Acetylation of Bombyx mori silk fibroin and their characterization in the dry and hydrated states using 13C solid-state NMR

BIOMAC-13897; No of Pages 10 International Journal of Biological Macromolecules xxx (xxxx) xxx

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Acetylation of Bombyx mori silk fibroin and their characterization in the dry and hydrated states using 13C solid-state NMR Tetsuo Asakura ⁎, Hironori Matsuda, Akira Naito Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Nakacho, Koganei, Tokyo 184-8588, Japan

a r t i c l e

i n f o

Article history: Received 18 October 2019 Received in revised form 5 November 2019 Accepted 13 November 2019 Available online xxxx Keywords: Acetylation of Bombyx mori silk fibroin 13 C solid-state NMR Hydration of silk

a b s t r a c t Acetylation of Bombyx mori silk fibroin (SF) was accomplished by chemical reaction of the side chain OH groups in Ser and Tyr residues with acetic anhydrate to increase hydrophobic character of SF. A combination of three kinds of 13C solid-state NMR techniques was used to elucidate the effect of acetylation in the dry and hydrated states for [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF powder. The mobilities of Tyr and Ala residues in the amorphous region of acetylated 13C-labeled SF powder increased slightly and only very small amounts of the Ala residue with high mobility was observed by hydration. On the other hand, there are essentially no effect of water on the β-sheet region of these residues and acetylated Ser residues. A similar tendency toward the effect of hydration for acetylated SF powder was observed for acetylated SF fiber. The remarkable improvement of the dimensional stability in water was attained although the tensile strengths of fibers were slightly lower by the acetylation of SF fiber. Thus, acetylation proved very effective to achieve water-proofing of SF materials and the effect of acetylation on the local structure and dynamics of SF could be clarified using 13C solid-state NMR. © 2019 Published by Elsevier B.V.

1. Introduction Silk fibroin (SF) fiber from Bombyx mori silkworm has been used for thousands of years as “queen of fibers” in luxury apparel field due to its appearance, soft touch, durability and so on [1]. More recently, SF have received a lot of interest as one of promising resources of biotechnology and biomaterials due to many inherently superior qualities such as their mechanical properties, environmental stability, biocompatibility, low immunogenicity and biodegradability [2–6]. The ease of fabrication into different forms such as fiber, film, gel, powder, sponge and so on is also the reason to use SF in the biomedical field. The biomaterials are generally used in a hydrated state and therefore, it is important to understand the structure and dynamics of SF in the hydrated state as well as in the dry state for the biomedical application [7,8]. In the field of textile, in order to suppress the effect of water on SF fiber as much as possible to maintain functional and dimensional stabilities for use them widely, various physical, chemical or enzymatic modification techniques have been applied to overcome some inferior textile performance of SF fiber, i.e., low water repellency, vulnerable to friction, low dimensional stability, prone to wrinkles, yellowish browning and so on [9–11]. Thus, it is also important to understand change in the structure and dynamics of the modified SF by hydration.

⁎ Corresponding author. E-mail address: [email protected] (T. Asakura).

Many analytical methods including Raman [12], IR [12–16], DSC [16–18], XRD [18], DMTA [19], NMR [20–29] and so on have been used to characterize SF in the hydrated state as well as the dry state. In general, water in the silk–water system can be divided into three categories: free water, freezing bound water, and non-freezing bound water [12–17,20,21] However, a complete picture of the structure and dynamics of SF is still not well understood at molecular level. Due to the heterogeneous structure and dynamics of SF in the hydrated state, several 13C solid-state NMR techniques which emphasize the mobile and immobile components in the materials are very effective. Recently, we used the combinations of 13C refocused insensitive nuclei enhanced by polarization transfer (13C RINEPT) NMR [30], 13C cross polarization/ magic angle spinning (13C CP/MAS) NMR [31] and 13C dipolar decoupled-magic angle spinning (13C DD/MAS) NMR for the purpose [24–27,32–34]. The 13C RINEPT in which the pulse sequence was developed for solution NMR is sensitive to the fast motion component (N105 Hz) of hydrated SF and other protein systems [35,36]. In contrast, 13 C CP/MAS NMR is sensitive to the very slow motion components (b104 Hz). Thus, if the presence of water causes an increase in the dynamics of SF materials, a loss in CP signal occurs and consequently such a mobile domain cannot be observed in the 13C CP/MAS NMR spectra [37–39]. In the dry state, only 13C CP/MAS NMR was used for the NMR observation of SF. On the other hand, 13C DD/MAS NMR spectra can be used to detect the mobile domains as well as the immobile domains. Therefore, the combinations of these three kinds of 13C NMR techniques, 13C RINEPT, 13C CP/MAS and 13C DD/MAS NMR, provide

