Synthesis, properties and molecular conformation of paramylon ester derivatives

Accepted Manuscript Synthesis, properties and molecular conformation of paramylon ester derivatives Hongyi Gan, Yukiko Enomoto-Rogers, Taizo Kabe, Daisuke Ishii, Takaaki Hikima, Masaki Takata, Tadahisa Iwata PII:

S0141-3910(17)30127-1

DOI:

10.1016/j.polymdegradstab.2017.05.011

Reference:

PDST 8230

To appear in:

Polymer Degradation and Stability

Received Date: 16 February 2017 Accepted Date: 16 May 2017

Please cite this article as: Gan H, Enomoto-Rogers Y, Kabe T, Ishii D, Hikima T, Takata M, Iwata T, Synthesis, properties and molecular conformation of paramylon ester derivatives, Polymer Degradation and Stability (2017), doi: 10.1016/j.polymdegradstab.2017.05.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Synthesis, properties and molecular conformation of

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paramylon ester derivatives Hongyi Gan1,2, Yukiko Enomoto-Rogers3, Taizo Kabe1,2,4, Daisuke Ishii1,2, Takaaki Hikima5, Masaki Takata2, and Tadahisa Iwata1,2

Science of Polymeric Materials, Graduate School of Agricultural and Life Sciences, The

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University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Materials Visualization Photon Science Group, RIKEN SPring-8 Center, 1-1-1 Kouto,

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Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan 3

Eco-renewable group materials Group, Structural Materials Research Institute, The National

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Institute of Advanced Industrial Science and Technology, 2266-98, Anagahora, Shimoshidami, Moriyama-ku, Nagoya, Aichi 463-8560 Japan Materials Structure Group I, Research and Utilization Division, Japan Synchrotron Radiation

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Research Institute (JASRI), 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan Research Infrastructure Group, RIKEN Harima Institute/SPring-8 Center, 1-1-1 Kouto,

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Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan

*To whom all corresponding should be addressed Tel: +81-3-5841-5266, Fax: +81-3-5841-1304 E-mail: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT

Paramylon, which is a β-(1,3)-D-glucan photosynthesized by Euglena, was chemically

(carbon numbers 2−12) were successfully prepared.

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modified by esterification. Various paramylon triesters with different alkyl chain lengths All of the paramylon triesters have

higher thermal degradation temperatures than that of neat paramylon. Moreover, it was found

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that the paramylon triesters with C2−C6 alkyl chains are crystalline polymers with melting temperatures from 281 °C to 114 °C, and those with C8−C12 alkyl chains are amorphous

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polymers, confirmed by both DSC and X-ray diffraction analysis. Paramylon triesters with C3−C12 alkyl chains could shape self-sustaining films by both solvent-casting and melt-quench methods with high optical transmittance and sufficient tensile strength or elongation at break. Thermal and mechanical properties of paramylon triesters can be

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controlled freely from hard to soft by substituted acyl length. In the cases of the crystalline paramylon triesters, highly oriented and crystallized films could be fabricated by the

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thermally stretched method, and their tensile strengths have been obviously improved. Well-oriented X-ray fiber diagrams of the stretched and crystallized films suggest that all of

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the paramylon triesters have rare 5-fold helix conformation of molecular chains in crystal.

KEYWORDS: Paramylon esters, films, thermal properties, mechanical properties, molecular conformation

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ACCEPTED MANUSCRIPT 1. Introduction

In recent years, environmental friendly bio-based plastics have attracted a lot of attentions because the manufacturing process of plastics derived from petroleum will accelerate global

attractive raw materials for bio-based plastics.

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warming. Polysaccharides consisting of glucose with α- or β-glucoside linkages are the most

Paramylon, which is photosynthesized from Euglena of microalgae, is a storage

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polysaccharide, which refers to a β-(1,3)-glucan.1 Figure 1 shows a picture of Euglena

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accumulated paramylon. Since considerable amounts of paramylon can be photoproduced by using readily cultured Euglenoid alga from CO2 and water,2 it is a prospective biomass. For polysaccharides, chemical modification, such as esterification, is an effective way to obtain thermoplastic polymeric materials.3 Generally, unmodified polysaccharides do not

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show plasticity because of their strong inter- and intra-molecular hydrogen bondings. In the esterification method, hydroxyl groups are substituted by ester groups, which inhibits

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formation of hydrogen bonds and increases the hydrophobicity. In addition, polysaccharides esters have a wide range of thermal properties and mechanical properties by adjusting the

