.22 mm, film thickness 0.25 µm). The oven temperature was ramped from 60 °C to 220 °C at 2 °C/min and then held isothermal at 220 °C for 20 min; SSL inlet temperature: 250 °C; FID temperature: 250 °C; carrier gas: helium (0.8 mL/min); split: 1/60; injected volume: 0.5 µL. Retention indices (RI) were determined against a series of n-alkanes with linear interpolation (Target Compounds software from PerkinElmer). Quantification of the constituents was performed according to the method described by[START_REF] Tissot | Rapid GC-FID quantification technique without authentic samples using predicted response factors[END_REF], (i) methyl octanoate (MO) was used as an internal reference; (ii) predicted relative response factors (PRRF) were calculated for each compound; and (iii) the actual amount of each constituent was deduced and finally expressed in g/100 g.4.6. Gas Chromatography-Mass Spectrometry in Electron Impact ModeEssential oils samples were analyzed with a Perkin-Elmer TurboMass detector (quadrupole), directly coupled to a Perkin-Elmer Autosystem XL, equipped with two fusedsilica capillary columns, DB-1 (50 m × 0.22 mm, film thickness 0.25 µm) and DB-WAX (50 m × 0.22 mm, film thickness 0.25 µm). Carrier gas, helium at 0.8 mL/min; split: 1:70; injection volume: 0.5 µL; injector temperature: 250°C; oven temperature programmed from 60°C to 220°C at 2°C/min and then held isothermal at 220 °C for 20 min. Ion source temperature: 250°C; energy ionisation: 70 eV; electron ionisation mass spectra were acquired over a 35-350 Da mass range.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Key-NOEs for isolated compounds 67, 72, 77, and 78.

  1 H Z-GRD Z8246/0066), in CDCl 3 . The 1 H NMR spectra were recorded with the following parameters, pulse width (PW), 4.3 ms; acquisition time, 2.6 s for 32 K data table with a spectral width (SW) of 6000 Hz (15 ppm). 13 C NMR spectra were recorded with the following parameters: pulse width (PW), 4 μs

(flip angle 45°); acquisition time, 2.7 s for 128 K data table with a spectral width of 24000 Hz (240 ppm); total repetition time, 2.8 s; CPD (composite pulse decoupling) mode decoupling; digital resolution, 0.183 Hz/point.

Table 1 .

 1 Chemical composition of essential oils of Elionurus tristis.

	Cyclocopacamphene (38)
	C 15 H 14 ; RI (DB-1/DB-WAX) 1365/1479; EI-MS 70 eV, m/z (rel. int.): 204 (18, M •+ ),
	189 (14, M •+ -CH 3 ), 175 (3), 162 (15), 161 (75, M •+ -C 3 H 7 ), 147 (10), 135 (17), 134 (20),
	133 (41), 122 (18), 121 (28), 120 (43), 119 (81), 107 (45), 106 (25), 105 (100), 95 (21), 94
	(45), 93 (49), 92 (24), 91 (66), 81 (21), 79 (35), 77 (29), 69 (19), 67 (17), 65 (13), 55 (28), 53
	(15), 43 (18), 41 (42). 1 H NMR (CDCl 3 , 400 MHz) and 13 C NMR (CDCl 3 , 100 MHz): see
	Table S1.
	2-epi-Prezizaene (50)
	C 15 H 14 ; RI (DB-1/DB-WAX) 1447/1636; EI-MS 70 eV, m/z (rel. int.): 204 (4, M •+ ),
	189 (22, M •+ -CH 3 ), 175 (8), 161 (24), 147 (21), 134 (35), 133 (93), 120 (19), 119 (51), 117
	(21), 108 (43), 107 (39), 105 (46), 95 (32), 93 (59), 91 (100), 81 (35), 79 (41), 77 (29), 69
	(21), 67 (30), 65 (14), 55 (31), 53 (18), 43 (10), 41 (32). 1 H NMR (CDCl 3 , 400 MHz) and 13 C
	NMR (CDCl 3 , 100 MHz): see Table S1.
	2-epi-Ziza-5-en-2-ol (67)
	Colorless oily liquid; [α] 20	D -28 (c 0.01, CH 3 OH); UV (MeOH) λ max (log ε) 192 (0.26)
	nm; C 15 H 24 O; RI (DB-1/DB-WAX) 1582/2103; HREIMS: m/z 220.1821 (calcd for C 15 H 24 O,
	220.1827); EI-MS 70 eV, m/z (rel. int.): 220 (4, M •+ ), 206 (5), 205 (36), 202 (8, M •+ -H 2 O),
	188 (7), 187 (51), 177 (6), 160 (12), 159 (89), 149 (7), 147 (10), 145 (10), 133 (16), 131 (11),
	129 (6), 128 (6), 121 (6), 120 (10), 119 (100), 117 (10), 115 (10), 107 (8), 105 (20), 95 (6), 93
	(15), 91 (27), 81 (7), 79 (12), 77 (13), 69 (6), 67 (7), 65 (7), 55 (11), 53 (7), 43 (33). 1 H NMR
	(C 6 D 6 , 500 MHz) and 13 C NMR (C 6 D 6 , 125 MHz): see Table 4.

