Changping Zheng

Huanhuan Qu
email: quhuanhuan@simm.ac.cn

Wenfeng Liao

Teodora Bavaro

Marco Terreni

Matthieu Sollogoub

Kan Ding
email: dingkan@simm.ac.cn

Yongmin Zhang
email: yongmin.zhang@upmc.fr

Chemoenzymatically synthesized GM3 analogues as potential therapeutic agents to recover nervous functionality after injury by inducing neurite outgrowth

Keywords: ganglioside GM3, chemoenzymatic synthesis, PC12 cells, neurite outgrowth

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Introduction

Glycosphingolipids (GSLs) are ubiquitous components on mammalian cell membranes, and a variety of studies demonstrated that they are involved in many cellular functions, including cell proliferation, adhesion, motility and differentiation [START_REF] Hakomori | Glycosphingolipids in cellular interaction, differentiation, and oncogenesis[END_REF][START_REF] Yamakawa | A reflection on the early history of glycosphingolipids[END_REF][START_REF] Roseman | Refections on glycobiology[END_REF]. The changes of GSLs structures associated with biological functions have been a matter of research recently because structural change is the starting point for understanding biological significance, and enzymatic or genetic mechanisms [START_REF] Jennings | Induction of meningococcal group B polysaccharide-specific IgG antibodies in mice by using an N-propionylated B polysaccharide-tetanus toxoid conjugate vaccine[END_REF].

Ganglioside GM3, the first and simplest member in the metabolic series of a GSLs family, contains a single terminal sialic acid, lactose and ceramide (Figure 1). It has been reported that ganglioside GM3 is implicated in some human serious diseases, such as cancers and diabetes [START_REF] Inokuchi | GM3 and diabetes[END_REF][START_REF] Hakomori | GM3 and cancer[END_REF]. Sialic acids are nine-carbon monosaccharides also involved in a wide range of biological phenomena. Being at the terminus of natural cell-surface glycoconjugates, sialic acids are ideally positioned to mediate carbohydrate-protein interactions in cell-cell recognition. Sialic acids also play important masking roles to prevent biological recognition. In addition, sialic acids act as receptors for some toxins, bacteria, and viruses [START_REF] Varki | Biological roles of oligosaccharides: all of the theories are correct[END_REF]. In spite of extensive efforts and notable progress, the chemical synthesis of sialosides in high yield with complete stereoselectivity remains a significant challenge [START_REF] Von Itzstein | The synthesis of novel sialic acids as biological probes[END_REF][START_REF] Boons | Recent advances in O-sialylation[END_REF][START_REF] Ress | Sialic acid donors: chemical synthesis and glycosylation[END_REF]. The presence of a destabilizing electron-withdrawing carboxylic group along with a tertiary anomeric center and the lack of a participating auxiliary often drive glycosylation reactions toward competitive elimination reactions resulting in poor stereoselectivity (β-anomer) and in the formation of a 2,3-dehydro derivative [START_REF] De Meo | Synthesis of glycosides of sialic acid[END_REF].

Neurite outgrowth is a key process in the development of functional neuronal circuits and regeneration of the nervous system after injury [START_REF] Higgins | Inducing neurite outgrowth by mechanical cell stretch[END_REF]. The addition of neurotrophic factors such as nerve growth factor (NGF) to rat PC12 pheochromocytoma cells leads to cell cycle arrest and differentiate into neuron-like cells with elongated neurites [START_REF] Greene | Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor[END_REF]. The phenotypic changes associated with NGF-induced differentiation include the biosynthesis of neurotransmitters, the acquisition of electrical excitability and the growth of axon-like extensions named neuritis through a process known as neuritogenesis [START_REF] Jocavina | Neuritogenesis and the nerve growth factor-induced differentiation of PC-12 cells requires annexin II-mediated plasmin generation[END_REF].

In the present study, we firstly used enzymatic hydrolysis and chemical procedures to prepare GM3 analogues, and novel GM3 analogues C3 and C4 (Figure 1) were efficiently synthesized. Then along with a series of known analogues C1, C2, C5, C6 and C7 (Figure 1) [START_REF] Kawashima | Efficient synthesis of chloro-derivatives of sialosyllactosylceramide, and their enhanced inhibitory effect on epidermal growth factor receptor[END_REF][START_REF] Qu | Synthesis and cytotoxicity assay of four ganglioside GM3 analogues[END_REF], we employed the PC12 cells as a model to study the effects of these analogues on neurite outgrowth.

Results and discussion

Synthesis of GM3 analogues

For synthesizing GM3 analogues C3 and C4, three key building blocks were needed (Figure 2). Firstly, for the sialic acid block, it was activated as the sialyl xanthates form based on literature [START_REF] Martichonok | Stereoselective α-sialylation with sialyl xanthate and phenylsulfenyl triflate as a promotor[END_REF]. Then the galactose block G1 was prepared by enzymatic hydrolysis using the published method [START_REF] Filice | Chemo-biocatalytic regioselective one-pot synthesis of different deprotected monosaccharides[END_REF][START_REF] Bavaro | Chemoenzymatic synthesis of neoglycoproteins driven by the assessment of protein surface reactivity[END_REF]. Finally, the 3-O-benzoyl-azidosphingosine block was synthesized from commercial D-(+)-Galactose as previous report with some modifications [START_REF] Schmidt | Synthesis of D-erythro sphinosines[END_REF][START_REF] Gao | Recent progress in chemical syntheses of sphingosines and phytosphingosines[END_REF]. In fact, the study of complex molecules such as glycoconjugated products is some time limited by the complex procedure required for the preparation of oligosaccharides by the classical orthogonal synthetic strategy. In particular, the preparation of different sugar building blocks having only one free hydroxyl group in the position desired for glycosylation is the most time consuming task. In this study, the enzymatic hydrolysis is firstly introduced to prepare GM3 building block G1. The enzyme is specific for removing C-6 position acetyl group from all acetylated galactose. By enzymatic hydrolysis, we can avoid multiple chemical procedures.

Further the key step was α-sialylation between sialyl xanthates and free 6-OH of all acetylated galactose residue G1. In our previous studies, we tried to optimize the -sialylation reaction between acceptors and the sialic acid donors. The reaction in the presence of benzenesulfenyl chloride (PhSCl) and silver trifluoromethanesulfonate (AgOTf) as promoters in a 2:1 mixture of CH 3 CN/CH 2 Cl 2 at low temperature afforded α sialoside in significantly higher yield [START_REF] Qu | Synthesis and cytotoxicity assay of four ganglioside GM3 analogues[END_REF]. So for the sialylation of galactose residue with free 6-OH, we directly selected this Martichonok and Whitesides method. As shown in Scheme 1, the α-sialylation reaction was carried out at -68 ℃ with PhSCl and AgOTf as promoters in the mixture of CH 2 Cl 2 :CH 3 CN (1:2) for 3 h. PhSCl was freshly prepared each time according to the previous method [START_REF] Martichonok | Stereoselective α-sialylation with sialyl xanthate and phenylsulfenyl triflate as a promotor[END_REF].

At last, the sialylation product G2 was absolute α isomer in the yield of 70%. In the 1 H NMR, for H-3eq of sialic acid residue, the chemical shift was smaller for α-glycosides than for β-glycosides. Next, for the α-sialosides G2, the acetyl group at the anomeric center was selectively removed by PhCH 2 NH 2 in the dry THF at RT for 24 h to provide the hemiacetal G3 as α and β mixture in Scheme 1. This crude product G3 was not separated and could be directly used for the next step. This hemiacetal was then treated with trichloroacetonitrile in the presence of 1,8-diazabicyclo [START_REF] Inokuchi | GM3 and diabetes[END_REF][START_REF] Jennings | Induction of meningococcal group B polysaccharide-specific IgG antibodies in mice by using an N-propionylated B polysaccharide-tetanus toxoid conjugate vaccine[END_REF],0] undec-7-ene (DBU) to give the trichloroacetimidate G4 in 70% yield for two steps in Scheme 1. The 1 H NMR spectrum showed that G4 is the α-trichloroacetimidate on the basis of the H-1' and H-2' coupling constant (J 1',2' = 3.7 Hz) of the galactose residue.

Conjugation of trichloroacetimidate G4 with 3-O-benzoyl-azidosphingosine was performed using BF 3 •Et 2 O as a promoter to provide the desired glycolipid G5 in 65% yield, as shown in Scheme 2. The β configuration of the newly introduced glycosidic linkage was confirmed from the 1 H NMR spectrum (J 1',2' = 8.0 Hz). We adopted the 3-O-benzoyl-azidosphingosine block to conjugate with sugar block, not directly using ceramide block. Then all the acetyl groups of compound G5 were removed by NaOMe/MeOH. After adding several drops of water, compound G6 was obtained.