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carbon number of the ester group, as seen from previous research such as cellulose esters,4 xylan esters,5 pullulan esters,6 and curdlan esters.7 In the cases of paramylon ester derivatives, mixed esterification can be performed by two-pot synthesis8 or one-pot synthesis9 using two different types of ester groups. Moreover, solvent-casting films and hot-pressed films have been prepared, and their physical properties have been characterized.10,11 As a result in previous research, paramylon esters with various physical properties have been successfully obtained.8-11 However, most of mixesters are 3

ACCEPTED MANUSCRIPT amorphous, and it is desirable to produce high tensile strength films and fibers with crystalline polymers. Furthermore, it is necessary to understand the relationship between the physical properties and the molecular and crystal structures.

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In this study, paramylon homoesters (triesters) with hydroxyl groups fully substituted by the same ester groups were prepared. Eight types of paramylon homoesters with different alkyl side-chain lengths from acetate to laurate (C2−C12) were prepared and their

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fundamental properties were characterized. Furthermore, based on the X-ray fiber diagrams

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obtained for thermally stretched and annealed films, the molecular structures of the

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paramylon triesters were also determined.

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ACCEPTED MANUSCRIPT 2. Material and Methods 2.1. Materials Paramylon was purchased from the Euglena Co. (Tokyo, Japan). Trifluoroacetic anhydride

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(TFAA), acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, octanoic acid, decanoic acid, and lauric acid were purchased from Wako Pure Chemicals (Tokyo, Japan),

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2.2. Preparation of the paramylon esters

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and they were used without further purification.

The procedure for preparation of the paramylon triesters is demonstrated in Scheme 1. The representative procedure for paramylon acetate is described below. A premixed solution of TFAA (80 mL) and acetic acid (40 mL) was stirred at 50 °C for 5 min and then

50 °C for 1.0 h.

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immediately added to pre-dried paramylon (2 g) in a flask.

The solution was stirred at

After cooling to room temperature, the solution was poured into a mixture The precipitate was filtered, washed with methanol,

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of water and methanol (2.0 L).

dissolved in chloroform, and reprecipitated in methanol, before finally being filtered, washed

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with methanol, and dried in vacuo to give solid paramylon acetate (PaAc, n = 2) (90% yield). For the other paramylon esters, appropriate carboxylic acids (40 mL or 40 g, n = 3−12) were used instead of acetic acid. The yields (%) of the obtained paramylon esters were as follows: paramylon propionate (PaPr, n = 3) 86%, paramylon butylate (PaBu, n = 4) 80%, paramylon valerate (PaVa, n = 5) 95%, paramylon hexanoate (PaHe, n = 6) 75%, paramylon octanoate (PaOc, n = 8) 94%, paramylon decanoate (PaDe, n = 10) 89%, and paramylon laurate (PaLa, n = 12) 87%. The degree of substitution (DS) of the paramylon esters was calculated from the 5

ACCEPTED MANUSCRIPT ratio of the integrated area of the methyl protons of the acyl group to the ring protons of glucose: DS = ([CH3]/3)/([ring-H]/7).

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2.3. Preparation of solvent-cast films

The paramylon triesters (0.25 g) were dissolved in chloroform (10 mL) and poured into The

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polytetrafluoroethylene (PTFE) plates (diameter 4 cm) after filtration through cotton. solvent was then evaporated in air at room temperature.

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vacuo for 1 day to remove the solvent.

The obtained films were dried in

2.4. Preparation of melt-quenched films

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Paramylon triester samples were placed between PTFE or polyimide sheets and pressed at 30−50 °C above their melting temperatures (Tm), and then quenched into ice water or at room

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3 MPa to 15 MPa.

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temperature. The film thickness was controlled to be ca. 100 µm by varying the pressure from

2.5. Preparation of thermally stretched films

The melt-quenched films of the paramylon triesters were cut into pieces with length 20 mm and width 5 mm. The cut films were fixed to a stretch machine with 2 points marked with certain distances, which were used to determine the stretch times. The films were stretched in an oven set at 10−20 °C above their glass transition temperatures, and then stretched film fixing in the stretching machine was further annealed in the oven at the crystallization 6

ACCEPTED MANUSCRIPT temperatures for 1 h. All of the paramylon triesters were stretched to 4 times with respect to their initial lengths.