22 O; RI (DB-1/DB-WAX) 1749/2433; HREIMS: m/z 218.1668 (calcd for

Table 2 .

 2 NMR data of 7-epi-Khusian-2-ol (72).

	C	13 C δ (ppm)	1 H	1 H δ (ppm) by HSQC	COSY 1 H-1 H	HMBC H → C	NOESY a
	1 45.1	1a	1.47-1.52 (m)	1b, 2, 5	2, 3, 5, 6	2
			1b	1.36 (dd, J = 12.9 5.4 Hz)	1a, 2	2, 5, 6, 10	-
	2 70.5	2	3.78 (ddd, J = 9.6 5.4 2.2 Hz) 1a, 1b, 4b	4	1a, 12, 14
	3 39.9	-	-	-	-	-
	4 23.5	4a	1.64-1.70 (m)	4b, 5	2, 3, 5, 11	4b, 5, 12
			4b	1.15-1.21 (m)	2, 4a, 5	2, 3, 5	4a, 12
	5 20.4	5	1.00-1.08 (m)	1a, 4a, 4b	1, 4, 6, 7,	4a, 13
	6 43.2	-	-	-	-	-
	7 42.7	7	1.31-1.36 (m)	8a, 8b, 13	5, 6, 8, 13	8a, 13
	8 32.2	8a	1.70-1.78 (m)	7, 8b, 9	7, 9, 10	7, 8b, 9
			8b	1.13-1.19 (m)	7, 8a, 9	7, 9, 13	8a
	9 22.6	9	1.39-1.47 (m)	8a, 8b, 10	8, 10, 11	8a, 15
	10 54.3	10	1.12-1.19 (m)	9	1, 5, 6, 9, 11, 14, 15 -
	11 34.9	-	-	-	-	-
	12 16.5	12	0.72 (s)	-	2, 3, 4, 11	2, 4a, 4b
	13 14.6	13	0.77 (d, J = 6.9 Hz)	7	6, 7, 8	5, 7
	14 26.8	14	0.78 (s)	-	3, 10, 11,	2
	15 21.7	15	0.80 (s)	-	3, 10, 11,	9

a Pure NOE correlations appear underlined. Hydrogens H1a/H1b, H4a/H4b and H8a/H8b could not be differentiated.

Table 3 .

 3 NMR data of 4,8-di-epi-Acorone (78).

	C	13 C δ (ppm)	1 H	1 H δ (ppm) by HSQC	COSY 1 H-1 H HMBC H → C	NOESY a
		59.6	1	1.64-1.67 (m)	-	2, 5, 6, 10, 11, 12	13
		215.5	-	-	-	-	-
		45.0	3a (anti)	1.57 (d, J = 18.6 Hz)	3b	2, 15	3b, 15
			3b (syn)	2.17 (dd, J = 18.6 8.3 Hz)	3a, 4	2, 4, 15	3a, 4, 6a, 6b
		32.2	4	1.64-1.69 (m)	3b, 15	2, 3, 5, 6, 10, 15	3b, 6b, 15
		50.5	-	-	-	-	-
		47.9	6a	1.89 (dd, J = 12.8 1.0 Hz)	6b	1, 4, 5, 7, 10	3b, 6b, 10a, 11, 12, 13
			6b	2.05 (dd, J = 12.8 2.8 Hz)	6a, 10b	4, 5, 7, 8, 10	3b, 4, 6a, 12
		209.7	-	-	-	-	-
		44.3	8	1.80 (m, J = 6.4 Hz)	9a, 14	7, 9, 14	9a, 14
		31.0	9a	1.43 (m)	8, 9b	7, 8, 10	8, 9b
			9b	1.01-1.06 (m)	9a, 10a	8, 10, 14	9a, 10a, 15
		31.5	10a	1.27-1.32 (m)	9b, 10b	4, 5, 6, 8, 9, 11	6a, 9b, 10b
			10b	1.13-1.19 (m)	6b, 10a	6, 8, 9	10a, 15
		25.3	11	1.57 (m)	12, 13	1, 2, 5, 12, 13	6a, 12, 13
		19.7	12	0.95 (d, J = 6.8 Hz)	11	1, 11, 13	6a, 6b, 11
		24.9	13	1.16 (d, J = 7.0 Hz)	11	1, 11, 12	1, 6a, 11
		14.7	14	1.01 (d, J = 6.5 Hz)	8	7, 8, 9, 10	8
		15.1	15	0.36 (d, J = 7.2 Hz)	4	3, 4, 5	3a, 4, 9b, 10b

a Pure NOE correlations appear underlined.

Table 4 .

