Chemical Synthesis of Multiblock Copolypeptides Inspired by Spider

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Chemical Synthesis of Multiblock Copolypeptides Inspired by Spider Dragline Silk Proteins Kousuke Tsuchiya* and Keiji Numata* Enzyme Research Team, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan S Supporting Information *

ABSTRACT: Novel multiblock polypeptides with a structure similar to the unique sequence observed in spider silk proteins (spidroins) were synthesized via a two-step chemical synthesis method, that is, chemoenzymatic polymerization, using papain followed by postpolycondensation. Two types of polypeptide fragments were prepared by chemoenzymatic polymerization: polyalanine as a hard block, which forms β-sheets in the spider silk fibers, and poly(glycinerandom-leucine) as a soft block. These two fragments were ligated by postpolycondensation using polyphosphoric acid as a condensing agent. Wide-angle X-ray diffraction (WAXD) and IR measurements revealed that the resulting multiblock polypeptides formed an antiparallel β-sheet structure with a degree of crystallinity similar to that of spider silk, which resulted in a fibrous morphology. This work provides the first example of a synthetic multiblock polypeptide mimicking the secondary structures of spider silk.

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silk-like materials using chemical synthesis methods. Some bioinspired polymers containing an oligopeptide or polypeptide backbone have been chemically synthesized to exploit the secondary and higher order structures assembled by the polypeptide blocks.9,10 The alternating sequence of polyAla and glycine-rich motifs is essential for achieving the mechanical properties of spider dragline silk because this sequence leads to higher-order structures based on β-sheet nanocrystal domains. However, the synthesis of multiblock polypeptides in one-pot chemical reactions, such as the ring-opening polymerization of Ncarboxyamino acid anhydrides (NCAs), which is a process that is conventionally used for di- or triblock polypeptides, is difficult.11−13 In this study, spider-silk-like multiblock polypeptides were prepared via the two-step synthetic process described in Figure 1. In the first step, chemoenzymatic polymerization was used to synthesize the oligopeptide fragments with a sequence similar to that of spidroins, that is, hard polyAla and soft glycine-rich segments. Recently, we demonstrated that amino acid esters can be polymerized using proteases as catalysts in aqueous media, resulting in a broad range of polypeptide materials.14−19 This chemoenzymatic polymerization proceeds in a facile, costeffective, and environmentally benign manner to provide polypeptide fragments. Consecutively, ligation of these fragments was performed to prepare the multiblock polypeptides, which exhibit a repetitive sequence similar to that of spider silk spidroins. Some techniques, including native chemical ligation20−22 and disulfide coupling,23−25 have been used as synthetic methods for multi-block-like polypeptide materials;

pider silks have attracted increasing attention for their potential applications in multiple fields, including biomaterials and high-strength materials, because they exhibit biocompatibility, biodegradability, lightweight property, high extensibility, and robust mechanical strength. Dragline silk, which is used by spiders to construct the frame of a spider web as well as to hang above the ground, exhibits excellent mechanical properties, such as high tensile strength and extensibility.1,2 The two proteins that mainly compose the dragline silk, major ampullate spidroins 1 (MaSp1) and 2 (MaSp2), possess unique amino acid sequences that establish these exceptional mechanical properties. The primary structures of the spidroins consist of C- and N-terminal domains and a highly repetitive middle domain that consists of alternating polyalanine (polyAla) and glycine-rich segments.3 The former assembles into an antiparallel β-sheet secondary structure that forms the crystal domains in the spider silks and imparts high tensile strength to the silk fiber. The latter is dominated by GGX (G: glycine; X: leucine, tyrosine, or glutamine) motifs with other minor residues, such as serine or valine, and its soft nature imparts elasticity to the spider dragline silk. Spider dragline silk has the potential to be used as a novel functional material in fields requiring biocompatibility and specific mechanical properties. However, harvesting natural spider silk fiber from spiders is difficult because of their low productivity and cannibalistic nature. Genetically recombinant DNA technology has been used to develop microbes capable of biosynthesizing spider-silk-like materials.4−8 Precise control in the synthesis of desired sequences can be achieved, although the costly biosynthesis is inappropriate for mass production. Chemical synthesis of silklike polypeptide materials would be beneficial for industrial manufacturing because it would render the process facile, costeffective, energy efficient, and atom-economical on a large scale. The literature contains no reports on the preparation of spider© XXXX American Chemical Society

Received: January 4, 2017 Accepted: January 10, 2017

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DOI: 10.1021/acsmacrolett.7b00006 ACS Macro Lett. 2017, 6, 103−106