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different perspectives on the dynamical behavior of SF in the dry and hydrated states. Moreover, conformation-dependent 13C solid-state chemical shift [40–42] coupled with 13C selective labeling of SF samples is extremely useful to selectively determine the several local conformations and as a result, the fraction of several conformations in an aminoacid-specific manner. We reported that 13C selectively labeled SF such as [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF was extremely useful because Ser residues in SF were present predominantly in the crystalline domain, Tyr residues predominantly in the non-crystalline domain and Ala residues in both domains [43]. Therefore, we could elucidate the local structure and dynamics of SF from the crystalline and non-crystalline domains independently using these solid-state NMR methods together with the 13C selective labeling of SF [24–27,29]. In this paper, we noticed that acetylation is one of the chemical modifications of SF among the various methods available for the modifications [11]. Acetylation is expected to achieve water-proofing of SF materials via acetylation of the OH groups of Tyr and Ser side chains of SF. However, there are no reports about the effect of acetylation on the change in the local structure and dynamics of SF by hydration at molecular level. Therefore, we elucidated changes in the structure and dynamics of [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF powder by acetylation in the dry and hydrated states using the combination of three kinds of 13C NMR techniques, 13C RINEPT, 13C CP/MAS and 13C DD/MAS NMR. Next, the fiber formation of acetylated SF was performed by solution spinning process in order to elucidate the effect of acetylation in the fiber form of SF [44]. The physical properties and dimensional stabilities of the acetylated SF fiber were compared with those of nonacetylated SF fiber. The difference in the structure of these two kinds of SF fibers in the dry and hydrated states was also elucidated using the combination of three kinds of 13C NMR techniques mentioned above. 2. Materials and methods 2.1. Preparations of regenerated powders of acetylated and non-acetylated [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SFs Seven Bombyx mori silkworms were reared in our laboratory. At the fifth instar larval stage, the 13C labeling of the SF was performed by oral administration of an artificial diet with 13C-enriched amino acids, as reported previously [24,25,27]. Briefly, the supplementary [3-13C] Tyr and [3-13C] Ser was mixed with 2.0 g of an artificial diet per day. The amount of labeled Tyr and Ser was 10 mg each on the fourth and fifth day of the fifth larval stage. The [3-13C]Ala was enriched by transamination from [3-13C] Ser in the silkworm [24,27]. To prevent amino acid transfer of Ser into Gly, 20 mg non-labeled Gly was also mixed with the artificial diet per day [45]. Thus, the total amount of [3-13C] Tyr and [3-13C] Ser was 20 mg per silkworm. The 13C-labeled amino acids, [3-13C] Tyr, and [3-13C] Ser (each 99% enrichment), used for labeling of SF, were purchased from Cambridge Isotope Laboratories, Inc., Andover, MA USA. The [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-cocoons were degummed with 0.5 w/v% Marseilles soap (Aikuma Senryo, Co. Japan) solution at 100 °C for 30 min and washed with distilled water to remove silk sericin [24]. The process was repeated three times. The degummed 13 C labeled SF fibers were dissolved in 9 M LiBr (Wako Pure Chemical Industries, Ltd., Japan) solution to a concentration of 10% w/v at 40 °C for about 30 min. and then dialyzed against deionized water for 4 days at 4 °C using a cellulose membrane. The aqueous solution was lyophilized and 13C-labeled SF sponge was obtained [46]. For acetylation, the 13Clabeled SF sponge (1.0 g) was dissolved in dimethylformamide (DMF) 40 ml with LiCl (1.6 g) at 80 °C with stirrer [11]. Then, acetic anhydrate (5 ml) was added and stirred at 80 °C for 6 h. The DMF solution was poured in 400 ml methanol (MeOH). The precipitate was collected by the centrifugal separation and washed a few times with MeOH. The precipitate suspension in distilled water was dried by the lyophilization, and then finally dried at 80 °C for overnight. The regenerated powder

of the acetylated 13C-labeled SF was obtained and named as regenerated C acetylated SF (RA13CSF) powder. The acetylation was also performed for regenerated natural abundant SF powder (RSF powder) according to a similar method and the regenerated acetylated SF powder was named as RASF powder. The regenerated 13C-labeled SF (R13CSF) powder without acetylation was prepared and dried it after immersion in methanol for 45 h. 13

2.2. 13C solution NMR observations of acetylated [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF, and acetylated and non-acetylated SF samples In order to characterize acetylated SF samples, we observed 13C solution NMR spectra of acetylated [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala-SF using a JEOL ECX-400 NMR spectrometer. The RA13CSF powder (50 mg) was dissolved in DMSO‑d6 (0.5 ml) and then the 13C NMR spectrum was measured with 1H decoupling mode at room temperature. For a comparison, 13C solution NMR spectra of RASF and RSF powders were also observed under similar condition. 2.3. Preparation of regenerated fibers of acetylated and non-acetylated SFs Because of limited amounts of 13C labeled SF, the preparation of the regenerated SF fibers was performed using only acetylated and nonacetylated natural abundant SF powders. The RASF powders were dissolved in hexafluoroisopropanol (HFIP) overnight at 40 °C, yielding 13.5% (w/w) solutions. The HFIP solutions were extruded through a stainless steel spinneret with 0.2 mm inner diameter using a mechanical spinning method into the MeOH coagulation bath at room temperature through the air gap of 10 mm [47–50]. The fibers were named as RASF fibers. According to the similar experimental condition, the regenerated fibers of non-acetylated SF were prepared and named as RSF fibers. These fibers were taken up with a roller in air at draw ratios of 3.0 and 3.5. 2.4. 13C solid-state NMR observations of acetylated and non-acetylated [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF powders in the dry and hydrated states The 13C solid-state NMR spectra of [3- 13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF powders with and without acetylation were observed in the dry and hydrated states using a Bruker Avance 400 NMR spectrometer with a 4-mm double resonance MAS probe and a MAS frequency of 8.5 kHz at room temperature. For the NMR observations in the hydrated state, the RA 13C SF powders were carefully inserted into a zirconia rotor to avoid drying of the hydrated sample and sealed with PTFE insert to prevent dehydration during the NMR measurements [24–27]. Typical experimental parameters for the 13C CP/MAS NMR experiments were 3.5 μs 1H 90° pulse, 1 ms ramped CP pulse with 71.4 kHz rf field strength, TPPM 1H decoupling during acquisition, 2176 data points, 8 k scans, and 4 s recycle delay. Lorentzian line broadening of 20 Hz was applied prior to Fourier transformation. Details of the NMR experimental conditions for the 13 C DD/MAS NMR experiments were described in our previous paper [24]. A recycle delay of 5 s and 13 C 90° pulse of 3.5 μs were used. Typical experimental parameters for the 13C RINEPT NMR experiments were 3.5 μs 1H and 3.5 μs 13C pulses, inter-pulse delay of 1/4 1 JCH (1 JCH = 145 Hz), refocusing delay of 1/3 1 J CH, TPPM 1 H decoupling during acquisition, 1438 data points, 4 k scans and 4 s recycle delay. There should be no peaks in the carbonyl region because no 1H nuclei are attached to the carbonyl carbons directly in the 13C RINEPT spectrum [30]. The 13C RINEPT, 13C CP/MAS and 13C DD/MAS NMR spectra were also observed in the dry and hydrated states for RASF and RSF fibers. The 13C chemical shifts were calibrated externally through the methylene peak of adamantane observed at 28.8 ppm with respect to TMS at 0 ppm. The fractions of Gly Cα peak, and two conformations, β-sheet and random coil of Tyr Cβ