 4 NMR data of 2-epi-Ziza-5-en-2-ol (67). Gas chromatograms on apolar column of the Aerial Parts and the Roots essential oils depicting compounds 38,

	C	13 C δ (ppm)	1 H	1 H δ (ppm) by HSQC COSY 1 H-1 H	HMBC H → C NOESY a
	1 58.2	-	-	-	-	-
	2 80.3	-	-	-	-	-
	3 38.9	3a (anti)	1.55 (m)	3b, 4a, 4b	4	4a, 4b, 10a, 12
			3b (syn)	1.67 (m)	3a, 4a	1, 2, 4, 5	4a
	4 25.7	4a	2.28-2.40 (m)	3a, 3b, 4b, 13	5	3a, 3b, 4b,
			4b	2.12 (dd, J = 17.0 9.0 Hz) 3a, 4a, 13	2, 5, 6	3a, 4a
	5 141.6	-	-	-	-	-
	6 129.2	-	-	-	-	-
	7 40.8	-	-	-	-	-
	8 47.8	8	1.81-1.86 (m)	9b, 10b, 11b	1, 6, 7, 10, 11	10b, 11b, 14,
	9 25.5	9a (exo)	1.80-1.85 (m)	9b, 10b, 11a, 11b	1, 7, 8, 10, 11, 14 9b, 10b
			9b (endo)	1.56-1.63 (m)	8, 9a, 10a, 10b, 11a, 11b -	9a, 10b, 15
	10 32.4	10a (exo)	1.77-1.83 (m)	9b, 10b, 11a, 11b	8, 9, 11	3a, 10b, 12
			10b (endo)	1.27-1.33 (m)	8, 9a, 9b, 10a, 11b	1, 5, 7, 8, 12	8, 9a, 9b, 10a
	11 34.7	11a (exo)	1.32-1.38 (m)	9a, 9b, 10a, 11b	1, 5, 7, 9	11b, 12
			11b (endo)	1.15-1.22 (m)	8, 9a, 9b, 10a, 10b, 11a	14	8, 11a
	12 22.3	12	1.13 (s)	-	1, 2, 3	3a, 10a, 11a
	13 13.3	13	1.46 (s)	4a, 4b	5, 6, 7	4a, 14, 15
	14 28.6	14	1.05 (s)	-	6, 7, 8, 15	8, 13
	15 25.3	15	0.99 (s)	-	6, 7, 8, 14	8, 9b, 13

a Pure NOE correlations appear underlined. Hydrogens H4a/H4b could not be differentiated Fig. 1.

50, 67, 69, 71, 72, 75, 76, 77, 78 Fig. 2.

  Structures of compounds characterized by NMR. Compounds 67, 69, 72, 77, and 78 were isolated by pc-GC.

Acknowledgments

Gabriel P. Garcia thanks the "Collectivité de Corse" for a Ph.D. grant. The authors are grateful to Robertet S.A. and especially Dr. H. Brevard and Dr. N. Barat for their technical assistance during HREIMS analysis

Supplementary Materials

The following data are available online. Tables S1 Order of elution and percentages are given on apolar column; except for those with an asterisk (*), percentages on polar column. RIa, RIp: retention indices measured on apolar (DB-1) and polar (DB-WAX) columns, respectively. a RI lit: retention indices according to Tepenoids Library Website [START_REF] König | Terpenoids and Related Constituents of Essential Oils Terpenoids[END_REF]). b RI lit: retention index according to [START_REF] Garcia | Integrated Analysis of the Bark oil from Cinnamosma madagascariensis by GC(RI), GC-MS and NMR. 13 C NMR data of Cyclocopacamphene and Cyclosativene[END_REF]. c RI lit: retention index according to [START_REF] Mevy | Composition and Some Biological Activities of the Essential Oils from an African Pasture Grass: Elionurus elegans Kunth[END_REF]. The relative proportions of each constituent were expressed in g/100 g. tr: traces (<0.05%); 2D-NMR = 2D-NMR spectra are provided in Supporting Information.


domain: chim spi

text:
A N Chichkov

Kanaev

P Gorbovyi

A P Diaz-Gomez

M Traore
email: traore@lspm.cnrs.fr

# L Museur

L Rozes

F Ribot

C Sanchez

A I Kuznetsov

B N Chichkov

A Kanaev

Alkoxysilane effect in hybrid material: A comparison of pHEMA-TiO2 and pMAPTMS-TiO2 nanoparticulate hybrids

Keywords: A. Hybrids, A. nanostructures, B. Sol-gel chemistry, B. Optical properties

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

Introduction

For over two decades, methods of preparing inorganic and hybrid nanomaterials based on "soft chemistry" have attracted strong interest both in academia and industry [1][2][START_REF] Kickelbick | Hybrid materials, synthesis, characterization, and applications[END_REF][START_REF] Nicole | [END_REF] . Hybrid materials advantageously mix structural flexibility and convenient processing of polymer with electronic properties of inorganic component. Consequently, they are promising candidates for potential application in many fields, such as electronics, optics, biology, etc.