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ACS Macro Letters

Leucine was used as the copolymer counterpart because it, along with alanine and glycine, is frequently found in the primary structure.3 Thepapain-catalyzed polymerization of glycine and leucine ethyl esters was conducted using various feed ratios (Table S1). The formation of poly(Gly-r-Leu) was confirmed by 1H NMR and MALDI-TOF mass spectrometry (Figures S3 and S4). Leucine-rich random polypeptides with an Mn of approximately 1000 g·mol−1 were obtained because the hydrophobic leucine is likely to polymerize faster because of its stronger affinity for papain.26 The postpolycondensation between polyAla (Mn = 810 g·mol−1) and poly(Gly-r-Leu) (Mn = 670 g·mol−1) was conducted using a condensing agent to synthesize multiblock polypeptides, polyAla-b-poly(Gly-r-Leu), as shown in Scheme 1. Because of the poor solubility of the polypeptide fragments in common solvents, a conventional condensation reaction using a carbodiimide condensing agent afforded only low-molecular-weight products (see Supporting Information). Therefore, the condensing agent was changed to PPA and the polycondensation was conducted at elevated temperature. The results of the polycondensation under various conditions are summarized in Table 1. The feed weight ratio of polyAla to poly(Gly-r-Leu) (A/GL) was varied from 50/50 to 14/86, and the resulting polymers showed higher molecular weights than the fragments, indicating that these soft and hard fragments were ligated into the block polypeptide. The weightaverage molecular weight (Mw) increased to 17200 g·mol−1 when N,N-dimethylacetamide (DMAc) was used as the solvent at 140 °C. 1H NMR spectroscopy revealed that all of the polymers exhibited a lower polyAla content compared to the corresponding feeds (Figure S5), which was likely caused by the lower solubility of polyAla fragments compared to poly(Gly-r-Leu). The GPC profiles of the resulting polymers showed a large peak shift to higher molecular weights compared to the polypeptide fragments, indicating that the ligation between polyAla and poly(Gly-r-Leu) occurred to afford multiblock copolymers (Figure S6). The multiblock polypeptides were characterized by wideangle X-ray diffraction (WAXD) analysis, as shown in Figure 2. Three sharp peaks derived from the (020), (210), and (211) planes in an antiparallel β-sheet structure were detected in the WAXD profile of polyAla, which exhibited a degree of crystallinity of 75.9% (Figure 2a).27 Poly(Gly-r-Leu) also showed diffraction peaks, which were possibly derived from an α-helix structure similar to that of polyleucine (Figure 2b).28 Peaks from both polyAla and poly(Gly-r-Leu) were detected in the profiles of the multiblock polypeptides (Figure 2c,d). The intensity of the peaks derived from poly(Gly-r-Leu) decreased substantially, and a large contribution from the polyAla β-sheet crystal was present, resulting in the degree of crystallinity ranging from 10 to 20%. These results indicate that the β-sheet crystal structure from polyAla blocks was formed in the amorphous-rich matrix of the multiblock polypeptides. Given that the degree of crystallinity of spider silks generally ranges from 15 to 25%,29 the content of β-sheet crystal domains in the

Figure 1. Synthetic strategy for multiblock polypeptides that resemble the spider dragline silk proteins, which possess alternately repeating polyalanine and glycine-rich sequences.

however, these approaches require appropriate reactive units at the terminals of the peptide fragments. In contrast, we conducted direct condensation between amino and ester groups using polyphosphoric acid (PPA) as a condensing agent to ligate the hard and soft peptide fragments prepared by chemoenzymatic polymerization. The two polypeptide building blocks of spider-silk-like block copolymers were synthesized by chemoenzymatic polymerization of amino acid esters using papain. PolyAla plays a role in the construction of the β-sheet structure, and poly(glycinerandom-leucine) [poly(Gly-r-Leu)] possesses a primary structure similar to the soft segment between polyAla sequences in the repetitive domain of the spidroin. The papain-catalyzed polymerization of alanine ethyl ester was performed according to a previously reported method.19 The polymerization was conducted in phosphate buffer/methanol at 40 °C and pH 8.0, and polyAla was obtained as a white powder by centrifugation of the precipitate. The chemical structure of polyAla was confirmed by 1H NMR and MALDI-TOF mass spectrometry. We estimated the Mn of polyAla to be 810 g·mol−1 by comparing the integral ratio of the signals between the αproton of repeating units and the terminal ethyl group in the 1 H NMR spectrum (Figure S1). In the MALDI-TOF mass spectrum of polyAla (Figure S2), a series of peaks derived from polyAla with an ethyl ester terminal were observed; the degree of polymerization (DP) was found to range from 5 to 11, which is comparable to that of the polyAla sequence in the spider dragline silks. The other fragment is the glycine-rich soft segment, which contributes to the extendibility of the spider silk. Random copolypeptides consisting of glycine and leucine were designed to imitate the soft segment and were synthesized via the aforementioned chemoenzymatic polymerization method.