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2.6. Nuclear magnetic resonance (NMR) measurements

H-NMR spectra were recorded with a JEOL JNM-A500 FT-NMR (500 MHz)

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spectrometer, using tetramethylsilane (TMS) as the internal standard. The chemical shifts (δ)

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and coupling constants (J) are reported in ppm and Hz, respectively.

2.7. Gel permeation chromatography (GPC) measurements

The number- and weight-average molecular weights (Mn and Mw) and polydispersity index

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values (Mw/Mn) were estimated by gel permeation chromatography (GPC) (RID-20A refractive index detector, Shimadzu) in chloroform at 40 °C. Shodex columns (K-806M, K-802) were used, and the flow rate was 0.8 mL/min. A calibration curve was constructed

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using polystyrene (PS) standards (Shodex).

2.8. Thermogravimetric analysis

Thermogravimetric analysis (TGA) was performed using a STA 6000 (Perkin Elmer) instrument under a nitrogen atmosphere.

Thermograms were obtained between 30 and

450 °C at a heating rate of 10 °C/min.

2.9. Differential scanning calorimetry measurements 7

ACCEPTED MANUSCRIPT Differential scanning calorimetry (DSC) thermograms were recorded on a DSC8500 (Perkin Elmer) under a nitrogen atmosphere.

The measurements were performed with a 2~6

mg sample of the paramylon ester cast film on a DSC pan.

The samples were first heated

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from 30 °C to 250 °C at a rate of 100 °C/min, cooled to -70 °C at a rate of 200 °C/min, and then scanned with heating from −70 °C to 250 °C at a rate of 10 °C /min.

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2.10. Wide-angle X-ray diffraction of the solvent-cast and melt-quenched films

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Wide-angle X-ray diffraction (WAXD) of the solvent-cast and melt-quenched films was performed with a Rigaku RINT 2000 system operating at 40 kV and 200 mA. The measurements were performed with a Bragg-Bretano type 2θ/θ goniometer in reflection mode.

Ni-filtered Cu-Ka radiation (λ = 0.15418 nm) was collimated in a 1/2° divergence

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slit, 1/6° scatter slit, and a 0.15 mm receiving slit. The scans were performed twice in the 2θ

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range 3−40° with a scan rate of 0.5°/min and a step size of 0.05°.

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2.11. Wide-angle X-ray diffraction of the stretched and annealed films at SPring-8

X-ray fiber diagrams of the stretched and annealed films were obtained by WAXD measurements at the BL45XU of SPring-8 (Harima, Japan), with a wavelength of 0.1000 nm. Each WAXD diagram was recorded by an PILATUS detector with a sample-to-detector distance of about 266 mm. A vacuum path was set up between the sample holder and the detector.

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ACCEPTED MANUSCRIPT 2.12. Tensile tests

The tensile tests were performed at room temperature using an EZ-test machine (Shimadzu, Japan).

The crosshead speed was 20 mm/min, and the initial gauge length was 10 mm.

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About five specimens (length 20−30 mm, width 2−4 mm) of the paramylon ester cast films

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were used for each measurement, and the data were averaged for each film.

3.1. Esterification of paramylon

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3. Results and Discussion

Paramylon ester derivatives were synthesized by using carboxylic acid/ TFAA reaction system. Figure 2 shows the 1H-NMR spectra of the representative paramylon esters. The

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peaks that represent methyl groups, methylene protons, and ring-protons are observed. The degree of substitution (DS) was determined to be 3 by the ratio of the peak area of the methyl

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protons in the ester group to that of the ring protons in the glucose unit, indicating that all of the hydroxyl groups in the glucose unit are completely substituted by the corresponding

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carboxyl groups.

The weight-average molecular weights and polydispersity values of the paramylon triesters range from (3.0 to 9.6)×105 and 1.8 to 2.6, respectively (see Table 1), which are suitable values for manufacturing plastic materials. The results of esterification of paramylon are summarized in Table 1.

3.2. Solvent-cast films and melt-quenched films of the paramylon triesters 9

ACCEPTED MANUSCRIPT Figure 3a shows photographs of the solvent-cast films of PaPr, PaBu, PaVa, and PaHe, respectively. All of the cast films are relatively transparent and colorless as shown in pictures. During the hot-pressing process, after melting above Tm, the film was immediately

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quenched into ice water or air and formed a melt-quenched film. However, PaAc could not form a film because of its brittle properties. As shown in Figure 3b, films with high transparency were successfully obtained from paramylon triester powders. From the

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ultraviolet-visible (UV-vis) analysis shown in Figure 4, all of the melt-quenched films have

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relatively high optical transmittance (close to 90 %) in visible rays.