Then the azide in the G6 was reduced with propanedithiol/triethylamine to afford the new analogue C3 in 85% yield. On the other hand, the azide group of G5 was reduced by triphenylphosphine in a mixture of toluene and water at 50 ℃ for 12 h to give an amino free derivative G7. Again, G7 was not separated and directly reacted with stearic acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in dry CH 2 Cl 2 to give the glycosyl ceramide G8 in 60% yield for two steps from G5.

The acetyl groups of compound G8 were subsequently removed by NaOMe/MeOH.

After adding several drops of water, ganglioside C4 was obtained with the yield of 95%.

Biological evaluation of GM3 analogues

In this study, we used two models to investigate the effects of the compounds on neurite outgrowth. Firstly, we employed no NGF induced model, and in this model, NGF was used as a positive control. Generally, the cells maintained in low-serum medium were treated with NGF (25 ng/ml), C1, C2, C3, C4, C5, C6 and C7 (10 μM).

After 72 h, the morphology of the cells was photographed under an inverted microscope and the representative micrographs were exhibited in Figure 3A, and the number of neurite bearing cells was counted and shown in Figure 3B. Interestingly, neuritogenesis was augmented robustly by compound C1 comparing to the blank group (Figure 3A-c), the percentage of neurite-bearing cells reached 26%, even higher than that of the NGF group at the tested concentration (Figure 3B). The compound C3 (Figure 3A-e), C5 (Figure 3A-g) and C7 (Figure 3A-i) could significantly promote neurite outgrowth, while the percentage of neurite-bearing cells was 9%, 6% and 8%, respectively (Figure 3B). While the compound C2 (Figure 3A-d), C4 (Figure 3A-f) and C6 (Figure 3A-h) had no obvious effects on neuritogenesis, as the proportion of neurite-bearing cells was closed to the blank group (Figure 3B). Several literatures had emphasized the important role of GM3 and glycolipids in neuritogenesis [START_REF] Cannella | Comparison of epi-GM 3 with GM 3 and GM 1 stimulators of neurite outgrowth[END_REF][START_REF] Ariga | The role of globo-series glycolipids in neuronal cell differentiation[END_REF]. For example, in mouse neuroblastoma Neuro2a cells, studies found that GM3 enriched microdomains were involved in signal transduction during neurite formation [START_REF] Prinetti | Glycosphingolipid-enriched Signaling Domain in Mouse Neuroblastoma Neuro2a Cells: mechansim of ganglioside-dependent neuritogenesis[END_REF]. PC12 cell line has been widely used as a model system for studying neuronal differentiation and neurosecretion [START_REF] Greene | Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor[END_REF][START_REF] Westerink | The PC12 cell as model for neurosecretion[END_REF]. In the present study, we firstly employed the PC12 cells as a model to evaluate the effects of the GM3 analogues on neuritogenesis. Our studies demonstrated that some GM3 analogues could induce neurite outgrowth. From the preliminary studies, it seems that GM3 analogues with free amino group NH 2 at the sphingosine are more prone to induce neurite outgrowth, despite of different sugar composition or linkages.

It has been reported that nervous system injuries affect many people every year, and due to its high incidence, nerve regeneration or repair, is becoming a rapidly growing field dedicated to the discovery of new ways to recover nerve functionality after injury [START_REF] Prang | The promotion of oriented axonal regrowth in the injured spinal cord by alginate-based anisotropic capillary hydrogels[END_REF][START_REF] Stabenfeldt | Thermoreversible laminin-functionalized hydrogel for neural tissue engineering[END_REF]. Neurite outgrowth is a key process in the development of functional neuronal circuits and regeneration of the nervous system after injury. The ability of GM3 analogues to induce neurite outgrowth can provide a valuable sight for developing potential therapeutic agents to recover nervous functionality after injury.

Conclusion

Taken together, two new GM3 analogues were synthesized by enzymatic hydrolysis and chemical procedures from simple and commercially available substrates and reagents. By chemoenzymatic synthesis, we can avoid multiple procedures. Further, we firstly employed PC12 cells model to demonstrate that some GM3 analogues could induce neurite outgrowth with or without NGF induction, which provides the valuable sights for potential nervous system treatment after injury.

Experimental section

General methods

All chemicals were purchased as reagent grade and used without further purification. All reactions were carried out under N 2 atmosphere and anhydrous conditions with freshly distilled solvents, unless otherwise noted. Reactions were monitored by thin-layer chromatography (TLC) on a precoated plate of silica gel 60 F254 (Merck) and detection by staining with sulfuric acid. Solvents were evaporated under reduced pressure and below 40 ℃ (water bath). Flash column chromatography was performed on silica gel 60 (230-400 mesh, Merck). 1 H NMR and 13 C NMR spectra were recorded at 400 MHz and 100 MHz with Bruker AVANCE DRX 400 spectrometer. The chemical shifts were referenced to the solvent peak, 7.26 ppm ( 1 H) and 77.16 ppm ( 13 C) for CDCl 3 , 3.31 ppm ( 1 H) and 49.00 ppm ( 13 C) for CD 3 OD, 2.50 ppm ( 1 H) and 39.52 ppm ( 13 C) for DMSO, at 25 ℃, and coupling constants were given in Hz. High-resolution mass spectra (HRMS) were recorded with a Bruker micrOTOF spectrometer in electrospray ionization (ESI) mode, using Tuning-Mix as reference. Optical rotations were measured at 589 nm (Na line) at 20 ℃ with a Perkine Elmer Model 343 digital polarimeter, using a 10 cm, 1 mL cell.

O -(Methyl 5 -acetamido -4,7,8,9 -tetra -O -acetyl -3,5 -dideoxy -D -glycero -α -D -galacto -2 -nonulopyranosylonate) -(2 → 6) -1,2,3,4 -tetra -O -acetyl -α -Dgalactopyranoside (G2)

A mixture of sialyl xanthates (1.03g, 1.73 mmol) and compound G1 (400 mg, 1.15 mmol) with 4Å powdered molecular sieves (2.2 g) were dissolved in the dry CH 3 CN (24 mL) and CH 2 Cl 2 (12 mL) stirring at RT for 1 h. Further, AgOTf (488 mg, 1.90 mmol) and DTBP (0.45 mL, 2.00 mmol) were added, and the mixture was cooled to -68 o C and kept protected from light. Then PhSCl (0.23 mL, 2.00 mmol) in dry CH 2 Cl 2 (1.2 mL) was added by running the solution down the cold wall of the reaction flask. Finally, the mixture was stirred for 3 h at -68 o C. After that, the mixture was diluted with a suspension of silica gel (6 g) in EtOAc (36 mL), filtered through celite, washed with saturated aqueous NaHCO 3 and water, dried with MgSO 4 , and concentrated under reduced pressure. The residue was chromatographed (Cy-EtOAc 1:3) to give the compound G2 as a white foam (659.4 mg, 70%). R f = 0.29 (Cy-EtOAc 1:3, twice). [α] D 20 = -11.9 (c 1.0 in CHCl 3 ). 1

O -(Methyl 5 -acetamido -4,7,8,9 -tetra -O -acetyl -3,5 -dideoxy -D -glycero α -D -galacto -2 -nonulopyranosylonate) -(2 → 6) -2,3,4 -tri -O -acetyl -α -Dgalactopyranosyl trichloroacetimidate (G4)

The solution of compound G2 (610 mg, 0.74 mmol) in 3 mL of dry THF was added PhCH 2 NH 2 (122 µL, 1.12 mmol). The mixture was stirred at RT for 24 h. The

O-(Methyl 5 -acetamido -4,7,8,9 -tetra -O -acetyl -3,5 -dideoxy -D -glyceroα -D -galacto -2 -nonulopyranosylonate) -(2 → 6) -(2,3,4 -tri -O -acetyl -β -Dgalactopyranosyl) -(1 → 1) -(2S, 3R, 4E) -2 -azido -3 -O -benzoyl -4 -octadecene -l,3 -diol (G5)

The compound G4 (114.6 mg, 0.124 mmol) and

O -(5 -Acetamido -3,5 -dideoxy -D -glycero

-α -D -galacto -2 - nonulopyranosylonic acid) -(2 → 6) -(β -D -galactopyranosyl) -(1 → 1) -(2S, 3R, 4E) -2 -amino -4 -octadecene -l,3 -diol (C3)
The compound G5 (40.0 mg, 0.034 mmol) in 6.7 mL of NaOMe/MeOH (0.04 M) was stirred at RT for 14 h. Then a few drops of water were added at 0 o C. After stirring at RT for 2 h, the mixture was neutralized by Amberlite IR 120/H + ion exchange resin. After filtration and concentration, the residue was dried in vacuo to afford crude intermediate G6, R f = 0.39 (EtOAc-iPrOH-H 2 O 4:2:1). The crude intermediate G6 in the anhydrous MeOH (1.8 mL) was added propane-1,3-dithiol (0.18 mL) and triethylamine (0.18 mL), and the mixture was stirred at RT for 4 days.