Considerable progress has been reached in elaboration of organic-inorganic hybrids and understanding of their physical properties. Between them, silica, titania, and zirconia hybrids attract particular interest for applications in 2-photon laser microstructuring due to their high refractive index and biocompatibility [5][6][7][8] . Furthermore, titania hybrids show interesting electron transport properties, consisting in the effective photoinduced charge separation across the large internal organic-inorganic interface [9][10][11] , which permits laser writing of 3D domains with modified physical properties 12 . More recently, it has been established that the modification of the refraction index of irradiated domains can attain ∆n=-0.005 13 , which may enable fabrication of optical waveguides in these materials. Titania hybrids, capable accumulating of up to 10 % Ti 3+ centers, were proposed for photochemical conversion and solar energy storage [14][15] .

A capacity of the titania solids to effectively separate and store charges has attracted much attention in the past [16][17][18] . This ability is related to the presence of photo-excited hole scavengers and positive electron affinity of Ti 4+ centers. It has been recognized that an adequate design of the material nanoscale interface can considerably increase the efficiency of charge separation and storage process. While the typical efficiency in the range of 1-10 % has been reported in early studies, late developments of the hybrids have promoted the process efficiency up to 50% 19 and even higher values [20][21] .

In consequence, the elaboration process is crucial for electronic properties of the hybrid materials. A control of the inorganic-organic interface and nanoscale morphology, including the inorganic particle sizes and aggregation state, permits to promote the photoinduced charge separation efficiency of these materials and increase their photonic sensitivity. Despite of the key functional role of the inorganic titania component, much attention has to be paid to the organic component, since recombination of photogenerated charges depend on their capacity to trap the photoexcited hole. It is therefore of high interest to compare photonic sensitivities of the materials prepared with different organics, which can or cannot produce strong interaction with the inorganic component.

In this paper, we report on the effect of alcoxysilane as a molecular precursor of the polymer matrix embedding TiO2 nanoparticles. A comparative study was carried out of organic-inorganic hybrid materials prepared of size-selective non-aggregated 5nm TiO2 nanoparticles with two different organic phases: 2-hydroxyethyl methacrylate (HEMA) and methacryloxypropyltrimethoxysilane (MAPTMS).

Experiment and sample preparation

Material preparation

The preparation schema adopted for pHEMA-TiO2 and pMAPTMS-TiO2 hybrids was previously described by Gorbovyi et al. 19 . In short, the size-selected 5 nm titanium-oxo-alkoxo (TOA) nanoparticles were generated in a thermostatic sol-gel reactor with rapid micromixing [22][23] , using titanium tetraisopropoxyde (TTIP) precursor (Acros Organics, 98% purity) and distilled water in 2-propanol (Acros Organics, 99.5% purity) solution and the hydrolysis ratio H=2.0 at 20.0 °C. The underlying sol-gel chemistry does not proceed via hydroxide intermediates, but results directly in well-defined titanium-oxo-alkoxo species through one-step hydrolysiscondensation transformation associated with profound restructuring of the precursor molecules 24 .

In our experimental conditions, the protected from external humidity nanocolloids are almost monodispersed and stable beyond one day 25 , which allows their relatively simple manipulations.

At the second step, the solvent exchange (2-propanol by HEMA or MAPTMS) was carried out overnight in vacuum. The both monomers were preliminary distilled in order to eliminate impurities and inhibitors of spontaneous polymerization. The procedure is performed in a roundbottom flask, equipped with magnetic stirrer and vacuum valve. A single stage membrane pump (KNF N 840.3FT.18) was used for pressure reduction down to 10 mbar. A LN2 trap was used to condensate the 2-propanol vapors. The exchange reactor temperature control was performed with a simple water bath (20 °C). The adjustment of the inorganic nanoparticle concentration was performed at this stage. By this method, the hybrid solutions with Ti concentrations from 0.15 mol/l (x1) to 3.0 mol/l (x20) have been prepared (Table 1).

The nanoparticulate precursors were stable in dry atmosphere, e.g. x1 sample could be conserved over 1 month.

At the final step, free radical polymerization of the methacrylate functions was produced thermally or photochemically from the prepared monomer-TiO2 solution. The thermal polymerization was conducted at 90 °C during 12 hours with an addition of the AIBN initiator.

Photopolymerisation was conducted with an addition of the Igacure-369 photoinitiator using two types of lasers: UV picosecond (355 nm, ~10 -11 s) and near-IR femtosecond (1030 nm, ~10 -13 s) lasers. Two different mechanisms of initiation were involved: while UV irradiation triggers onephoton polymerisation process, the near-IR femtosecond laser pulses induce two-photon polymerization. The particle size (2R) and the scattered light intensity (I) were measured at the nucleation and solvent exchange stage in situ with a monomode optical fiber probe of He-Ne laser by photon-correlated spectroscopy method, using 16-bit 255-channels digital correlator (PhotoCor Instruments). The observation volume defined by a mutual positioning of two monomode optical fibres is small enough (~10 -6 cm 3 ) to prohibit multiple scattering events. The measurements (I,R)

were carried out in the automatic sampling mode with the data accumulation over 60 s, which permits to reduce noisy data due to rare dust particles.