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peak in the 13C DD/MAS (hydrated), 13C CP/MAS (hydrated) and 13C CP/MAS (dry) NMR spectra were determined by the peak deconvolution analysis by assuming Gaussian lineshapes [24,25,27,29,32–34].

2.5. Physical property measurements of regenerated fibers of acetylated and non-acetylated SFs The physical property of RASF fiber prepared by solution spinning method was determined and compared with that of RSF fiber. Because of limited amounts of the 13C labeled SF, the preparation of the SF fibers was performed using only acetylated and non-acetylated natural abundant SF powders. The stress-strain curves of RASF and RSF fibers with draw ratios of 3.0 and 3.5 were measured in the dry condition as follows. The both edges of the fibers were mounted on the Scotch tape with a base length of 20 mm and fixed with ethyl cyanoacrylate, respectively [48–50]. Before starting the tensile test, the diameters of these fibers were measured with an optical microscope (KEYENCE BIOREVO BZ9000, Japan). The stress-strain curves were measured using an EZGraph tensile testing machine (EZ-Graph, SHIMADZU Co. Ltd. Japan) at room temperature with a 5 N lead cell. The rate of crosshead was 3 mm/min on samples of 20 mm length. Each value was obtained by averaging at least 6 measurements. The breaking strength (MPa) measured as the highest stress value attained during the test was calculated by dividing the cross-sectional area of the fiber. The elongation at break (%) was measured as the change in length divided by the initial length.

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2.6. Determination of water absorption rates of regenerated fibers of acetylated and non-acetylated SF fibers About 35 mg of the dry powder was weighed and pressed at 20 MPa for 5 min using the powder compacting machine for infrared spectroscopy, and obtained the tablet of the diameter of ca. 10 mm and the thickness of ca. 1 mm. The weight, A in mg, of the dry tablet was measured. The tablet was immersed in water for 30 min, and then quickly wiped the water on the surface of the tablet and the weight B of the wet tablet was measured. The water absorption rate of the tablet was calculated by the equation of [(B − A) / A] × 100 (%).

2.7. Dimensional stabilities measurement of regenerated fibers of acetylated and non-acetylated SF fibers by water immersion treatment The dimensional stabilities of RASF and RSF fibers with draw ratios of 3.0 and 3.5 in water were measured as follows [29]. A bundle of 24 fibers were loaded by a 7.84 mN weight installed at the bottom edge of the bundle. The length of the bundle in the dry state, Ldry0, was measured at room temperature. After soaking the SF fiber bundles in water for 30 min, the length in the hydrated state, Lhydrated 1, was measured. We defined the primary contraction rate of the fiber as the change in length when the fibers were first immersed in water, expressed by the following formula, (Lhydrated1 − Ldry0) / Ldry0. Then the SF fiber bundles were dried at room temperature for 30 min and Ldry2, was measured. This hydration and drying treatment were repeated three times. The averaged value over (Lhydrated3

Fig. 1. 13C solution NMR spectra of the aliphatic region (10–70 ppm) of (a-1) acetylated [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF, (a-2) acetylated SF and (a-3) non-acetylated SF, and the aromatic and carbonyl regions (110–180 ppm) of (b-2) acetylated SF and (b-3) non-acetylated SF. The peak assignments were included together with DMSO‑d6 peak at 39.5 ppm used as the chemical shift reference.