A white precipitate was formed. After filtration and washing with MeOH, the filtrate was concentrated under reduced pressure. The residue obtained was flash-chromatographed, eluting with CHCl 3 -MeOH 3:1 to yield C3 (21.5 mg, 85% for two steps from G5) as a white amorphous solid. R f = 0.33 (EtOAc-iPrOH-H 2 O 4:2:1).

[α] D 20 = -2.1 (c 1.0 in CHCl 3 -MeOH 1:1).

Neurite outgrowth assay

The experiments were conducted as the previous study [START_REF] Li | GLCE regulates PC12 cell neuritogenesis induced by nerve growth factor through activating SMAD/ID3 signalling[END_REF]. Briefly, PC12 cells were seeded into a 12-well plate pre-coated with 50 ng/ml poly-L-lysine (Sigma-Aldrich, USA) at a density of 2 × 10 4 cells/well. The cells were allowed to attach for 24 h, then treated with DMEM containing 1% horse serum and 0.5% FBS for 24 h. In the non-NGF-induced experiments, PC12 cells were treated by the indicated compounds at the concentration of 10 μM or treated by NGF (25 ng/ml) alone, subsequently cultured for another 72 h. In the NGF-induced assay, NGF (25 ng/ml) was added into the culture medium accompanied with the compounds, then maintained for further 72 h. PC12 cell morphology was observed by the inverted microscope (Olympus, Japan) and determined by counting the proportion of cells containing at least one neurite twice as long as the cell body diameter, at least over 200 cells per sample were scored (Image J software, NIH, USA).

Figure 1 .

 1 Figure 1. Chemical structure of ganglioside GM3 and its seven analogues, including compound

Figure 2 .

 2 Figure 2. Three key building blocks for synthesizing analogues C3 and C4: sialyl xanthate block, galactose residue G1 block and 3-O-benzoyl-azidosphingosine block.

Figure 3 .

 3 Figure 3. Compound C1, C3, C5 and C7 could induce the neuritogenesis of PC12 cells. (A): PC12 cells were seeded in a 12-well plate, incubated overnight and then were maintained in a low-serum medium (DMEM supplemented with 1.0% horse serum and 0.5% FBS) for 24 h, followed by the treatment with the compound C1 (c), C2 (d), C3 (e), C4 (f), C5 (g), C6 (h), C7 (i) at the concentration of 10 μM or 25 ng/ml NGF (j) or without (a) for an additional 72 h. The cells were photographed by a phase contrast microscope, and the representative micrographs of each group were exhibited. (B): Quantification of PC12 cells bearing one neurite at least twice the length of the cell body diameter in each group were shown. Results are presented as means ± S.D., scale bar, 5 μm, and experiments were repeated at least three times.***, p<0.001; *, p<0.05.

Figure 4 .

 4 Figure 4. Compound C1 and C5 could promote NGF-induced PC12 cells neuritogenesis. (A): The representative micrographys of neuritigenesis of PC12 cells in each group were presented. In this assay, PC12 cells were plated in a 12-well plate, 12 h later, the culture medium was changed by the low-serum medium and the cells were maintained for further 24 h, followed by the treatment of the compound C1 (b), C2 (c), C3 (d), C4 (e), C5 (f), C6 (g) and C7 (h) accompanied by NGF for an additional 72 h. The morphology of the cells were photographed by the inverted microscope. (B): The proportion of PC12 cells bearing one neurite at least twice the length of the cell body

  solvent was evaporated and the residue was taken into CH 2 Cl 2 and washed progressively with 1.0 M HCl, saturated aqueous NaHCO 3 and water. The organic layer was dried over MgSO 4 and concentrated to give the crude intermediate G3, R f = 0.43 (EtOAc). The crude intermediate was directly engaged in the following step without further purification. Then the intermediate G3 was dissolved in the dry CH 2 Cl 2 (19.6 mL), and 2 mL of trichloroacetonitrile was added to the solution. After that, 192 µL of DBU was added dropwise at -5 o C, and the mixture was stirred at -5 o C for 5 h. After concentration, the residue was purified by flash column chromatography (Cy-EtOAc 1:4) to yield compound G4 as pale yellow foam (514.1 mg, 70% for two steps from G2). R f = 0.48 (EtOAc). [α] D 20 = +7.8 (c 1.0 in CHCl 3 ).

4. 6 .

 6 O -(Methyl 5 -acetamido -4,7,8,9 -tetra -O -acetyl -3,5 -dideoxy -D -glyceroα -D -galacto -2 -nonulopyranosylonate) -(2 → 6) -(2,3,4 -tri -O -acetyl -β -Dgalactopyranosyl) -(1 → 1) -(2S, 3R, 4E) -2 -octadecanamido -3 -O -benzoyl -4octadecene -l,3 -diol (G8)To a solution of compound G5 (40 mg, 0.034 mmol) in 3.9 mL of toluene and 0.16 mL of water was added 22.0 mg of triphenylphosphine. The mixture was stirred at 50 o C for 12 h. After concentration, the crude residue G7, R f = 0.26 (EtOAc), was directly used for the next step. The intermediate G7, stearic acid (32.9 mg, 0.12 mmol), EDC•HCl (34.2 mg, 0.18 mmol) in 5.4 mL of CH 2 Cl 2 were stirred at RT for 24 h. Then the mixture was washed with water, dried over MgSO 4 and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (Cy-EtOAc 1:3) to afford compound G8 (37.2 mg, 60% for two steps from G5) as an amorphous solid. R f = 0.33 (Cy-EtOAc 1:3). [α] D 20 = -17.5 (c 1.0 in CHCl 3 ).

4. 7 .

 7 O -(5 -Acetamido -3,5 -dideoxy -D -glycero -α -D -galacto -2nonulopyranosylonic acid) -(2 → 6) -(β -D -galactopyranosyl) -(1 → 1) -(2S, 3R, 4E) -2 -octadecanamido -4 -octadecene -l,3 -diol (C4)A solution of compound G8 (30 mg, 0.021 mmol) in 4.1 mL of NaOMe/MeOH (0.04 M) was stirred at RT for 14 h. A few drops of water were added at 0 o C. Then the mixture was stirred at RT for 2 h, and further neutralized by Amberlite IR 120/H + ion exchange resin. After filtration and concentration, the residue obtained was flash-chromatographed, eluting with CHCl 3 -MeOH 3:1 to yield C4 (20.2 mg, 95%) as a white amorphous solid. R f = 0.36 (EtOAc-iPrOH-H 2 O 4:2:1). [α] D 20 = +6.1 (c 1.0 in CHCl 3 -MeOH 1:1). 1 H NMR (400 MHz, CD 3 OD): δ 5.74-5.67 (m, 1H, H-5''), 5.49-5.43 (m, 1H, H-4''), 4.41 (d, J=9.8 Hz, 1H, H-1'), 4.09-4.00 (m, 2H, Ha-1'', H-3''), 3.99-3.95 (m, 1H, H-3'), 3.87-3.80 (m, 5H, H-4, H-5, H-4', Ha-6', Hb-1''), 3.70-3.51 (m, 9H, H-

  H NMR (400 MHz, CDCl 3 ):

	δ 6.29 (d, J=3.7 Hz, 1H, H-1'), 5.42 (t, J=9.8 Hz, 1H, H-3'), 5.32-5.27 (m, 3H, H-7,
	H-8, NH), 5.19 (d, J=9.3 Hz, 1H, H-5'), 5.06 (dd, J=10.2, 3.6 Hz, 1H, H-2'),
	4.88-4.81 (m, 1H, H-4), 4.25 (dd, J=11.3, 3.2 Hz, 1H, Ha-9), 4.07-4.00 (m, 4H, H-4',
	H-5, Ha-6', H-6), 3.93 (dd, J=11.2, 3.3 Hz, 1H, Hb-9), 3.78 (s, 3H, COOCH 3 ), 3.36
	(dd, J=11.4, 1.9 Hz, 1H, Hb-6'), 2.61 (dd, J=13.4, 4.7 Hz, 1H, H-3eq), 2.15 (s, 3H,
	OAc), 2.14 (s, 6H, 2 × OAc), 2.07 (s, 3H, OAc), 2.02 (s, 9H, 3 × OAc), 1.98 (s, 3H,
	OAc), 1.92 (t, 1H, J=9.8 Hz, H-3ax), 1.86 (s, 3H, NAc). 13 C NMR (100 MHz, CDCl 3 ):
	δ 173.49 (C=O), 172.40 (C=O), 171.89 (C=O), 171.80 (C=O), 171.67 (C=O), 171.61
	(C=O), 171.52 (C=O), 171.48 (C=O), 170.96 (C=O), 169.18 (C=O), 100.09 (C-2),
	90.82 (C-1'), 73.46, 71.06, 70.56, 69.32, 69.18, 68.95, 68.54, 68.15 (C-6, C-4', C-3',
	C-2', C-4, C-8, C-5', C-7), 63.93 (C-9), 63.53 (C-6'), 53.51 (COOCH 3 ), 50.01 (C-5),
	38.91 (C-3), 22.73 (CH