3.3. Thermal properties of the paramylon triesters

Figure 5 shows the 5 % mass reduction temperatures (Td_5%) of paramylon and the

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paramylon triesters obtained by TGA analysis. The Td_5% values of all of the paramylon triesters are above 300 °C and they are about 50 °C higher than that of neat paramylon, which

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reveales improvement of the thermal stability by the esterification process. However, there is no obvious tendency by adjusting the alkyl side-chain lengths.

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The thermal behaviors of the cast films of the paramylon triesters was analyzed by DSC (Figure 6a). As shown in the first heating scans, an endothermic peak is present in the thermograms of the paramylon triesters with C2−C6 side chains. Thus, PaAc, PaPr, PaBu, PaVa, and PaHe are considered to possess melting points (Tm). Comparing with general engineering plastics, Tm of PaAc (281 °C) is higher than that of polyethylene terephthalate (PET, Tm = 269 °C). Furthermore, PaPr, PaBu, and PaVa have Tms of 221 °C, 207 °C, and 196 °C, respectively, which are higher than that of polypropylene (PP, Tm = 175 °C). 10

ACCEPTED MANUSCRIPT Considering that the Tm of all of the crystalline paramylon triesters are significantly lower than the thermal degradation temperature of around 330 °C, these ester derivatives have relatively good thermal plasticity during the molding process, which is conducted above the

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melting temperature. The melting point can be controlled by substituted alkyl side-chain lengths. However, the heats of transition of PaOc, PaDe, and PaLa were not detected. Therefore, PaOc, PaDe, and PaLa are considered to be amorphous polymers. In the cases of

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glass transition temperatures (Tg), only PaAc, PaPr, and PaBu can be clearly observed in the

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second heating scans. PaAc and PaPr have glass transition temperatures above 100 °C (168 °C and 112 °C, respectively), indicating that they have higher thermal stability, compared with polystyrene and polycarbonate of typical oil-based amorphous polymers.

triesters

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3.4. WAXD measurements of the cast films and melt-quenched films of the paramylon

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Figure 7a shows the WAXD curves of the cast films of the paramylon triesters. For PaAc and PaPr, there are several sharp diffraction peaks. For the other paramylon triesters, there is

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a broad peak from 15° to 25°. This long-range order belongs to the amorphous halo. In the cases of PaBu and PaVa, there are still crystal diffraction peaks although their intensities are low. For PaHe, there is no reflection indicating the existence of crystals, even though a Tm is observed in DSC (Figure 6a). It can be considered that crystals formed during the heating process of the DSC measurement. Based on these results, the crystallinity decreases with increasing alkyl side-chain lengths. The peak in the low angle region (3−10°) belongs to the periodic structure of the paramylon polymer backbone, which shifts to lower angle with 11

ACCEPTED MANUSCRIPT increasing alkyl side-chain lengths. This phenomenon has also been reported for cellulose esters4 and pullulan esters6 etc. since the intermolecular distance increases with increasing chain length of the ester group.

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Figure 7b shows the WAXD patterns of melt-quenched films of the paramylon triesters. As can be seen, sharp diffraction peak observed in the cast films have disappeared, which confirms that through the hot press and quench process, the obtained melt-quenched films are

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amorphous.

3.5. DSC measurements of the melt-quenched films of the paramylon triesters

DSC measurements of the melt-quenched films of the paramylon esters with C3−C6 side chains were performed using the same heating procedure as that used for the cast films. As

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shown in Figure 6b, an endothermic peak appears at 215−177 °C during the first run of the DSC measurements despite the X-ray diffraction patterns indicating that those samples are

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amorphous. However, exothermic peaks are also simultaneously observed before the melting point. This result indicates that the melt-quenched films in the amorphous state are

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crystallized during the heating process. In the cases of PaHe, there is no heat of transition observed, suggesting that the rate of crystallization of PaHe is too low to form a crystal in a short time. This result also confirms the lower crystallinity of PaHe than PaPr, PaBu, and PaVa.