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− Ldry2) / Ldry2, (Lhydrated3 − Ldry4) / Ldry4, (Lhydrated5 − Ldry4) / Ldry4 and (Lhydrated5 − Ldry6) / Ldry6 was defined as the secondary contraction rate. 3. Results and discussion 3.1. 13C solution NMR spectra of acetylated and non-acetylated SF samples Fig. 1 shows the 13C solution NMR spectra of (a-1; 10–70 ppm) acetylated [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala-SF, and (a-2; 10–70 ppm), (b-2; 110–180 ppm) acetylated naturally abundant SF and (a-3; 10–70 ppm), (b-3; 110–180 ppm) non-acetylated naturally abundant SF in DMSO‑d6 together with the assignment [51]. The chemical shift data are listed in Table 1. Because of 13C labelings of Ser and Tyr Cβ carbons in SF, these peaks were obtained with sufficient S/N ratio in the spectrum (a-1) contrary to the spectrum (a-2). Increase in the Ala Cβ peaks (a-1) was also attained by transamination from [3-13C] Ser carbons in the silkworm [24,27]. By acetylation to the OH group of Ser residue, Ser Cβ peak shifted to lower field by 2.0 ppm and SerCα peak shifted to higher field by 4.1 ppm due to the polarization of the Ser Cα-Cβ bond. The relative ratio of the Ser Cβ peaks at 63.6 ppm and 61.6 ppm in the spectrum (a-1) gives the acetylation ratio of the Ser residue and could be evaluated to be 95%. The acetylation of the Tyr OH group was observed in the spectrum, (b-2). By acetylation of SF, the Tyr Cζ and Cε peaks shifted to higher field by 7.3 ppm and lower field by 6.3 ppm, respectively due to the polarization of the Cζ and Cε bond in the aromatic group of Tyr residue as shown in the spectra (b-2) and (b-3). The acetylation for Tyr residue was considered to be almost completed as evaluated from the Yε or Yζ peak intensities in the spectrum (b-2) although the S/N ratios were low. The methyl peak of the acetyl group was observed clearly at 21.0–21.2 ppm in the spectra (a-1) and (a-2). The amino acid composition of Ala, Ser and Tyr residues in SF was reported as 30.0%, 12.2% and 4.8%, respectively [43,51,52], and therefore, the ratio of the area of acetyl methyl and Ala methyl peaks is expected to be 1 (0.122 × 0.95 + 0.048) :1.83 (0.30), which almost coincides with the ratio, 1:1.96 evaluated from the spectrum (a-2). The acetyl methyl peak was split into two peaks, i.e., the upper field peak at 21.0 ppm could be assigned to acetyl methyl one of Ser residue and the lower field peak at 21.2 ppm to acetyl methyl one of Tyr residue from the relative peak intensity and the amino acid composition. In the spectrum (a-1), the fraction of the acetyl methyl peak in the acetyl plus Ala methyl peaks could be evaluated to be 12%, which is remarkably lower than the fraction evaluated from the spectrum (a-2), 35%. This is due to 13C labeling of [3-13C]Ala carbon by transamination from [3-13C]Ser carbon as mentioned above. The C_O peak of the acetyl group was observed at 169.3 ppm at the spectrum (b-2). Thus, we

Fig. 2. 13C solid-state NMR spectra of acetylated [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF powders together with assignments. (a) 13C RINEPT (hydrated), (b) 13C DD/ MAS (hydrated), (c) 13C CP/MAS (hydrated) and (d) 13C CP/MAS (dry) NMR spectra.

could obtain almost completely acetylated SF samples by acetylation condition described in the Materials and methods. 3.2. 13C solid-state NMR spectra of acetylated [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF powders in the dry and hydrated states Fig. 2 shows the 13C solid-state NMR spectra of RA13CSF powder in the dry and hydrated states, i.e., (a) 13C RINEPT, (b) 13C DD/MAS, (c) 13C CP/MAS spectra in the hydrated state, and (d) 13C CP/MAS spectrum in the dry state. The spectral assignments are Ala Cβ, Tyr Cβ, Gly Cα, Ala Cα, Ser Cα, Ser Cβ, Τyr aromatic carbons and C_O carbons to lower field as shown in the spectrum, (d) as reported previously [24–27]. The appearance of the 13C peaks in the 13C RINEPT spectrum means very fast isotropic motion (N105 Hz) of the observed 13C nuclei. A very small sharp peak was observed at 16.8 ppm which was assigned to Ala Cβ peak with random coil conformation [24–27,53–55]. The peak intensity is considerably smaller than those expected amounts of Ala residues in the sample [53–55]. Thus, there are small amounts of Ala residues with very fast motion (N105 Hz) in the hydrated state. The 13 C DD/MAS NMR spectrum (b) was quite different from the 13C RINEPT spectrum. One large peak at about 110 ppm was assigned to the spacer, Teflon and not from the SF sample [26]. The 13C nuclei with both fast and slow motions could be observed in the 13C DD/MAS

Table 1 13 C solution NMR chemical shifts (in ppm) of acetylated and non-acetylated SF samples in DMSO‑d6.a Residue

Carbon

A (Ala)

Cα Cβ Cα Cα Cβ Cα Cβ Cγ Cδ Cε Cζ CH3(S) CH3(Y) C=O

G (Gly) S (Ser) Y (Tyr)

Acetyl

a

13

C chemical shift (ppm)

Acetylated

Non-acetylated

49.0 18.0 42.4 52.2 63.6 – 36.6 – 130.6 121.6 149.1 21.0 21.2 169.3

49.0 18.0 42.4 56.3 61.6 – 36.6b 128.1 130.2 115.3 156.4 – – –

The error in the chemical shift was evaluated to be ±0.03 ppm from the digital resolution.

b Determined from the Tyr Cβ chemical shift of non-acetylated [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF samples.

Fig. 3. Expanded 13C solid-state NMR spectra of acetylated [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF powders together with assignments. (a) 13C RINEPT (hydrated), (b) 13C DD/MAS (hydrated), (c) 13C CP/MAS (hydrated) and (d) 13C CP/MAS (dry) NMR spectra.