3 , NAc), 21.22 (CH 3 , OAc), 20.91 (CH 3 , OAc), 20.80 (CH 3 , OAc), 20.73 (CH 3 , OAc), 20.70 (CH 3 , OAc), 20.68 (CH 3 , OAc), 20.61 (CH 3 , OAc), 20.43 (CH 3 , OAc). ESI-HRMS (m/z) calcd for C 34 H 47 NO 22 Na [M+Na] + : 844.2487, found: 844.2469.

  OAc), 20.73 (CH 3 , OAc), 20.70 (CH 3 , OAc), 20.68 (CH 3 , OAc), 20.61 (CH 3 , OAc), 20.43 (CH 3 , OAc). ESI-HRMS (m/z) calcd for C 35 H 48 N 2 Cl 3 O 21 Na [M+Na]

	1 H NMR (400 MHz, CDCl 3 ): δ 8.74 (s, 1H, NH), 6.40 (d, J=3.7 Hz, 1H, H-1'), 5.42 (t,
	J=9.8 Hz, 1H, H-3'), 5.32-5.27 (m, 3H, H-7, H-8, H-5'), 5.06 (dd, J=10.2, 3.6 Hz, 1H,
	H-2'), 4.88-4.81 (m, 1H, H-4), 4.25 (dd, J=11.3, 3.2 Hz, 1H, Ha-9), 4.07-4.00 (m, 4H,
	H-4', H-5, Ha-6', H-6), 3.93 (dd, J=11.2, 3.3 Hz, 1H, Hb-9), 3.78 (s, 3H, COOCH 3 ),
	3.36 (dd, J=11.4, 1.9 Hz, 1H, Hb-6'), 2.61 (dd, J=13.4, 4.7 Hz, 1H, H-3eq), 2.16 (s,
	3H, OAc), 2.14 (s, 6H, 2 × OAc), 2.07 (s, 3H, OAc), 2.02 (s, 6H, 2 × OAc), 1.98 (s,
	3H, OAc), 1.92 (t, 1H, J=9.8 Hz, H-3ax), 1.86 (s, 3H, NAc). 13 C NMR (100 MHz,
	CDCl 3 ): δ 172.40 (C=O), 171.89 (C=O), 171.80 (C=O), 171.67(C=O), 171.61 (C=O),
	171.52 (C=O), 171.48 (C=O), 170.96 (C=O), 169.18 (C=O), 161.17 (C=NH), 100.09
	(C-2), 92.50 (C-1'), 90.00 (CCl 3 ) 73.46, 71.06, 70.56, 69.32, 69.18, 68.95, 68.54,
	68.15 (C-6, C-4', C-3', C-2', C-4, C-8, C-5', C-7), 63.93 (C-9), 63.53 (C-6'), 53.51
	(COOCH 3 ), 50.01 (C-5), 38.91 (C-3), 22.73 (CH 3 , NAc), 20.91 (CH 3 , OAc), 20.80
	(CH 3 ,

+ : 960.1713, found: 960.1701.

  Hz, 3H, CH 3 ).13 C NMR (100 MHz, CDCl 3 ): δ 172.40 (C=O), 171.89 (C=O),

	2.20 (m, 2H, H 2 -6''), 2.13 (s, 3H, OAc), 2.12 (d, J=2.2 Hz, 6H, 2 × OAc), 2.05 (s, 3H,
	OAc), 2.02 (s, 3H, OAc), 2.01 (s, 3H, OAc), 1.99 (s, 3H, OAc), 1.94 (t, J=11.2 Hz,
	1H, H-3ax), 1.86 (s, 3H, NAc), 1.36 (m, 2H, H 2 -7''), 1.24 (s, 20H, 10 × CH 2 ), 0.86 (t,
	J=6.8 171.80 (C=O), 171.67(C=O), 171.61 (C=O), 171.52 (C=O), 171.48 (C=O), 170.96
	(C=O), 169.18 (C=O), 166.51 (PhC=O), 138.24 (C-5''), 133.53 (C, CH aromatic),
	130.48 (C aromatic), 130.15 (2C, CH aromatic), 128.90 (2C, CH aromatic), 125.08
	(C-4''), 100.09 (C-1'), 97.53 (C-2), 75.05 (C-3''), 73.46 (C-3'), 71.06 (C-4'), 70.56
	(C-6), 69.81 (C-2'), 69.32 (C-4), 69.18 (C-5'), 68.95 (C-1''), 68.54 (C-8), 68.15 (C-7),
	64.05 (C-2''), 63.93 (C-6'), 63.53 (C-9), 53.51 (COOCH 3 ), 50.01 (C-5), 38.94 (C-3),
	32.38 (C-6''), 30.17, 30.16, 30.12, 29.82 (11 × CH 2 ), 22.73 (CH 3 , NAc), 20.91 (CH 3 ,
	OAc), 20.80 (CH 3 , OAc), 20.73 (CH 3 , OAc), 20.70 (CH 3 , OAc), 20.68 (CH 3 , OAc),
	20.61 (CH 3 , OAc), 20.44 (CH 3 , OAc), 14.57 (CH 3 ). ESI-HRMS (m/z) calcd for
	C 57 H 82 N 4 O 23 Na [M+Na]
	3-O-benzoyl-azidosphingosine
	(90.2 mg, 0.21 mmol) in 6 mL of dry CH 2 Cl 2 was stirred with 4Å powdered
	molecular sieves (500 mg) at RT for 1 h. The mixture was then cooled to -15 o C, and
	BF 3 •Et 2 O (78 µL, 0.62 mmol) was added dropwise, stirred for 3 h at -15 o C, and then
	filtered through Celite. The filtrate was washed with saturated aqueous NaHCO 3 and
	water, dried over MgSO 4 and concentrated under reduced pressure. The residue was
	applied to a flash chromatography eluted with Cy-EtOAc 1:5 to give the product G5
	(96.0 mg, 65%) as an amorphous solid. R f = 0.58 (EtOAc). [α] D	20 = -14.5 (c 1.0 in
	CHCl 3 ). 1 H NMR (400 MHz, CDCl 3 ): δ 8.02-7.99 (m, 2H, Ar-H), 7.58-7.54 (m, 1H,
	Ar-H), 7.46-7.42 (m, 2H, Ar-H), 5.85 (dd, J=14.2, 7.3 Hz, 1H, H-5''), 5.59-5.48 (m,
	2H, H-3'', H-4''), 5.36-5.23 (m, 5H, H-4, H-7, H-8, NH, H-3' ), 5.12 (d, J=9.59, 1H,
	H-2'), 4.92-4.84 (m, 1H, H-4'), 4.73 (d, J=8.0 Hz, 1H, H-1'), 4.50 (m, 1H, Hb-9),

4.27 (dd, J=12.4, 2.7 Hz, 1H, Ha-9), 4.08-3.99 (m, 3H, Ha-6', H-2'', H-5), 3.94-3.87 (m, 2H, H-5', Hb-6'), 3.79 (m, 1H, Ha-1''), 3.76 (s, 3H, COOCH 3 ), 3.65 (dd, J=10.8, 4.3 Hz, 1H, Hb-1''), 3.40 (d, J=9.1 Hz, 1H, H6), 2.59 (dd, J=12.7, 4.6 Hz, 1H, H-3eq), + : 1213.5268, found: 1213.5286.