The ligands exchange during solvent exchange and C=C bonds opening during polymerization stages were monitored by Raman spectroscopy. The Raman spectra were measured with high-resolution micro-Raman spectrometer (HR800 HORIBA Jobin Yvon) operating at 633 nm wavelength with spectral and spatial resolution of 0.25 cm -1 and ~5 µm, respectively. The scattered light was collected in a backscattering configuration and recorded on a Peltier-cooled CCD camera.

The NMR 17 O, 29 Si spectra in liquid and solid forms have been performed using Bruker

Avance III 300 (300.13 MHz) spectrometer equipped with fiber BBFO 5mm. No additional deuterated solvents were used for sample preparation. Liquid (before organic polymerization) and solid (polymerized) hybrid samples were characterized with 17 O NMR method. The spectra acquisitions were performed at the following conditions: spin-echo (D1-90°-τ-180°-τ-AQ), where period of 90° pulse was 15.2 µs; delay echo was 100 µs, interpulse delay was 0.1 s and acquisition time was 4.1 ms (TD=4 K, SW=12300 ppm or 50000 Hz). 28800 scans were accumulated (~50min / exp). MAPTMS-TiO2 samples were additionally characterized with the RMN 29 Si (59.62 MHz) method. The time proton "inverse gated" decoupling was performed. Impulse angle was 30° (~3 µs), acquisition time was 0.69 s (TD=16 K, SW=200 ppm ou 12000 Hz) and recycling time was 5 s. 1024 scans were accumulated (t~100 min) per acquisition. Laser-induced two-photon polymerization (2PP) has been carried out using second harmonic of Yb:glass femtosecond laser oscillator (λ=515 nm) delivering pulses of 150 fs duration with the repetition rate of 10 MHz. 2PP is a direct laser writing technique, which allows fabrication of complex computer-controlled 3D structures with a resolution below 100 nm [27][28] . It is based on laser initiation of polymerization reactions inside transparent photocurable materials. Due to nonlinear nature of the laser-material interaction, the polymerization process can be strongly localized in the focal point of the laser beam, which produces small polymerized voxels inside the non-polymerized material. Scanning the laser beam focus allows fabrication of arbitrary form computer-controlled polymerized structures. After removing of the non-polymerized part of the material, free-standing or attached to a substrate polymerized microstructures can be obtained.

Measurements

Big box-like structures were polymerized with the laser beam loosely focused by a 120 mm lens inside a thick flat layer of the hybrid material. These relatively large structures were produced in order to check photochromic response of the polymerized hybrids. In order to verify the ability of two-photon polymerization to reach submicronic spatial resolution, woodpile-like structures were fabricated with the tightly focused laser beam by 100X oil immersed microscope objective (Zeiss, Plan Apochromat, NA=1.4).

Results and Discussion

Structure and composition

The intensity auto-correlation function (ACF) contains information about the diffusion coefficient and hydrodynamic radius of the inorganic nanoparticles in monomer solutions.

The measured ACF is expresses by (2) ( ) =

(2) ( )

(2) (0) = ( ). ( + ) 2 ( ) = 1 + -2 2 (1)
where = sin(θ/2) is the scattering wave vector at the observation angle θ and light wavelength λ, D is the particle diffusion coefficient and τ is the correlation time. Since = (η, n and T are respectively the dynamic viscosity, refraction index and temperature of colloid and k is the Boltzmann constant), The exponential factor in Equation 1 can be presented as:

(!, θ, #)$ %& ' ( )
, where f(T,θ,λ) is the constant related to the experimental geometry and temperature. Tracing the experimental points in frame (g 2 , ' (

) ) permits easier comparison of the particles radii.

We used the refraction index / dynamic viscosity of HEMA and MAPTMS solutions (20°C), As expected, the TOA nanoparticles conserve their initial size 2R=5 nm in HEMA solutions after the solvent exchange. On the other hand, their size in MAPTMS solution increases with CTi; however, this size dependence is strongly non-linear, and in the range of small concentrations CTi≤0.75 mol/l the size is almost constant. We therefore argue that this diameter of ~7. and (OTi3-OTi2) coordinations. There is an intensification of the OTi4 coordination in solid samples (Figure 3b and3d), which can be related to a temperature increase 29 during the polymerization process. Two other bands situated at -30 and 70 ppm and characteristic of Si-OH and Si-O-Si condensed species, were observed in MAPTMS hybrids spectra. In addition, the broad signal in the 200-300 ppm range can be assigned to Si-O-Ti bridges 30 . Complementary, liquid-state 29 Si NMR measurements of the MAPTMS-TiO2 hybrids presented in Figure 4 support this conclusion. Several studies have been realized on functionalization of TiO2 nanoparticles with MAPTMS.