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NMR spectrum and therefore most of the observed peaks were due to the 13C nuclei with slow motion in the hydrated state. Actually, the 13 C CP/MAS NMR spectra (c) and (d) detected slow motion with frequency b 104 Hz, showed a similar pattern with that of 13C DD/MAS NMR spectrum although there are still different parts among these spectra. The aromatic Tyr peaks could be observed only in the 13C CP/ MAS NMR spectrum (d) in the dry state whose chemical shifts indicated that the Tyr OH groups were acetylated from the comparison with the spectrum, Fig. 1(b-2). Fig. 3 shows the expanded 13C solid-state NMR spectra (0–80 ppm) of RA13CSF powder in the dry and hydrated states, i.e., (a) 13C RINEPT, (b) 13C DD/MAS, (c) 13C CP/MAS spectra in the hydrated state, and (d) 13C CP/MAS spectrum in the dry state. We already reported the

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conformation-dependent chemical shifts of Ala Cβ, Tyr Cβ and Ser Cβ peaks as well as Gly Cα peak in the 13C solid-state NMR spectra of R13CSF fiber in the dry and hydrated states [24–27,29]. Namely, the assignment is 16.8 ppm (random coil), 19.8 ppm (β-sheet A) and 21.7 ppm (β-sheet B) for Ala Cβ peak, and 61.5 ppm (random coil), 64.0 ppm (β-sheet A) and 65.5 ppm (β-sheet B) for Ser Cβ peak [27,54,55]. Thus, each β-sheet peak is further divided into two peaks due to the difference in the packing states, A and B. On the other hand, in the spectra of the acetylated SF powder, it is difficult to assign the β-sheet peak to different packing state in Fig. 3 because the acetyl methyl peak (12% of total methyl peak) was overlapped with β-sheet peak of Ala Cβ carbon at about 20 ppm. In this paper, we assigned that the β-sheet peak of Ala Cβ carbon and acetyl methyl peak were assumed

Fig. 4. Deconvolutions of Gly Cα (42.6 ppm) and Tyr Cβ (40.4 ppm (β-sheet) and 36.2–36.4 ppm (random coil)) peaks of acetylated [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF powders in the (a) 13C DD/MAS (hydrated), (b) 13C CP/MAS (hydrated) and (c) 13C CP/MAS (dry) NMR spectra.

Please cite this article as: T. Asakura, H. Matsuda and A. Naito, Acetylation of Bombyx mori silk fibroin and their characterization in the dry and hydrated states using 13C solid-state NMR..., , https://doi.org/10.1016/j.ijbiomac.2019.11.116

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Table 2 The chemical shifts (ppm) and half-height-widths (×102 Hz) used for deconvolutions of Gly Cα and Tyr Cβ peaks of acetylated [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF powders in the (a) 13C DD/MAS (hydrated), (b) 13C CP/MAS (hydrated) and (c) 13C CP/MAS (dry) NMR spectra shown in Fig. 4 together with the fractions determined from the deconvolution analysis.

(a) (b) (c) a

Chemical shift (ppm)

Half-height-width (×102 Hz)

Fractions (%)

Gly Cα

Gly Cα

Gly Cα

42.60 ± 0.43 42.60 ± 0.41 42.60 ± 0.36

Tyr Cβ β-Sheet

r.c.

40.40 ± 0.56 40.40 ± 0.53 40.40 ± 0.45

36.40 ± 0.77 36.20 ± 0.86 36.20 ± 0.67

2.88 ± 0.22 2.88 ± 0.21 3.24 ± 0.18

Tyr Cβ β-Sheet

r.c.

3.78 ± 0.28 3.78 ± 0.27 4.02 ± 0.23

5.18 ± 0.39 6.06 ± 0.43 6.06 ± 0.34

20.9 ± 0.4 30.4 ± 0.6 26.1 ± 0.5

Tyr Cβ β-Sheet

r.c.

31.8 ± 0.6 (40.2)a 35.6 ± 0.7 (51.1) 29.2 ± 0.6 (39.5)

47.3 ± 0.9 (59.8) 34.1 ± 0.6 (48.9) 44.7 ± 0.9 (60.5)

The values in parentheses mean the fraction (%) when the β-sheet and random coil fractions evaluated from the Tyr Cβ peaks were assumed to be 100%.

to be 20 ppm without further detailed assignment reflecting difference in the packing state. In addition, the conformation-dependent peak assignment did not try for the Ser Cβ peak although the main peak could be assigned to β-sheet judging from the shape of the Ala Cβ peak of acetylated SF powder. Therefore, as shown in Fig. 4, we tried to determine only the fractions of Gly Cα peak, and two conformations, β-sheet and random coil of Tyr Cβ peak from the (a) 13C DD/MAS (hydrated), (b) 13C CP/MAS (hydrated) and (c) 13C CP/MAS (dry) NMR spectra by the peak deconvolution by assuming Gaussian lineshapes [24,25,27,29]. The fractions were listed in Table 2 together with the chemical shifts and half-height-widths used for the deconvolutions. From a comparison of the spectra (b) and (c), it is noted that the 13C CP/MAS NMR spectra of Tyr residues changed considerably, that is, the fractions of random coil decreased and β-sheet increased by hydration. This indicated that the mobilities of Tyr residues increased due to diffusion of water molecules in the random coil domain. The fractions of random coil and β-sheet of Tyr Cβ peak in the 13C DD/MAS NMR spectrum (a) of the sample in the hydrated state were almost the same as those of Tyr peak in the 13C CP/MAS NMR spectrum (c) in the dry state. Almost all of the Tyr residues in β-sheet come from residues in the sequences (GAGAGY)n and (GAGYGA)n. as reported previously [56] and therefore Tyr residues located in the other region except for the β-sheet region are partially hydrated by water. 3.3. Comparison of the 13C solid-state NMR spectra of acetylated and nonacetylated [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF powders in the dry and hydrated states Fig. 5 shows the expanded 13C solid-state NMR spectra (0–80 ppm) of R13CSF powders in the dry and hydrated states, i.e., (a) 13C RINEPT, (b) 13C DD/MAS, (c) 13C CP/MAS spectra in the hydrated state, and (d) 13C CP/MAS spectrum in the dry state, respectively. There are remarkable differences in the 13C CP/MAS NMR spectra between RA13CSF (Fig. 3) and R13CSF (Fig. 5) powders in the dry states. For example, the upfield shift of Ser Cα peak in Fig. 3(d) compared with Fig. 5 (d) is due to acetylation of Ser OH group as shown in Fig. 1(a-1) and (a-2). The fraction of β-sheet structure seems to be increased for RA13CSF powder compared with R13CSF powder as was observed in the Ala Cβ peak even if overlapping of 12% methyl peak of acetyl group was taken into account for the peak at 20 ppm assigned to βsheet structure in Fig. 3(d). A clearer observation about increase of βsheet structure is obtained from the intensity of lower field β-sheet peak of Tyr Cβ carbon in the chemical shift range of 30–45 ppm which was assigned to Tyr Cβ plus Gly Cα carbons. The original 13C labeled SF powders before acetylation are the same between two SF fibers. The increase of β-sheet fraction in acetylated SF fiber may come from two possibilities, that is, acetylation of SF fiber and/or difference in the insolubilization process. Namely, β-sheet formation of R13CSF powder was performed by immersion in MeOH for 45 h, but that of RA13CSF powder was performed in the process of acetylation. By considering these points, we concentrate to elucidate change in the spectra by hydration for each sample and compare difference in the hydration effect on the structure and dynamics between RA13CSF and R13CSF powders using 13C solid-state NMR in the dry and hydrated states.