  6, H-7, H-8, Ha-9, Hb-9, H-2', H-5', Hb-6', H2''),3.35 (s, 1H, NH), 2.71 (dd, J=12.1, 4.9 Hz, 1H, H-3eq), 2.18 (t, J=6.8 Hz, 2H, CH 2 C(O)), 2.06-2.01 (m, 2H, H 2 -6''), 2.00 (s, 3H, NAc), 1.79 (t, J=12.1 Hz, 1H, H-3ax), 1.58 (m, 2H, CH 2 CH 2 C(O)), 1.29 (s, 50H, 25 × CH 2 ), 0.90 (t, J=7.4 Hz, 6H, 2 × CH 3 ). 13 C NMR (100 MHz, CD 3 OD): δ 174.16 (C=O), 169.18 (C=O), 168.65 (C=O), 134.18 (C-5''), 128.44 (C-4''), 100.09 (C-1'), 97.53 (C-2), 75.05 (C-3''), 73.46 (C-3'), 71.06 , 30.16, 30.12, 29.82 (CH 2 ), 22.71 (CH 3 , NAc), 15.47 (2 × CH 3 ). ESI-HRMS (m/z) calcd for C 53 H 97 N 2 O 16 [M-H] + : 1017.6916, found: 1017.6934.PC12 cells were obtained from the ATCC (Manassas, VA, USA). The cells were maintained in DMEM (Dulbecco's modified Eagle's medium; HyClone, USA), supplemented with 10% (v/v) heat-inactivated horse serum (Gibco, USA) and 5% (v/v) FBS (Gibco, USA). The cells were cultured in a humidified incubator at 37 o C with 5% CO 2 atmosphere.

	(C-4'), 70.56 (C-6), 69.81 (C-2'), 69.32 (C-4), 69.18 (C-5'), 68.95 (C-1''), 68.54
	(C-8), 68.15 (C-7), 64.36 (C-2''), 63.93 (C-6'), 63.53 (C-9), 50.97 (C-5), 40.12 (C-3),
	30.174.8. Biological evaluation
	4.8.1. Cell culture

μm, and experiments were repeated at least three times.***, p<0.001; *, p<0.05.

Acknowledgments

We thank the China Scholarship Council (CSC) for a Ph.D. fellowship to Changping ZHENG. Financial supports from the Centre National de la Recherche Scientifique (CNRS) and the Université Pierre et Marie Curie (UPMC) in France, National Natural Science Foundation of China (NSFC) (21402224) and Shanghai Science and Technology Development Funds (14YF1407800) are gratefully acknowledged.

Supporting information

Supplementary data of NMR spectra associated with this article can be found in the online version.


John Susainathan

Florent Eyma

Emmanuel De Luycker

Arthur Cantarel

Bruno Castanie
email: bruno.castanie@insa-toulouse.fr

Experimental investigation of impact behavior of wood-based sandwich structures

Keywords:

Low carbon emission and sustainable development are shared goals throughout the transportation industry. One way to meet such expectations is to introduce lightweight materials based on renewable sources. Sandwich panels with plywood core and fiber reinforced composite skins appear to be good candidates. Additional properties of wood such as fire resistance or thermal and acoustic insulation are also essential for many applications and could lead to a new interest for this old material. In this paper, Sandwich panels with two different types of plywood and four different skins (aluminum and glass, CFRP, or flax reinforced polymer) are tested under low-velocity/low energy impacts and their behavior is discussed.

Introduction

Sandwich structures are lightweight composite structures that have been widely used in numerous sectors, such as the automotive, aerospace, marine and energy industries, due to their several advantages: high specific bending strength and stiffness, excellent damping, and thermal insulation [START_REF] Zenkerts | The handbook of sandwich construction[END_REF][START_REF] Bucci | Experimental investigation on Iroko wood used in shipbuilding[END_REF]. Low carbon emission and sustainable development are shared goals in the transportation industry and one way of achieving them is to implement lightweight materials based on renewable materials. Sandwich panels with plywood core and fiber reinforced composite skins appear to be good candidates, particularly as certain additional properties of wood such as fire resistance or thermal and acoustic insulation are also essential for many applications. Plywood is still used in the construction of homemade airplanes and, until the 1990s was employed in the design of acrobatic aircraft like the Mudry CAP10. It is perhaps less well known that, in the 1960s, a car designed for the "Le Mans" race by the famous English engineer Frank Costin had a plywood structure for a total mass of only 450 kg. So, a combination of plywood and other materials seems to be relevant and was first investigated statically by the authors [START_REF] Susainathan | Manufacturing and quasi-static bending behavior of wood-based sandwich structures[END_REF][START_REF] Prakash | Susainathan development and characterization of wood based ecostructure[END_REF]. Wood based sandwich structures with high specific properties, low costs and good energy dissipation capability are promising candidates for impact and crash applications in the transportation sector [START_REF] Prakash | Susainathan development and characterization of wood based ecostructure[END_REF][START_REF] Abrate | Dynamic failure of composite and sandwich structures[END_REF][START_REF] Balıkoglu | Compression after low velocity impact tests of marine sandwich composites: effect of intermediate wooden layers[END_REF][START_REF] Shin | An experimental study of low-velocity impact responses of sandwich panels for Korean low floor bus[END_REF]. The buckling of tracheid cells in wood at micro scale is similar to the structural buckling of honeycomb cell walls at macro scale and enables maximum energy dissipation [START_REF] Vural | Dynamic response and energy dissipation characteristics of balsa wood: experiment and analysis[END_REF][START_REF] Aminanda | Experimental analysis and modeling of the crushing of honeycomb cores[END_REF]. Hence, the implementation of new sandwich structures requires significant efforts to understand their behavior. In particular, sandwich structures are vulnerable to various impact loads and may be exposed to different impacts during their service life [START_REF] Prakash | Susainathan development and characterization of wood based ecostructure[END_REF]. These impacts may result in significant damage, such as local cell wall buckling or core crushing, and debonding between skin and core, so damage in the skin can intensively compromise the integrity of the structure [START_REF] Abrate | Dynamic failure of composite and sandwich structures[END_REF][START_REF] Balıkoglu | Compression after low velocity impact tests of marine sandwich composites: effect of intermediate wooden layers[END_REF][START_REF] Shin | An experimental study of low-velocity impact responses of sandwich panels for Korean low floor bus[END_REF][START_REF] Vural | Dynamic response and energy dissipation characteristics of balsa wood: experiment and analysis[END_REF][START_REF] Aminanda | Experimental analysis and modeling of the crushing of honeycomb cores[END_REF][START_REF] Castanié | Modelling of low energy/ low velocity impact on Nomex honeycomb sandwich structures with metallic skins[END_REF] and especially the compression after impact strength [START_REF] Balıkoglu | Compression after low velocity impact tests of marine sandwich composites: effect of intermediate wooden layers[END_REF][START_REF] Castanié | Core crush criteria to determine the strength of sandwich composite structures subjected to compression after impact[END_REF]. So the analysis of plywood based sandwich structures under impact is a priority.