3.6. Tensile tests of the cast films and melt-quenched films of the paramylon triesters

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ACCEPTED MANUSCRIPT Figure 8a and Figure 8b show the stress−strain curves of the cast films and melt-quenched films of the paramylon triesters, respectively. The cast films of PaAc and PaPr could not be set to instrument because of their brittleness. With increasing alkyl side-chain lengths, the

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tensile strength and Young’s modulus decrease while the elongation at break tends to increase. Table 2 summarizes the tensile strengths, elongation at break values and Young’s moduli of the tested films. The cast films of PaBu, PaVa, and PaHe have tensile strengths of 20.6, 7.9,

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and 7.5 MPa, respectively, which are comparable with those of starch esters with

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corresponding ester groups (starch butyrate 17.9 MPa, starch valerate 14.1 MPa, starch hexanoate 8.1 MPa).12

There are only slight differences in the tensile strengths and elongation at break values of the cast films and melt-quenched films with corresponding ester groups, except for PaPr and

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PaVa. In the cases of PaPr, the melt-quenched film has high tensile strength of 30 MPa despite that the cast film of PaPr being very brittle. Furthermore, the melt-quenched film of PaVa has high elongation at break of ca. 350 % while that of the cast film is ca. 60 %. These

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differences seem to be because of the difference in the crystallinities of the cast films and

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melt-quenched films.

3.7. Preparation and properties of the thermally stretched films of the crystalline paramylon triesters

According to the DSC and WAXD measurements, paramylon triesters with C2−C6 side chains have crystal structures. Since PaAc is unable to form a melt-quenched film, a thermally stretched film was not produced. Therefore, thermally stretched PaPr, PaBu, PaVa, 13

ACCEPTED MANUSCRIPT and PaHe films were prepared. The PaPr, PaBu, and PaVa films were easily stretched until 4 times with respect to their initial lengths, and then further annealed for 1 h to fix the stretched molecular chains. In the cases of PaHe, since low crystallinity was revealed by WAXD and

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DSC measurements, the annealing time was set at 12 h. Figure 9 exhibits stress−strain curves of the thermally stretched films produced from PaPr, PaBu, PaVa, and PaHe. Table 2 summarizes the mechanical properties of the solvent-cast

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films, melt-quenched films and thermally stretched films of the paramylon triesters. The

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tensile strengths of the thermally stretched films are markedly higher than those of the cast films and melt-quenched films with corresponding side-chain groups. The tensile strength of the thermally stretched PaPr film (ca. 70 MPa) is to the same as that of PET. The mechanical properties of the films of the paramylon triesters can be significantly improved by modifying

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the arrangement of the molecular chains using the thermally stretching method. Furthermore, the mechanical properties can be controlled by the length of the ester groups and the

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stretching procedure.

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3.8. Molecular structures of the paramylon triesters

Figure 10 shows X-ray fiber diagrams of the thermally stretched films of PaPr, PaBu, PaVa, and PaHe. Strong and well-organized X-ray diffractions are observed in all of the X-ray fiber diagrams, suggesting that the molecular chains and crystals are oriented along the stretching direction. The degree of crystallinity of PaPr, PaBu, PaVa, and PaHe are 39%, 14%, 8%, and 6%, respectively. In addition, the degree of orientation values of PaPr, PaBu, PaVa, and PaHe are 97%, 94%, 96%, and 94%, respectively. 14

ACCEPTED MANUSCRIPT There is one strong diffraction on the 5th layer of the meridian. This result indicates that all of the paramylon triesters have rare 5-fold screw symmetry along the molecular axis.13 It is well known that cellulose triacetate, tributyrate, and trivalerate have 2-fold screw symmetry,

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and cellulose tripropionate has 3-fold symmetry. Cellulose has β-1,4-glycoside linkages and paramylon has β-1,3-gricoside linkages. This different glycoside linkage might affect the molecular conformation of the ester derivatives.

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Further molecular structure and crystal structure analysis of these ester derivatives should

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be performed.

4. Conclusions

A series of paramylon triesters with different alkyl side-chain lengths have been prepared,

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and their thermal properties, mechanical properties, and molecular structures have been investigated. The thermal stability improves because the Td_5% values of the paramylon

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triesters are ca. 50 °C higher than that of neat paramylon. The paramylon triesters with C2−C6 alkyl side-chains are crystalline polymers while those with C8, C10, and C12 side

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chains are amorphous polymers. For the crystalline paramylon triesters, PaAc and PaPr have melting points above 200 °C and glass transition temperatures above 100 °C, which are higher than those of general engineering plastics such as Nylon and PET. The mechanical properties of the paramylon triester films change from hard to soft with increasing alkyl side-chain length. Furthermore, by making highly oriented and annealed thermally stretched films of the crystalline paramylon esters, the tensile strength clearly improves. The well-oriented X-ray fiber diagrams of the stretched and crystallized films indicate that all of 15

ACCEPTED MANUSCRIPT the paramylon triesters have unusual 5-fold screw symmetry in the molecular chains. Detail molecular structure and crystal structure will be determined in future.