Junlabhut et al. 31 have elaborated optical sensitive thin films by Ag nanoparticles doped SiO2/TiO2 hybrid deposition via sol-gel spin coating technique, Sedghi et al. 32 have synthesized hybrids by modifying the surface of the TiO2 nanoparticles with MAPTMS and Ali [START_REF] Ali | Reference Module in Materials Science and Materials Engineering[END_REF] has

Ti O Ti Si O Ti N N - - - -

/

summarized synthesis methods and characterization techniques of sol-gel hybrid coatings. The formation of organic-inorganic interface has not been clearly addressed in these studies. Our particular preparation of the nanoparticulate hybrids may explain the formation of silica shell.

The integration of the TOA nanoparticles in HEMA has been previously studied 9,19 . These HEMA-TiO2 hybrids were classified according to Sanchez [START_REF] Mabilleau | [END_REF] as belonging to Class II related by strong ion-covalent bonds between the organic and inorganic moieties.

In contrast to HEMA where ~40 % of Ti atoms receive OEMA ligands, the organic part of MAPTMS are not grafted on the TOA nanoparticles surface during the solvent exchange stage.

We can conclude that conditions are created for the inorganic nanoparticles isolation from the polymer network. On the other hand, solid-state 17 O NMR spectrum of the polymerized hybrids presented in Figure 3d proves the formation of Si-O-Ti bonds, which relative number density increases by about 10 times. Consequently, the condensation process is induced by heating. At the same time, the fact that relative number of Si-O-Si bonds does not change during polymerization signifies that new hydrolyzed Si-OH species do not appear. The origin of these Si-O-Ti bonds may be explained by the formation of silica layer at the particle surface.

An additional comparison between HEMA-TiO2 and MAPTMS-TiO2 hybrids can be made by Raman spectroscopy. This method is based on observation of intensities of the characteristic peaks of C=C at 1407 and 1641 cm -1 , associated with C=CH2 stretching and C=C aliphatic stretching vibrations of the organic monomers 19,[START_REF] Mabilleau | [END_REF] . The thermal polymerization of the hybrid samples was carried out at 90°C until the process saturation attained after 24 hours. It was observed that additional polymerization at 120 °C during 3 hours does not improve the monomerpolymer conversion rate but induces yellow coloration of samples.

The residual fraction of the C=C double bonds in HEMA-TiO2 and MAPTMS-TiO2 solid samples is shown in Figure 5 as a function of Ti concentration (CTi). We observe that an addition of the inorganic component does not affect the polymerization (almost 100 %) at relatively low concentrations CTi≤0.75 mol/l and considerably decreases the polymerization extent at higher Ti concentrations. Polymerization of the concentrated hybrid solutions does not result in complete C=C bonds opening. This has been earlier attributed to steric limitations in this nanoparticulate system 9,19 . Longer thermal polymerization (above 12 hours) and at higher temperatures (120 °C) only moderately (~5%) improves the polymerization extent. One can however see that the residual non-polymerized component is stronger in HEMA-TiO2 (50%) compared to MAPTMS-TiO2 (30%) hybrids, while in pure polymeric samples 100% double bonds conversion has been observed at the same conditions. Since TOA nanoparticles with similar size and concentration are used as the inorganic component of these hybrids, the difference was attributed to the organic-inorganic interface. The higher content of the non-polymerized monomers in HEMA compared to MAPTMS hybrids supports our conclusion about the stronger bonding between its organic and inorganic components. Indeed, the monomers grafted onto the TOA nanoparticle surface possess much lower flexibility. Moreover, two types of sites can trigger polymerization: bulk monomers and those grafted on the TOA surface. This will reduce steric compatibility of the assembled chains and polymerization extent. In contrast, MAPTMS monomers not, grafted onto the TOA nanoparticle surface because of the silica shell, will conserve sufficiently high flexibility making the polymerization process more complete and its steric hindrance, due to the presence of the inorganic component, less pronounced.

An additional remark concerns mechanical quality of the HEMA and MAPTMS monolithic samples. While HEMA-TiO2 samples were highly homogeneous and optically transparent, cracks appeared in bulk of the MAPTMS-TiO2 samples after the preparation. The bulk stress due to the silica shell interlayer between the organic (MAPTMS) and inorganic (TiO2) components seems to explain this fact.

Photonic sensitivity

The p h o t o n i c s e n s i t i v i t y of the hybrids w a s o b t a i n e d b y t h e m e t h o d explained in

Refs 10,19 . The following major parameters were determined: the photo-induced charge separation efficiency (η) and the charge storage capacity (Q). The first one represents the initial slope of the darkening curve * = +, -. /0 +, 123,4567683

, while the second one represents the ratio between the charged photoinduced and total number of Ti centers in the unitary material volume: 9 = , -. /0 , -.