Impact tests are generally classified as low (< 10 m/s), medium (10-50 m/s) or high velocity (50-1000 m/s) impacts [START_REF] Abrate | Impact on composite structures[END_REF]. In this paper, we will focus on low energy/low velocity impact, which corresponds to common uses of structures and may be sensitive for innovative structures. Much research has focused on low velocity impacts on conventional composite and sandwich structures [START_REF] Abrate | Dynamic failure of composite and sandwich structures[END_REF][START_REF] Castanié | Modelling of low energy/ low velocity impact on Nomex honeycomb sandwich structures with metallic skins[END_REF][START_REF] Castanié | Core crush criteria to determine the strength of sandwich composite structures subjected to compression after impact[END_REF][START_REF] Abrate | Impact on composite structures[END_REF][START_REF] Heimbs | Foldcore sandwich structures and their impact behaviour: an overview[END_REF][START_REF] Belingardi | Energy absorbing sacrificial structures made of composite materials for vehicle crash design[END_REF][START_REF] Kolopp | Experimental study of sandwich structures as armour against medium-velocity impacts[END_REF][START_REF] Ude | Impact damage characterization in reinforced woven natural silk/epoxy site face-sheet and sandwich foam and honeycomb[END_REF][START_REF] Wang | Exp. study of the medium velocity impact response of sandwich panels with different cores[END_REF][START_REF] Chai | A review of low-velocity impact on sandwich structures[END_REF][START_REF] Raju | Impact damage resistance and tolerance of honeycomb core sandwich panels[END_REF] while wood-based sandwich structures have been little investigated. Toson et al. [START_REF] Toson | Finite element modeling of Balsa wood structures under severe loadings[END_REF] pointed out that balsa wood presents significant interest as a core material in sandwich panels because of its transversely isotropic behavior, i.e., it is stiffer and stronger in the fiber direction (axial) than in the radial and tangential directions. Atas [START_REF] Atas | The impact response of sandwich composites with cores of balsa wood and PVC foam[END_REF] compared the impact responses of composite skinned sandwich structures with balsa wood -HD (high density) or PVC foam cores, and revealed that sandwich structures with balsa wood gave better results in terms of energy absorption capability and impact induced damage than sandwich structures with conventional polymeric foam cores. In similar way, Shin et al. [START_REF] Shin | An experimental study of low-velocity impact responses of sandwich panels for Korean low floor bus[END_REF] analyzed impact responses of composite skinned sandwich structures with various cores, such as HD balsa wood and aluminum honeycomb, and claimed that the energy absorption of wood based sandwich structures was comparable with that of aluminum honeycomb sandwich structure. Hachemane et al. [START_REF] Hachemane | Sandwich composites impact and indentation behaviour study[END_REF] performed an experimental characterization of a jute/epoxy-cork sandwich structure exposed to impact and indentation. Petit et al. [START_REF] Petit | Impact and compression after impact experimental study of a composite laminate with a cork thermal shield[END_REF] used cork as a thermal shield and analyzed the impact behavior of Sandwich panels and laminates. It was shown that the thermal shield significantly modified the failure patterns and created an effect of shift in damage creation. Mezeix et al. [START_REF] Mezeix | Experimental analysis of impact and post-impact behaviour of inserts in Carbon sandwich structures[END_REF] tested inserts in sandwich structures using a drop-weight device and analyzed the impact response and failure patterns. The residual strength after impact was very high in comparison to the large reductions habitually observed after impact tests. Abdalasam [START_REF] Abdalsalm | Impact damage analysis of balsa wood sandwich composites Thesis USA[END_REF] compared the low velocity impact response between end and regular grain balsa wood core sandwich with glass epoxy skin and found that a sandwich offered better energy absorption when it had a regular rather than an end grain balsa core. However, end grain balsa core can withstand higher impact loads than regular balsa core thanks to its higher stiffness. Energy absorption, impact load and failure modes are strongly dependent on the orientation of the wood core grain [START_REF] Abdalsalm | Impact damage analysis of balsa wood sandwich composites Thesis USA[END_REF]. Wang et al. [START_REF] Wang | Experimental study of the medium velocity impact response of sandwich panels with different cores[END_REF] analyzed the medium velocity impact response of sandwich structures with different cores such as cores of balsa wood, cork, polypropylene honeycomb and polystyrene foam. He claimed that, among the five panels, the sandwich panel with the HD balsa core yielded the best results in terms of specific energy absorption because of its lower density compared to the other core materials. In summary, a review of the results regarding the impact response of sandwich structures confirms that structures with plywood core have been little studied. Therefore, the Sandwich panels with plywood cores that were manufactured and tested statically in [START_REF] Susainathan | Manufacturing and quasi-static bending behavior of wood-based sandwich structures[END_REF] are analyzed under low energy impacts here. Considering the results mentioned above, the precise aim of our work was to compare the materials currently used for cargo bay floors, namely aramid honeycomb having carbon and glass composite skins, with wooden sandwich structures developed in the laboratory. A 10 mm plywood core was used in order to be able to compare the effects on the impact behavior of skins made out of aluminum alloy, and composites reinforced with glass, carbon or flax fibers. These materials were impacted at energy levels of 5 J, 10 J, and 15 J using a drop-weight impact test, and a comparison based on the force-displacement response and failure modes of the panels is presented. The damage resistance and failure modes of wood based sandwich structures under low energy impact will be described on the basis of post impact tomography analysis [START_REF] Li | Impact of internal structure on water-resistance of plywood studied using neutron radiography and X-ray tomography[END_REF][START_REF] Paris | Quantitative wood-adhesive penetration with X-ray computed tomography[END_REF][START_REF] Corigliano | Experimental and theoretical analyses of Iroko wood laminates[END_REF][START_REF] Crupi | Internal damage investigation of composites subjected to low-velocity impact[END_REF].

Materials and methods

Specimens

The manufacturing method and the specimens are described in [START_REF] Susainathan | Manufacturing and quasi-static bending behavior of wood-based sandwich structures[END_REF] and are briefly recalled here. The core materials were plywood structures, named plywood A and plywood B. Both plywood structures were made up of poplar and okoume plies bonded together using Melamine Urea Formaldehyde (MUF) glue. The stacking sequences and thicknesses of plywood A & B are shown in Fig. 1. The two cores had the same thickness (about 10 mm) in order to minimize the effects of the geometry on the bending stiffness of the sandwich, and make comparisons easier.

Skins were made of aluminum sheet (1xxx) or fiber reinforced polymer composite, containing carbon, glass or flax. The skin materials were chosen as representative of the different types of face sheets used in sandwich construction. Eight different configurations of wood based sandwich structures were manufactured according to Table 1. A reference material, Nomex honeycomb sandwiched between carbon or glass reinforced skins, which is currently used in cargo-bay floors in some AIRBUS aircraft, was also considered for qualitative comparison with the above eight configurations. Large plates 500 × 500 mm 2 were manufactured and then cut into 150 × 100 mm 2 squares for impact testing as per AIRBUS standard AITM 1-0010.

Impact testing

Impact tests were performed using a drop weight apparatus (Fig. 2) followed by tomography analysis. The principle of the falling weight is to drop an instrumented mass, guided in a tube, onto a sample plate held by a clamping window. In our test, the main components were:

• A mass of 2.08 kg. This value was set so as to achieve the desired impact energy with speeds of up to 5 m/s;

• A load sensor located under the mass, to measure the force between the impactor and the specimen during the impact;

• A hemispherical impactor 16 mm in diameter;

• An optical sensor measuring the speed of the impactor immediately before impact;

• A support window, of internal dimensions 125 × 75 mm 2 , on which the specimen was positioned (standard specimen dimensions: 100 × 150 mm 2 ). These dimensions were determined based on Airbus standards AITM 1-0010;

• A clamping window having inner dimensions identical to those of the lower window (125 × 75 mm 2 ) to hold the specimen during the impact;

• A kickback system to prevent multiple shocks on the specimen (same as in [START_REF] Mezeix | Experimental analysis of impact and post-impact behaviour of inserts in Carbon sandwich structures[END_REF]). The load measured was not a real impact load, as a mass was present between the laminate and the sensor. The real impact load was calculated according to the following expression:

= ⎡ ⎣ ⎢ - ⎤ ⎦ ⎥ -- F m m m F F ( ) ( ) real t t h f i f i ( ) ( 1)
where m t = mass of impactor, m h = mass of head, F f = measured load,

F real = real impact load F f (filtered load) = + - F F ( )/2 i i ( ) ( 1)
and F i = measured load.

The filtered load is calculated on a sliding average of 40 points for an acquisition frequency of 200 kHz. The system for measuring the velocity of the impactor used an optical sensor (Laser), the output signal from which depended on the reflection of the emitted beam, and thus on the reflective surfaces engraved on the drop weight head (see Fig. 1b). Initially, the acceleration was calculated using the following equation.

= - - a F m m g ( ) i i t h
Knowing the distance and the time recorded by the laser between two grooves, it is easy to determine the initial velocity at impact. From the initial velocity at impact and the acceleration, instantaneous velocity and displacement were calculated by numerical integration. The same numerical method was used to calculate the energy absorbed from a plot of displacement and real impact load. The results of a low velocity impact test at different impact energies, 5 J, 10 J and 15 J, corresponding to energy levels generally encountered in the industry when tools fall onto on a floor, for example, were thoroughly investigated. We then considered the orientation of the top plies of the plywood core as a reference to distinguish the longitudinal and transverse directions. The length and breadth directions were the same for all specimens. The test matrix for the impact test study is summarized in Table 2. The comprehensive results are available in ref [START_REF] Prakash | Susainathan development and characterization of wood based ecostructure[END_REF]. The tomography analysis was performed using typical values given hereafter: Voltage 131 kV current 61 µA, no beam filter, Focal spot size: 8 µm (8 W), Image resolution: 1008 * 1008, size of voxel: x = 5.01 µm and y = 5.01 µm.

Experimental results and discussion

Plywood structure

Firstly, it was observed that crack initiation always occurred in the plywood close to pre-existing damage in the plywood. These pre-existing damages are due to the peeling cutting process used to obtain wood plies from the log. This process generates pre-cracks in the radial/ longitudinal plane that may propagate in the same direction or rotate to propagate in the tangential/longitudinal plane because of transverse shear [START_REF] Denaud | Wood peeling process monitoring: a comparison of signal processing method to estimate veneer average lathe check frequency[END_REF]. A tomography analysis on damaged samples of plywood A and B is shown in Fig. 3(a) &(b). In the case of plywood structures, a permanent indentation occurred at all energy levels through deformation due to plasticity or fracture of wood ply. Maximum deformation occurred at the top ply due to crushing. Only one third of the sample was concerned at 5 J but permanent deformation occurred in up to one half of the sample for a 10 J impact test. For 15 J tests, perforation occurred and the impactor hangs in the plywood which creates the second peak. In summary, for both plywoods (A and B), we identified fiber fracture, transverse shear, crushing, permanent indentation and deformation as predominant failure modes, with propagation to one half of the plywood core with increasing energy levels.