There are remarkable differences in the Ser Cβ peaks between two samples by hydration. Namely, the chemical shift of main Ser Cβ peak did not change and the line shapes did not change significantly among Fig. 3(b), (c) and (d), too for RA13CSF powder. However, the line shapes changed remarkably, that is, a broad peak in Fig. 5(d), a slightly sharper main peak with a small sharp peak at the upper field in Fig. 5(c) and the intensity of the latter sharp peak increased in Fig. 5(b) for R13CSF powder. Such a sharp peak could be observed as the 13C RINEPT main peak (Fig. 5(a)), but no peak in Fig. 3(a). The Ser residues located in the other region except for the β-sheet region of R13CSF powder were partially hydrated by water and the mobilities of such Ser residues are expected to be very high [24,25,27]. On the other hand, the acetylated Ser residues did not hydrate by water at all in RASF powder. There are also significant differences in the broad Tyr Cβ peaks between two samples by hydration. The relative intensity of the Tyr Cβ random coil peak at 36.2–36.4 ppm decreases due to a loss in CP signal in the 13C CP/MAS NMR spectrum when the mobility of the side chain of Tyr residue increases by hydration [32–34]. This was observed in both RA13CSF and R13CSF powders as shown in Fig. 3(d) and (c), and also Fig. 5(d) and (c), respectively. However, the degree of decrease in the Tyr Cβ random coil peak is greater in R13CSF powder compared with that of RA13CSF powder. In addition, s small Tyr Cβ peak was observed in the 13C RINEPT spectrum of R13CSF powder (Fig. 5(a)), but could not be observed in the spectrum of RA13CSF powder (Fig. 3(a)). This means that acetylation of Tyr and Ser residues decreases hydration of Tyr residue in the non-crystalline domain of RA13CSF powder slightly. Changes in the Ala Cβ peaks, especially sharp peaks at 16.8 ppm assigned to random coil conformation were also smaller for RA13CSF powder (Fig. 3) compared with those for R13CSF powder (Fig. 5) among four kinds of

Fig. 5. Expanded 13C solid-state NMR spectra of non-acetylated [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF powders after methanol treatment. (a) 13C RINEPT (hydrated), (b) 13C DD/MAS (hydrated), (c) 13C CP/MAS (hydrated) and (d) 13C CP/MAS (dry) NMR spectra.

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T. Asakura et al. / International Journal of Biological Macromolecules xxx (xxxx) xxx 13 C solid-state NMR spectra. This means that acetylation of SF powder reduces the hydration effect of Ala residues.

3.4. Stress-strain curves of acetylated and non-acetylated SF fibers Next, the fiber formation of acetylated SF was tried by solution spinning process in order to elucidate the effect of acetylation in the fiber form of SF [44]. In our previous papers [47–50], we reported the stress-strain curves of regenerated SF fibers prepared by using HFIP as a spinning solvent and methanol as a coagulation solvent by changing the draw ratios from 1.5 to 3.0 in the preparation process. The tensile strength (MPa) and elongation-at-break (%) of the regenerated SF fibers were 162 ± 44, 60 ± 30 for 1.5×, 293 ± 47, 28 ± 6 for 2.0×, 306 ± 61, 25 ± 4 for 2.5× and 408 ± 80, 21 ± 3 for 3.0×, respectively. Namely, with increasing the draw ratios, the tensile strength increased and elongation-at-break decreased. The tensile strength of the regenerated fibers (3.0×) was slightly higher than those of native SF fibers where the tensile strength (MPa) and elongation-at-break (%) were 398 ± 51 and 27 ± 0.8, respectively. Therefore, in this paper, the acetylated and non-acetylated SF fibers were prepared by using HFIP as a spinning solvent and methanol as a coagulation solvent by changing the draw ratios from 3.0 to 3.5.

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Table 3 The physical properties of the acetylated and the non-acetylated SF samples.