. The experimental dependences of the number of trapped electrons (per unit cross section) in the cell : !; 3+ = = >!; 3+ ?(@) A 0 B@ as a function of the UV laser irradiation doze are shown in Figure 6 for HEMA-TiO2 and MAPTMS-TiO2 hybrids with the titania concentration CTi = 1.5M. They demonstrate better photosensitivity of the HEMA based hybrids compared to MAPTMS. Both the storage capacity and charge separation of the HEMA hybrids are significantly higher. Comparison of the charge separation efficiencies in both hybrids as a function of Ti concentration is shown in Figure 7. In agreement with the proposed model of photo-induced processes in TiO2 gels and hybrids 10 , our present results evidence the efficiency of photoinduced charge separation in MAPTMS hybrids ~1.3 %, which does not significantly vary with the concentration of the inorganic component, TOA nanoparticles. Although no significant changes with the TOA nanoparticle concentration were found in HEMA hybrids, the charge separation efficiency was much higher, ~25 %. This strong difference can be assigned to the electronic coupling between the organic and inorganic components.

The charge separation process in nanoparticulate hybrid materials takes place at their large organic-inorganic interface. It is therefore reasonable to attribute the observed differences in the charge separation efficiencies between HEMA-TiO2 and MAPTMS-TiO2 hybrids to the type of the interface. In contrast, we did not succeed in fabrication of stable stand-along solid hybrid structures from HEMA-TiO2 solutions. In this case, shown in Figure 8 (a) and (b), we were capable fabricating toughness structures inside the hybrid solutions, which readily decompose at the development stage due to the ductility of the sample.

Attempts to reinforce these structures were unsuccessful, because even small fluctuations in the laser fluence exploded the structures as depicted in inset in Figure 8 (b). This structural fragility of pHEMA-TiO2 hybrids can be explained by their strong electron storage effectiveness 38 . We notice that at similar fluence conditions, MAPTMS-TiO2 hybrid structures with higher mechanical properties were easily realized .

We notice that using one-photon polymerisation triggered by UV-visible lamps both HEMA-TiO2 and MAPTMS-TiO2 bulk solids were easily obtained. The obtained structures keep their photochromic properties inherent to the bulk hybrids.

Conclusion

The presented study is devoted to the comparison of structural organization and photonic sensitivities of two organic-inorganic hybrid materials based on TOA (TiO2) nanoparticles included in different polymer phases, pHEMA and pMAPTMS.

We show that the TOA particle size and aggregation state are not affected by their concentration in HEMA solutions. In contrast, the TOA nanoparticles are bigger in MAPTMS environment and their concentration increase beyond 1.0 mol/l (Ti) results in aggregation. The The monomer-to-polymer conversion in both hybrids with relatively small Ti concentration of CTi≤0.75 mol/l attains 100%. In contrast, the steric effect restricts the monomer conversion for higher Ti concentrations. The lower polymerization extent in HEMA-TiO2 hybrids (compared to MAPTMS-TiO2) is explained by lower flexibility of the grafted OEMA ligands, which serve to be a second center (after bulk monomers) for polymeric chains. On the other hand, stronger interaction between inorganic and organic components results in better mechanical stability of the HEMA-TiO2 hybrid solids.

The charge separation efficiency of 25% and long-term storage capacity of 12% were measured in HEMA-TiO2 hybrids, while in MAPTMS-TiO2 hybrids these values were considerably lower (1.3% and 4%, respectively) because of the presence of the silica shell. 3D solid structures were realized from MAPTMS-TiO2 hybrid solutions by two-photon laser polymerization. At the same time, structuring of HEMA-TiO2 hybrids was not successful and the produced structures were easily damaged during recording. This is explained by their high electron storage effectiveness leading to the material overheating and thermal explosion in course of the laser 2PP process. Moreover, because of the lower polymerization extent and insufficient cross-linking of the polymeric chains, fine HEMA-TiO2 structures are fragile for the development. Consequently, from the practical point of view HEMA-TiO2 hybrid shows better suitability for 2D/3D micropatterning related to photoinduced Ti 4+ -Ti 3+ reduction, whereas MAPTMS-TiO2 hybrids are useful for 2D/3D micromachining, related to two-photon laser polymerization.

  of the photonic sensibility of prepared hybrids were carried out in pumpprobe experiments. The pump, third-harmonics (λ=355 nm) of Nd:YAG laser (Ekspla), delivered 25-ps pulses at the repetition rate of 10 Hz. The UV-laser beam with the diameter of 5 mm interacts with the sample at normal incidence, and both incident and transmitted beams are analysed by two µJ-sensitive calorimeters C1 and C2 (Coherent). The UV-laser fluence <10 mJ/cm 2 was used to avoid any irreversible material damage. The probe cw-laser fiber coupled diode beam at 640 nm wavelength (Thorlabs) monitored the photoinduced absorption of the hybrid samples in the central part (D=1 mm) of the pump UV-laser interaction zone. The knowledge of the absorption cross-section at 640 nm σ=1.3•10 -18 cm 2 26 enables direct quantitative measurements of the concentration of trapped electrons. The data acquisition is controlled using LabView software.

respectively 1 .