The impact response in terms of the energy absorbed at different energy levels (5 J, 10 J and 15 J) is presented in Fig. 4 a, b and c respectively. The influence of permanent indentation on absorbed energy is illustrated in Fig. 4 d andTable 3. In this table, the relative absorbed energy column represents the ratio between the absorbed energy and the measured impact energy in percent. The specific absorbed energy is obtained from the ratio of absorbed energy to the density. It is found that the force-displacement histories of plywood structures exhibit common trends, such as linear increase of force as the impactor contacts the panel, and a plateau that indicates crushing through radial compression of cell walls. Then, due to loss of stiffness caused by fiber fracture, transverse shear or debonding, the peak force starts to decrease after attaining the peak value.

In terms of absorbed energy, the two plywood materials gave similar results with a slightly better performance for plywood A at higher energy levels due to its slightly longer plateau. Also, permanent indentation in plywood A was smaller than in plywood B due to its greater number of interfaces, which led to better transverse behavior. Both plywood structures showed comparable energy absorption capabilities and lower indentation when compared to our reference material of aramid honeycomb/carbon and glass skin. However, in terms of specific energy absorption, aramid honeycomb/carbon and glass skin yielded higher results due to its lower density. It is important to note that, at 15 J, all these structures were totally destroyed/perforated by the impactor (see Table 3). The integrity of the structure was then compromised. Hence, the indentations, damage and integrity loss of the structure at higher energy levels would be unacceptable in most applications.

Plywood structure with aluminum skin

With aluminum skins, a permanent indentation was observed for all impact energies due to the ductile property of this material and the smaller elastic spring back effect as shown in Fig. 5. Delamination between skin and core was not observed under the impactor but very slightly around it. In the case of 5 J impact, crushing, fiber fracture and transverse shear were observed in one third of the depth of the plywood core, i.e. down to the top okoume 0°ply. At 10 J, the same failure modes were observed on half the depth of the plywood core, down to the poplar 0°ply. At 15 J, the delamination and damage area increased around the impactor. The same phenomenon as above, with maximum deformation, occurred down to two thirds of the depth of the plywood core, i.e. to the middle poplar 90°ply in plywood -A, as shown in Fig. 5.

The force-displacement history of plywood with aluminum skin at different energy levels (5 J, 10 J and 15 J) is shown in Fig. 6 a, b andc and compared with our reference material of aramid honeycomb with carbon and glass skin. Globally, the shape shows that there is a little part of crushing (no plateau in the curve) for this sandwich. The mechanism of absorption is mainly plasticity of the skins. We found that plywood structures with aluminum skin had energy absorption capabilities comparable to those of our reference material (see Fig. 6 a and b and Table 4). However, in terms of specific energy absorption, aramid honeycomb/carbon and glass skin yielded higher results due to its low density. At higher energy levels, the rate of indentation increased rapidly, causing moderate indentation without perforation of the aluminum skin, due to its ductile behavior, whereas aramid honeycomb was perforated and lost its structural integrity.

Plywood structure with composite skins

Failure patterns with carbon composite skins

With carbon fiber composite as shown in Fig. 7 a and b, small permanent indentation was observed under the impactor and top carbon skin, due to crushing of the core and elastic spring back of the skin. It also caused indentation and a damage area on the ply below the top skin. The amount of damage and indentation area on the top skin were lower than in the wooden ply below the top skin. At 5 J, delamination was observed in the carbon fiber composite skins and at the interface between the core and the skin. Fiber fracture and crushing were observed in the top fifth of the plywood core. At 10 J, delamination inside the skin and between the impacted skin and the core increased. Crushing, fiber fracture and deformation were observed in a quarter of the depth of the plywood core, down to the top okoume 0°p ly. At 15 J, all the above failure modes spread into the top third of the plywood core, down to the top poplar 90°ply in plywood -B (see Fig. 7b).

Failure pattern with glass composite skins

With glass fiber reinforced composite skins as shown in Fig. 8 a and b, the phenomena were quite different, with no delamination between the top skin and the core, thanks to their perfect adhesion. The delamination thus occurred at the ply below the top skin. Elastic spring back of the composite skin also caused damage at the poplar 90°ply and fiber fracture at the top okoume 0°ply below the skin, which led to higher deformation at the third ply from the top (poplar 90°) than at the second one. At 5 J, slight indentation and crushing occurred under the impactor, delamination and fiber fracture were observed in the glass fiber skin, and fiber fracture was observed in one tenth of the plywood core, i.e. in a quarter of the cross section. In the case of 10 J, crushing occurred in a one quarter of the plywood core, delamination was observed between the skins and the core, but fiber and transverse shear fracture occurred in one quarter of the plywood core, down to the top okoume 90°ply. At 15 J, fiber fracture occurred in the composite skin, and transverse shear, crushing and maximum deformation occurred in one third of the plywood core, down to the top poplar 0°ply in plywood A (Fig. 8a &b).

Failure pattern with flax composite skins

Considering flax fiber reinforced composite skins, the damage modes were slightly different, as shown in Fig. 9. At 5 J, delamination between the skin and the core, and moderate indentation occurred under the impactor due to elastic spring back of the composite skin. Fiber fracture was observed on the one tenth of the plywood core, down to the top okoume 0°ply, while crushing appeared on one third of the plywood core, down to the top poplar 90°ply. In the case of 10 J, delamination increased under the impactor, crushing was observed on 40% of the plywood core (down to the top poplar 90°ply) and fiber fracture appeared in the flax composite top skin. At 15 J, fiber fracture and indentation was found in one half of the depth of the plywood core, down to the middle okoume 0°ply in plywood B. Delamination increased and fiber fracture occurred in the flax skin.

Energy absorbed and indentation of plywood with composite skins

Regarding energy absorption and indentation, the impact responses of plywood with composite skin at 5 J, 10 J and 15 J in terms of force vs displacement and ratio of absorbed energy to indentation are shown in Fig. 10 and also in Table 5. In the case of carbon skins, the higher stiffness of the skins and the lack of internal damages resulted in small indentation and the smallest absorbed energy as shown with shap shapes of the curves. This composite also resisted the highest impact load but with more delamination in the skin and extreme crushing of the plywood core due to its elastic spring back and the poor adhesion attained in the case of vacuum molding, where air can be trapped during vacuum creation. Regarding the glass fiber skins, absorbed energy was comparable to that for flax, with also a smaller indentation than for carbon because of the higher strength of the skins and perfect adhesion obtained with the samples manufactured by thermo-compression as compared to plywood with carbon skin manufactured by both vacuum molding and thermo-compression. Despite a lower indentation than for the reference material, the ratio of absorbed energy to indentation (Fig. 11) was not much higher for the flax composite skin structure.

In summary, the plywood structure with glass fiber skins appears to provide a good compromise between absorbed energy (close to the flax skin material) and permanent indentation (see Table 5). Regarding the influence of the process, very little effect was observed on the absorbed energy but a smaller permanent indentation appeared in the thermopressed case than in the vacuum molding ones. This was certainly due to better adhesion of the skins with this process.

Summary

The impact response of the eight different wood based sandwich structures and our reference materials are presented in Fig. 12 in terms of a force-displacement plot for the 15 J case only. In general, the initial slope of force-displacement varies with the skin materials, thus indicating variation in stiffness of the different wood based sandwich structures. As expected, the skin properties influence the impact behavior of these sandwich structures even at lower displacement values. Concerning plywood structures, plywood A is found to yield slightly better results than plywood B in terms of absorbed energy and indentation, due to its longer plateau. Its higher number of interfaces causes better transverse behavior, which leads to smaller permanent indentation than in plywood B. Both plywood structures have comparable energy absorption capabilities and lower indentation in comparison to the reference material (see Tables 345). In terms of specific energy absorption, despite the good results of the materials under test, the reference material yields the highest results due to its low density. However, at 15 J, all these materials undergo perforation with heavy loss of structural integrity, which are incompatible with most applications. The plywood also gives good results regarding specific energy absorption because of its low density.