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Acknowledgement This work was partially supported by a Grant-in-Aid for Scientific Research of Japan (A) (No. 26248044) and the Advanced Low Carbon Technology Research and Development

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Program (ALCA) of the Japan Science and Technology Agence (JST). The synchrotron

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radiation experiments were performed at SPring-8 with the approval of RIKEN (Proposal Nos. 20150080 and 20160020) at the BL45XU beamline, and some synchrotron radiation

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experiments were performed at BL03XU.

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ACCEPTED MANUSCRIPT References

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[1] Clarke, AE, Stone, BA. Structure of the paramylon from Euglena gracilis. Biochimica et Biophysica Acta. 1960;44(1):161-163. [2] Barsanti L, Vismara R, Passarelli V, Gualtieri P. Paramylon (beta-1,3-glucan) content in wild type and WZSL mutant of Euglena gracilis. Effects of growth conditions. Journal of Applied Phycology. 2001;13(1):59-65. [3] Edgar KJ, Buchanan CM, Debenham JS, Rundquist PA, Seiler BD, Shelton MC, et al. Advances in cellulose ester performance and application. Progress in Polymer Science. 2001;26:1605-1688. [4] Crepy L, Miri V, Joly N, Martin P, Lefebvre, J-M. Effect of side chain length on structure and thermomechanical properties of fully substituted cellulose fatty esters. Carbohydrate Polymers. 2011;83:1812-1820. [5] Fundador NGV, Enomoto-Rogers Y, Takemura A, Iwata T. Syntheses and characterization of xylan esters. Polymer. 2012;53:3885-3893. [6] Enomoto-Rogers Y, Iio N, Takemura A, Iwata T. Synthesis and characterization of pullulan alkyl esters. European Polymer Journal. 2015;66:470-477. [7] Marubayashi H, Yukinaka K, Enomoto-Rogers Y, Takemura A, Iwata T. Curdlan ester derivatives: Synthesis, structure, and properties. Carbohydrate Polymers. 2014;103:427-433. [8] Shibakami M, Tsubouchi G, Hayashi M. Thermoplasticization of euglenoid beta-1,3-glucans by mixed esterification. Carbohydrate Polymers. 2014;105:90-96. [9] Shibakami M, Tsubouchi G, Sohma M, Hayashi M. One-pot synthesis of thermoplastic mixed paramylon esters using trifluoroacetic anhydride. Carbohydrate Polymers. 2015;119:1-7. [10] Shibakami M, Tsubouchi G, Sohma M, Hayashi M. Preparation of transparent self-standing thin films made from acetylated euglenoid beta-1,3-glucans. Carbohydrate Polymers. 2015;133:421-428. [11] Shibakami M, Sohma M. Synthesis and thermal properties of paramylon mixed esters and optical, mechanical , and crystal properties of their hot-pressed films. Carbohydrate Polymers. 2017;155:416-424. [12] Sagar AD, Edward WM. Properties of Fatty-Acid Esters of Starch. Journal of Applied Polymer Science. 1995;58:1647-1656. [13] Cochran W, Crick FHC, Vand V. The Structure of Synthetic Polypeptides. I. The Transform of Atoms on a Helix. Acta Cryst. 1952;5:581-586.

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ACCEPTED MANUSCRIPT Table 1. Results of esterification of paramylon and properties of paramylon esters.

a

Mw×105 -a -a 3.4 5.8 8.1 8.5 8.9 9.2 9.6

Mw/Mn -a -a 1.8 1.8 2.1 2.1 1.9 1.9 1.8

DS 3 3 3 3 3 3 3 3

Td_5% (oC) 283 327 340 337 330 328 330 324 333

Tg (oC) -b 168 112 76 -b -b -b -b -b

Tm (oC) -b 281 221 207 196 114 -b -b -b

Not measured due to its insolubility in the GPC solution (CHCl3). Not detected by DSC measurement.