Powder Water absorption rate/% Fiber Draw ratio Diameter/μm Breaking strength/MPa Elongation at break/% Primary contraction/% Secondary contraction/%

Acetylated

Non-acetylated

25.7

48.8

3x 26.3 ± 0.2 277 ± 9 17 ± 5 8.9 ± 0.6

3.5x 24.3 ± 0.2 377 ± 14 14 ± 4

4.7 ± 0.4

3x 53.4 ± 0.3 384 ± 8 18 ± 2 18.8 ± 0.4 14.1 ± 0.2

3.5x 51.0 ± 0.3 430 ± 10 15 ± 2

Fig. 6 shows the stress-strain curves of (a) acetylated SF fibers with draw ratios, 3× and 3.5×, and (b) non-acetylated SF fibers with draw ratios, 3× and 3.5×. The tensile strength and elongation-at-break of these fibers were summarized in Table 3 together with the diameters. The diameters (μm), tensile strengths (MPa) and elongation-at-breaks (%) of non-acetylated fibers were 53.4, 384 ± 8, 18 ± 2 (3.0×) and 51.0, 430 ± 10, 15 ± 2 (3.5×), respectively, which were almost a similar trend to the previous data reported by us [50]. On the other hand, those values

Fig. 6. Stress-strain curves of regenerated fibers of (a) acetylated and (b) non-acetylated SFs after stretching (3×) and (3.5×).

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T. Asakura et al. / International Journal of Biological Macromolecules xxx (xxxx) xxx

Fig. 7. Dimensional stabilities of regenerated fibers of (a) acetylated and (b) non-acetylated SF after immersing in water repeatedly. L0:Ldry0, L1:Lhydrated1, L2:Ldry2, L3:Lhydrated3 L4:Ldry4 L5: Lhydrated5 L6:Ldry6.

Fig. 8. Expanded 13C solid-state NMR spectra of regenerated fibers of A. acetylated and B. non-acetylated SFs. (a) 13C RINEPT (hydrated), (b) 13C DD/MAS (hydrated), (c) 13C CP/MAS (hydrated) and (d) 13C CP/MAS (dry) spectra.

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T. Asakura et al. / International Journal of Biological Macromolecules xxx (xxxx) xxx

of acetylated fibers were 26.3, 277 ± 9 and17 ± 5 (3.0×), and 24.3, 377 ± 14 and 14 ± 4 (3.5×), respectively. The tensile strength increases remarkably by change in the draw ratio from 3× to 3.5× as well as the case of non-acetylated SF fiber. Thus, compared to non-acetylated fibers, the diameters were almost half and the tensile strengths were lower although the elongation-at-breaks were almost the same for acetylated fibers. In addition, the variation in data became a little larger for acetylated SF fiber. Because of the increased hydrophobic character of acetylated SF fiber compared with that of non-acetylated SF fiber, the acetylated fiber extruded from the nozzle tended to float immediately in the MeOH coagulation bath. 3.5. Dimensional stabilities of acetylated and non-acetylated SF fibers after water immersion treatment As listed in Table 3, the water absorption rate was 48.8% for nonacetylated SF powder and 25.7% for acetylated SF powder. Thus, the water absorption decreased remarkably by acetylation as is expected. In order to suppress the effect of water on SF fiber and to keep the dimensional stability, acetylation of the SF fiber is considered to be one of the effective treatments [11]. Thus, the dimensional stabilities were examined for the acetylated SF fibers. As described in the Materials and methods, we defined the primary contraction rate of the fiber as the change in length when the fibers were first immersed in water, expressed by the following formula, (Lhydrated1 − Ldry0) / Ldry0. After the fibers were dried again, the change in length was also observed repeatedly after repeating dry and hydrated process of the fibers, and the averaged value was defined as the secondary contraction rate. These two contraction rates were used to evaluate the dimensional stabilities of the fibers. Fig. 7 shows changes in length of the (a) acetylated and (b) non-acetylated fibers after these water treatment experiments. The primary and secondary contraction ratios were 18.0% and 14.1 ± 0.2 for non-acetylated SF fiber, and 8.9% and 4.7 ± 0.4 for acetylated SF fiber. Thus, these remarkably decreases of the two contraction rates suggest that the acetylation is a very effective treatment to keep the dimensional stabilities of the fibers in water. 3.6. 13C solid-state NMR spectra of acetylated and non-acetylated SF fibers in the dry and hydrated states As mentioned above, the combination of 13C RINEPT NMR, 13C CP/ MAS NMR and 13C DD/MAS NMR analyses is very effective to elucidate the structure and dynamics of acetylated and non-acetylated SF fibers in the dry and hydrated states. We applied them for natural abundance SF fibers. Although it is difficult to discuss about amorphous domain such as Tyr residue in SF because of low S/N ratio, the NMR analyses of two fibers are still effective. Fig. 8 shows the 13C solid-state NMR spectra (0–80 ppm) of A, acetylated and B, non-acetylated SF fibers in the dry and hydrated states, i.e., (a) 13C RINEPT, (b) 13C DD/MAS, (c) 13C CP/MAS spectra in the hydrated state, and (d) 13C CP/MAS spectrum in the dry state. When we compared these naturally abundant spectra with those of 13C labeled spectra as shown in Figs. 3 and 5, it is noted that difference in the effect of water on the structure and dynamics between acetylated and non-acetylated 13C SF powder was still kept in the naturally abundant SF spectra of the fibers. Namely, small peaks of Ala Cα, Gly Cα, and Ser Cα and Ser Cβ carbons could be observed in Figs. 8(B)(a) and (b), but these peaks could not be observed in Fig. 8 (A)(a) and (b). The Ala Cβ sharp peak (random coil) could be observed in both Fig. 8(A) and (B) although the peak intensity of the 13C RINEPT spectrum (Fig. 8(A)(a)) was very small. The small acetyl methyl peak was also observed in Fig. 8(a). 4. Conclusion Acetylation is one of the excellent chemical modification methods to increase hydrophobicity of SF. Actually, a remarkable improvement of