 1 45 / 0.0059 Pa•s and 1.43 / 0.00260 Pa•s. The ACF curves of freshly prepared HEMA-TiO2 and MAPTMS-TiO2 solutions are shown in Figure 1 (a) and (b). The decay of curve (b) is clearly longer compared to (a), which evidences larger TOA nanoparticles in the MAPTMS solution. The fit of the experimental curves following monoexponential decay with Eq. (1) results in the mean particles radii 2.5 and 3.8 nm in HEMA and MAPTMS solutions, respectively, after the exchange stage. The larger size of the TOA nanoparticles in MAPTMS solution may be a trace of their agglomeration. Since the agglomeration state is a sensitive function of the nanoparticle number density in the solution, their size is expected to increase with the titania precursor concentration (CTi).

Figure 1 .

 1 Figure 1. Characteristic ACF of oxo-TiO2 nanoparticles after solvent exchange in the x10

Figure 2 .

 2 Figure 2. Initial radius R0 of oxo-TiO2 nanoparticles after 2-propanol solvent exchange with

  Figure 3 (a) and (c) for liquid solutions and (b) and (d) for polymerized solids.

Figure 3 .

 3 Figure 3. 17 O-NMR spectra of liquid HEMA-TiO2 (a), solid pHEMA-TiO2 (b), liquid

Figure 4 .

 4 Figure 4. 29 Si NMR spectrum of MAPTMS-TiO2 hybrids solutions, which confirms the

Figure 5 .

 5 Figure 5. Residual monomer ratio after thermal polymerisation of HEMA-TiO2 (a) and

Figure 6 .

 6 Figure 6. Photo-darkening kinetics of HEMA-TiO2 (a) and MAPTMS-TiO2 (b) hybrids X10

Figure 7 .

 7 Figure 7. Charge separation efficiency in HEMA-TiO2 and MAPTMS-TiO2 hybrids (excitation

Figure 8 .

 8 Figure 8. 2-photon laser structuring of HEMA-TiO2 (a,b) and MAPTMS-TiO2 (c,d) hybrids

17 O

 17 NMR measurements indicate the preferential formation of Si-O-Si bonds compared to Ti-O-Si bonds in MAPTMS solutions. This is explained by the formation of a silica shell around the TOA nanoparticles prohibiting direct organic monomer grafting on the nanoparticle surface.

Table 1 :

 1 Hybrid samples compositions. The last column shows relative volume increase of

		monomer solutions after the solvent exchange stage (impregnation of TOA
		nanoparticles).
	Sample TiO2 colloid	HEMA : Ti	MAPTMS : Ti	CTi, mol/l	∆V/V
		volume, ml	molar ratio	molar ratio	(hybrids)	HEMA / MAPTMS
	x1	5	54.8:1	28.2 :1	0.15	0 / 0
	x2	10	27.3 :1	13.9 :1	0.30	0 / 0
	x5	25	11 :1	5.65 :1	0.75	0 / 0
	x10	50	5.5 :1	2.82 :1	1.50	0 / 0.04
	x15	75	3.7 :1	1.88 :1	2.25	0 / 0.08
	x20	100	2.7 :1	1.41 :1	3.00	0 / 0.11
	2.2 Material characterization

Acknowledgments

. This work has been supported by I-20110047 EC project within EU contract ELISA-226716. The authors acknowledge the financial support of C'Nano IdF and IFR Paris-Nord Plaine of France. This scientific work has also been supported by the Grant RSF No. 16-13-10528 (for investigations of nanoparticle -organic interactions).

additionally isolates nanoparticle prohibiting the interface charge transfer in pMAPTMS-TiO2 hybrids.

Several authors showed the detrimental effect of the silica shell. Guerrero-Martinez et al 35 showed the insulationg nature of silica by the influence of silica layer in the response of core shell under light irradiation for catalysis application. El Toni et al 36 showed also the decrease of the photocatalytic activity of TiO2 by the presence of a layer of silica. Moreover, Viravathana et al 37 showed the impact of a layer of silica on titania with the objective of changing the refractive index. Our present results are in agreement with these observations.

Laser two-photon structuring

In order to test the ability of the synthesized hybrids for 2D/3D structuring, different microstructures have been fabricated using two-photon laser polymerization technique. This method consists in triggering radical organic polymerization in a small volume defined by the beam waist in the focal point. Two-photon polymerization was induced in liquid hybrid solutions, followed by the chemical developments in order to remove residual non-polymerized organic monomers.

Non-colored transparent structures of different shapes were obtained by this way from MAPTMS-TiO2 hybrid solutions, see SEM images in Figure 8 (c) and(d).

Big box-like structures were produced using a laser beam loosely focused by 120 mm lens inside a thick flat layer of the hybrid solution. This kind of structures was used in order to check photochromic properties of the polymerized hybrids. In order to verify hybrids abilities for twophoton polymerization with micro-and nano-scale resolutions, woodpile-like structures have been fabricated. In this case, the laser beam was tightly focused using a 100× immersion oil microscope objective (Zeiss, Plan Apochromat, NA=1.4). These results demonstrate good