For plywood structures with aluminum skins, compared to plywood structures alone, there is an absence of plateau and peak force oscillation occurs due to the high strength and stiffness of the skin. This structure has comparable energy absorption and better resistance to indentation than the reference material but is not as good in terms of specific energy absorption, because of the high density of aluminum skin. Moreover, this structure results in a deeper indentation than in any of the other structures with composite skin, which can be undesirable in some applications.

The ratios of absorbed energy to indentation are plotted in Figs. 4(d absorptions at three energy levels for different sandwich structures are shown in Fig. 13. Two different processes, thermo-compression and vacuum molding with prepreg, were used to manufacture our panels of plywood structure with carbon/epoxy and glass/epoxy composite skins. In general, we found that the plywood with glass skin fabricated by the thermo-compression process had minimum defects, and better adhesion and structural integrity than that obtained by vacuum molding, as a result of the high operating temperature, the absence of trapped air, and the pressure used in the thermo-compression process.

In terms of energy absorption, carbon fiber reinforced composite shows somewhat weak results but with small indentation due to the greater stiffness of the skins. It also resists the highest impact load of all the plywood structures. However, it suffers from higher delamination in the skin and extreme crushing of the plywood core due to its elastic spring back effect and poor adhesion between skin and core, which results from the insufficient pressure used in the case of vacuum molding or the presence of trapped air in the case of thermo-compression.

With glass fiber reinforced composite skins, the behavior is quite different. The perfect adhesion and the spring back effect of the skin prevents delamination but decohesion occurs in the first ply of the plywood core. This results in an absorbed energy comparable to that observed with flax fibers, and smaller indentation than with carbon fibers because of the higher strength of the skins. Whatever the impact energy, this material is the best compromise between absorbed energy and indentation.

When flax fiber reinforcement is used in the skins, the composite behaves similarly to plywood with aluminum skins in terms of absorbed energy and specific energy absorption but shows smaller indentation as the plastic deformation is less than for aluminum. There is minimum delamination and debonding between skin and core due to the moderate elastic spring back effect and the better adhesion obtained through the thermo-compression process as compared to plywood with carbon skin fabricated by either the vacuum molding or the thermocompression process.

Finally, regarding specific energy absorption (see Fig. 13), the reference material, aramid honeycomb with carbon and glass composite skins, yields the highest results because of its low density but undergoes perforation and loses its structural integrity for high energy impacts (15 J). In comparison, in the case of plywood with aluminum skin, crushing is transformed into indentation without perforation. Regarding newly developed wood based sandwich structures; a very interesting compromise can be obtained for flax skin structures thanks to their low weight and their high energy absorption.

Conclusions

In the nine materials tested, both the plywood structures demonstrated comparable energy absorption capabilities and lower indentation in comparison to the reference material. However, plywood A yielded slightly better results than plywood B in terms of absorbed energy and indentation due to its longer plateau and its larger number of interfaces. Regarding plywood structures with skin, the plywood structure with glass fiber skin fabricated by the thermo-compression process seems to present a good compromise between absorbed energy (close to that of the material with flax reinforced skins) and permanent indentation (close to that of the structure with carbon reinforced skins). This results from its higher strength and the better adhesion provided by the thermo-compression process (noteworthy whatever the material considered). It also exhibits better results in terms of bonding or adhesion with the core as compared to plywood with carbon skin manufactured by vacuum molding with application of high pressure. Following the plywood with glass skin, the plywood with flax skin, which is bio-based, offers a good compromise between energy absorption and specific energy absorption due to its lower density. In conclusion, the development of these structures with plywood cores seems to be a good solution regarding impact concerns. These materials, which are more resistant, more functional and more environmentally friendly, could replace the ones currently used for cargo-bay floors in the aeronautics industry, for example. The only limitation remains the weight of these structures which is 2.5 times that of currently used materials. Nevertheless, for a floor intended for assembly on a final assembly line (i.e. outside a flying structure), this combination of properties associated with a 20 times lower cost seems to be a promising solution for certain potential applications. Nevertheless, a pending question remains the recyclability of these materials. Prospects are also being considered for the development of new materials and their use in crash box applications in the automotive industry.

Fig. 1 .

 1 Fig. 1. Plywood A and B stacking. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2 .

 2 Fig. 2. (a) Drop-weight impact test set-up and aluminum wood specimen installed, (b) Impactor head. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3 .

 3 Fig. 3. Failure modes of impacted plywood core at 10 J. (a) Plywood A, (b) Plywood B. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4 .

 4 Fig. 4. Force-displacement plot for plywood structure (a) 5 J, (b) 10 J, (c) 15 J, and (d) Ratio of Absorbed energy to indentation (J/mm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5 .Fig. 6 .

 56 Fig. 5. Tomography images of impacted plywood with aluminum skin -Failure modes at 15 J. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7 .Fig. 8 .Fig. 9 .Fig. 10 .

 78910 Fig. 7. Tomography images of impacted plywood with Carbon skins -Failure modes at 15 J, (a) plywood A, (b) plywood B. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

  ), 6(d) and 11 and are noted in Tables3-5. The specific energy

Fig. 12 .Fig. 11 .

 1211 Fig. 12. Force-displacement plot for the different wood based sandwich structures at 15 J. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 13 .

 13 Fig. 13. Specific energy absorption for the 8 configurations tested plus the reference one. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1

 1 Specimens manufactured.

	Core	Skin	Process	Density	Thickness (mm)	Process specification
	Plywood A	-	-	0.461	10	-
	Plywood B	-	-	0.433	10	-
	Plywood A	Aluminum	-	0.678	11	-
	Plywood A	Glass	Vacuum bag molding -Prepreg	0.638	12	At 160 °C for 3 h
		Carbon		0.569		At 90 °C for 30 min then at 125 °C for 1 h
	Plywood B	Flax	Thermo-compression -Prepreg	0.488	12	At 120 °C with pressure of 4 bar for 1 h
		Carbon		0.614		At 90 °C for 30 min then at 120 °C for 1 h, all with pressure of 4 bar
		Glass		0.609		At 160 °C with pressure of 4 bar for 3 h
	Aramid honeycomb	Carbon & Glass	-	0.233	10	-

Table 2

 2 Test matrix of wood based sandwich structures for drop-weight impact test.

	Materials		Impact			No. of samples	Thickness of skin (mm)	Area density (kg/m 2 )	Density (kg/m 3 )
			5 J	10 J	15 J
	Plywood -A		1	1	1	3	-	4.6	461
	Plywood -B		3	3	3	9	-	4.3	433
	Plywood -A/Aluminum		3	3	3	9	0.5	7.5	678
	Vacuum Molding	Plywood -A/Glass	1	1	1	3	1	7.7	638
		Plywood -A/Carbon	1	1	1	3	0.78	6.6	569
	Thermo-compression	Plywood -B/Flax	1	1	1	3	0.78	5.6	488
		Plywood -B/Carbon	1	1	1	3	0.78	7.1	614
		Plywood -B/Glass	1	1	1	3	1	7.3	609
	Aramid HC/carbon and glass	1	1	1	3	0.89	2.4	233

Table 3

 3 Absorbed energy and indentation results for Plywood A and B.

	Materials	Relative	Specific energy	Depth of indentation (mm)
		absorbed	absorption (J)
		energy (%)
		5	10 15 5	10	15	5	10	15
	Plywood -A	51 81 99 5	19	32	1.56	1.87	-
	Plywood -B	67 70 98 8	16	35	1.65	2.30	-
	Aramid HC/carbon	93 93 88 21 41	59	3.35	5.57	-
	and glass

Table 4

 4 Absorbed energy and indentation results for Plywood A with aluminum skins.

	Materials	Relative	Specific	Depth of indentation (mm)
		absorbed	energy
		energy (%)	absorption (J)
		5	10 15 5	10 15 5	10	15
	Plywood -A/Al	72 83 81 5	13 18 1.89	2.65	3.03
	Aramid HC/carbon	93 93 88 21 41 59 3.35	5.57	-
	and glass

Table 5

 5 Absorbed energy and indentation results for plywood structures with composite skin.

	Materials		Relative absorbed energy (%)		Specific energy absorption (J)		Depth of indentation (mm)
			5	10	15	5	10	15	5	10	15
	Vacuum molding	Plywood -A/Carbon	51	55	65	5	10	17	0.49	0.49	0.68
		Plywood -A/Glass	58	75	70	5	13	17	0.13	0.38	0.47
	Thermo-compression	Plywood -B/Carbon	53	49	54	5	8	13	0.36	0.36	0.77
		Plywood -B/Glass	54	68	75	5	11	20	0.13	0.16	0.24
		Plywood -B/Flax	68	72	79	7	15	25	0.87	1.45	2.44
	Aramid HC/carbon and glass	93	93	88	21	41	59	3.35	5.57	-