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Mn×105 -a -a 1.9 3.2 3.9 4.0 4.5 4.8 5.4

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Name Paramylon PaAc PaPr PaBu PaVa PaHe PaOc PaDe PaLa

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Table 2. Results of tensile tests of paramylon ester films.

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TFAA/acid 50 oC, 1~2 h

Paramylon esters

Paramylon

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Carbon number: 2 Acetate PaAc

Carbon number: 6 Hexanoate PaHe

Carbon number: 3 Propionate PaPr

Carbon number: 8 Octanoate PaOc

Carbon number: 4 Butyrate PaBu

Carbon number: 10 Decanoate PaDe

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Carbon number: 12 Laurate PaLa

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5

7

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Carbon number: 5 Valerate PaVa

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Scheme 1. Preparation of paramylon ester derivatives.

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10 μm

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Figure 1. A picture of Euglena accumulated paramylon

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ACCEPTED MANUSCRIPT (a)

a

(b)

a

b a

a

ring-proton

ring-proton

b H2O

e

a

c

(d)

b

d

k

b-c

ring-proton

h

e

c

d

f

a

b

b-i a

a

ring-proton

d

k

ppm

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e

ppm

g

i j

CDCl3 as solvent

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(c)

ppm

TFA-d as solvent

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ppm

CDCl3 as solvent

j

CDCl3 as solvent

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Figure 2. 1H-NMR spectra of (a) PaAc, (b) PaPr, (c) PaHe, and (d) PaLa.

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PaPr

PaBu

PaVa

PaPr

PaBu

PaVa

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(b)

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(a)

PaHe

PaHe

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Figure 3. Photographs of (a) cast-film and (b) melt-quenched film of paramylon esters.

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PaPr

PaHe

PaVa

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PaBu

Figure 4. UV-vis spectra of melt-quenched films

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of paramylon esters.

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100 Paramylon PaAc PaPr

60

PaBu PaVa PaHe PaOc

40

PaDe PaLa

20

Paramylon 0 100

150

200 250 300 350 Temperature (oC)

400

450

500

SC

50

RI PT

Weight (%)

80

AC C

EP

TE D

M AN U

Figure 5. TGA thermogram of paramylon esters.

6

ACCEPTED MANUSCRIPT (a) Cast-film

(b) Melt-quenched film

1st run

1st run

2nd run PaAc Tg=163 oC

Tc=198 oC Tg=112 oC

PaPr Tm=221 oC

Tm=114 oC

PaBu

PaVa

PaOc

PaHe PaOc PaDe

PaDe

PaLa

PaBu

Tm=183 oC

Tc=105 oC

PaVa

Tm=177 oC PaHe

SC

PaLa

Tm=215 oC

Tc=145 oC

RI PT

PaHe

Tc=122 oC Endotherm

Tm=196

PaVa

oC

PaPr

PaPr

EP

TE D

M AN U

Figure 6. DSC thermograms of (a) cast-film and (b) melt-quenched film of paramylon esters.

AC C

Endotherm

PaBu Tm=207 oC

PaAc

Endotherm

Tm=281

oC

7

ACCEPTED MANUSCRIPT

(a) Cast-film

PaAc

RI PT

PaPr

PaBu PaVa PaHe PaOc PaDe

PaPr PaBu PaVa PaHe

TE D

M AN U

(b) Melt-quenched film

SC

PaLa

Figure 7. X-ray diffraction diagrams of (a) cast-film

AC C

EP

and (b) melt-quenched film of paramylon esters.

8

ACCEPTED MANUSCRIPT

(a) Cast-film

PaHe

PaVa

PaOc

M AN U

(b) Melt-quenched film PaPr

PaLa

SC

PaDe

RI PT

PaBu

PaBu

PaVa

TE D

PaHe

Figure 8. Stress-strain curves of (a) cast-film and

AC C

EP

(b) melt-quenched film of paramylon esters.

9

ACCEPTED MANUSCRIPT

PaPr

RI PT

PaBu

PaVa

SC

PaHe

M AN U

Figure 9. Stress-strain curves of thermally-stretched

AC C

EP

TE D

and annealed films of paramylon esters.

10

(a) PaPr Carbon number: 3

(b) PaBu Carbon number: 4

RI PT

ACCEPTED MANUSCRIPT

(c) PaVa Carbon number: 5

(d) PaHe Carbon number: 6

AC C

EP

TE D

M AN U

SC

Figure 10. X-ray fiber diagrams of thermally-stretched and annealed films of paramylon esters.

11