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the dimensional stability of SF in water was accomplished by acetylation in this work. However, the microscopic interaction between acetylated SF and water is not currently well understood on a molecular level. For the first time, we could clarify the structure and dynamics of the acetylated SF from the crystalline and non-crystalline domains independently in the presence of water using the combination of three kinds of 13 C NMR techniques, 13C RINEPT, 13C CP/MAS and 13C DD/MAS NMR together with the 13C selective labeling of SF. The mobilities of Tyr and Ala residues in the amorphous region of acetylated 13C-labeled SF powder increased slightly and only very small amounts of the Ala residue with high mobility was observed by hydration. On the other hand, there are essentially no effect of water on the β-sheet region of these residues and acetylated Ser residues. A similar tendency toward the effect of hydration for acetylated SF powder was observed for acetylated SF fiber. However, the information on the hydration structure of acetylated SF-water system and also the dynamics of water molecules hydrated to the acetylated SF are lacking. Hereafter, we will try to clarify the former hydration structure using latest ultrafast-1H MAS experiments [57,58] and also the latter dynamics of water molecules using 2H solution relaxation NMR study of 2H2O in the acetylated SF-2H2O system [25,26]. Acknowledgement T.A. acknowledges support by a Japan Society for the Promotion of Science (JSPS) KAKENHI, Grant-in-Aid for Scientific Research (C), Grant Number JP19K05609. References [1] T. Asakura, Y. Suzuki, S. Kametani, Silk, Encycl. Polym. Sci. Technol. (2018) https:// doi.org/10.1002/0471440264.pst339.pub2. [2] B. Kundu, N.E. Kurland, S. Bano, C. Patra, F.B. Engel, V.K. Yadavalli, S.C. Kundu, Silk proteins for biomedical applications: bioengineering perspectives, Prog. Polym. Sci. 39 (2014) 251–267, https://doi.org/10.1016/j.progpolymsci.2013.09.002. [3] L.-D. Koh, Y. Cheng, C.-P. Teng, Y.-W. Khin, X.-J. Loh, S.-Y. Tee, M. Low, E. Ye, H.-D. Yu, Y.-W. Zhang, M.-Y. Han, Structures, mechanical properties and applications of silk fibroin materials, Prog. Polym. Sci. 46 (2015) 86–110, https://doi.org/10.1016/j. progpolymsci.2015.02.001. [4] A.E. Thurber, F.G. Omenetto, D.L. Kaplan, In vivo bioresponses to silk proteins, Biomaterials 71 (2015) 145–157, https://doi.org/10.1016/j.biomaterials.2015.08.039. [5] A.B. Tamara, E. DeSimone, T. Scheibel, Biomedical applications of recombinant silkbased materials, Adv. Mater. 30 (2018), 1704636. https://doi.org/10.1002/adma. 201704636. [6] C. Holland, K. Numata, J. Rnjak-Kovacina, F.P. Seib, The biomedical use of silk: past, present, future, Adv. Healthc. Mater. (2018), 1800465. https://doi.org/10.1002/ adhm.201800465. [7] T. Asakura, T. Miller, Biotechnology of Silk, Springer Netherlands, Dordrecht, 2014https://doi.org/10.1007/978-94-007-7119-2. [8] T. Asakura, T. Tanaka, R. Tanaka, Advanced silk fibroin biomaterials and application to small-diameter silk vascular grafts, ACS Biomater. Sci. Eng. (2019) https://doi.org/ 10.1021/acsbiomaterials.8b01482. [9] M.J. John, R.D. Anandjiwala, Recent developments in chemical modification and characterization of natural fiber-reinforced composites, Polym. Compos. 29 (2008) 187–207, https://doi.org/10.1002/pc.20461. [10] G. Li, H. Liu, T. Li, J. Wang, Surface modification and functionalization of silk fibroin fibers/fabric toward high performance applications, Mater. Sci. Eng. C. 32 (2012) 627–636, https://doi.org/10.1016/j.msec.2011.12.013. [11] M. Haque, M. Zaman, M.H. Rahaman, M. Hossain, M. Maniruzzaman, Thermal and tensile mechanical behavior of acetic anhydride treated silk fibres, Int. J. Mater. Sci. Appl. 3 (2014) 106–110, https://doi.org/10.11648/j.ijmsa.20140303.16. [12] A. Percot, P. Colomban, C. Paris, H.M. Dinh, M. Wojcieszak, B. Mauchamp, Water dependent structural changes of silk from Bombyx mori gland to fibre as evidenced by Raman and IR spectroscopies, Vib. Spectrosc. 73 (2014) 79–89, https://doi.org/10. 1016/j.vibspec.2014.05.004. [13] C. Mo, P. Wu, X. Chen, Z. Shao, The effect of water on the conformation transition of Bombyx mori silk fibroin, Vib. Spectrosc. 51 (2009) 105–109, https://doi.org/10. 1016/j.vibspec.2008.11.004. [14] F. Paquet-Mercier, T. Lefèvre, M. Auger, M. Pézolet, Evidence by infrared spectroscopy of the presence of two types of β-sheets in major ampullate spider silk and silkworm silk, Soft Matter 9 (2013) 208–215, https://doi.org/10.1039/C2SM26657A. [15] X. Hu, D. Kaplan, P. Cebe, Dynamic protein–water relationships during β-sheet formation, Macromolecules 41 (2008) 3939–3948, https://doi.org/10.1021/ ma071551d. [16] K. Numata, T. Katashima, T. Sakai, State of water, molecular structure, and cytotoxicity of silk hydrogels, Biomacromolecules 12 (2011) 2137–2144, https://doi.org/10. 1021/bm200221u.

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Please cite this article as: T. Asakura, H. Matsuda and A. Naito, Acetylation of Bombyx mori silk fibroin and their characterization in the dry and hydrated states using 13C solid-state NMR..., , https://doi.org/10.1016/j.ijbiomac.2019.11.116