Scheme 1. Synthesis of Multiblock Polypeptide by Post-Polycondensation of Polypeptide Fragments

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ACS Macro Letters Table 1. Synthesis of PolyAla-b-Poly(Gly-r-Leu) by Polycondensation of Polypeptide Fragments runa

feed A/GLb

solv

temp (°C)

time (h)

yieldc (%)

Mnd

Mwd

Mw/Mnd

polyAlae (wt %)

1 2 3 4 5 6 7 8 9

50/50 33/67 33/67 14/86 33/67 10/90 50/50 33/67 10/90

NMP NMP NMP NMP NMP NMP DMAc DMAc DMAc

120 120 120 120 140 140 140 140 140

24 24 48 48 48 48 48 48 48

64.7 68.6 70.7 77.9 46.7 49.1 65.1 61.2 63.3

2000 2300 2900 2600 2500 2400 4600 4600 3500

3600 3900 5200 4100 5200 4400 11,300 17,200 6800

1.85 1.72 1.82 1.58 2.11 1.85 2.48 3.77 1.98

24 17 16 8.5 18 6.4 26 21 4.2

a

Polymerization was conducted using polyAla/poly(Gly-r-Leu) (100 mg in total) and PPA (0.15 g) in NMP or DMAc (1 mL) under nitrogen. Weight ratio of polyAla to poly(Gly-r-Leu). cWater-insoluble precipitate was collected by centrifugation. dDetermined by GPC eluted with NMP containing LiBr (10 mM) using polystyrene standards. eWeight ratio of polyAla segments in polyAla-b-poly(Gly-r-Leu) determined by 1H NMR spectra. b

Figure 3. (a) Illustration of the secondary structure of the multiblock polypeptide and (b−d) AFM topographic images of polyAla-bpoly(Gly-r-Leu) with different polyAla contents (b and c: 26 wt %, run 7 in Table 1; d: 21 wt %, run 8 in Table 1).

1535 cm−1 assignable to antiparallel β-sheet structures appeared for the nanofiber of polyAla-b-poly(Gly-r-Leu), in accordance with the peaks of the natural dragline silk. The profiles of the amide II region slightly differed due to the lack of minor amino acid residues such as tyrosine and glutamine in polyAla-bpoly(Gly-r-Leu). This agreement in the IR spectrum strongly supports the results of WAXD analysis. Together with the AFM observation of the nanofibers, we confirmed the formation of antiparallel β-sheet structures in the nanofiber, similar to the spider dragline silks. In conclusion, multiblock polypeptides containing polyalanine and glycine-rich sequences, which are similar primary structures to the spider silk proteins, were synthesized using a two-step chemical synthesis method. Hard and soft polypeptide fragments, polyAla and poly(Gly-r-Leu), respectively, were prepared by chemoenzymatic polymerization using papain and then subjected to the postpolycondensation using PPA to afford the multiblock polypeptides. The ligation of these fragments was confirmed by GPC analysis, and the resulting polymer exhibited weight-average molecular weights as high as 17000 g·mol−1. WAXD measurements revealed that the resulting multiblock polypeptides formed antiparallel β-sheets and amorphous domains that coexisted in the multiblock polypeptide bulk samples, indicating that the ligation of polyAla and poly(Gly-r-Leu) afforded a structure that resembles the secondary structures of native spider silk proteins. Further tuning of the sequences in the fragments and, more

Figure 2. WAXD profiles of (a) polyAla, (b) poly(Gly-r-Leu), and polyAla-b-poly(Gly-r-Leu) with polyAla content of (c) 26 wt % (run 7 in Table 1), and (d) 21 wt % (run 8 in Table 1).

synthesized multiblock polypeptides is comparable to that of native spider silks. Finally, the potential of the multiblock polypeptides to form fibril structures was demonstrated using atomic force microscopy (AFM). The multiblock polypeptides were deposited onto mica substrates and observed by AFM. The AFM topographic images for the two polyAla-b-poly(Gly-rLeu) polypeptides with different polyAla content are shown in Figure 3. The block copolymer with a degree of crystallinity similar to that of natural spider silk exhibits nanofibril-like structures (Figure 3b), whereas large aggregates were observed for the more amorphous copolymer (Figure 3c). These results indicate that the primary structure of the multiblock polypeptides strongly affects their nanoscale morphology. The IR spectra of the nanofiber was compared with that of native spider dragline silk fibers derived from Nephila clavata (Figure S7). The amide I (1700−1600 cm−1) and II (1575−1480 cm−1) regions in the IR spectrum give useful information about the secondary structures of spider silks, because the side chains of amino acid units barely affect the peaks in these regions according to previous reports.5,7 The strong peaks at 1630 and 105

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(21) Hackeng, T. M.; Griffin, J. H.; Dawson, P. E. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 10068−10073. (22) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science 1994, 266, 776−779. (23) Humphrey, J. M.; Chamberlin, A. R. Chem. Rev. 1997, 97, 2243−2266. (24) Amador, R.; Moreno, A.; Valero, V.; Murillo, L.; Mora, A. L.; Rojas, M.; Rocha, C.; Salcedo, M.; Guzman, F.; Espejo, F.; Nũnez, F.; Patarroyo, M. E. Vaccine 1992, 10, 179−184. (25) Patarroyo, M. E.; Amador, R.; Clavijo, P.; Moreno, A.; Guzman, F.; Romero, P.; Tascon, R.; Franco, A.; Murillo, L. A.; Ponton, G.; Trujillo, G. Nature 1988, 332, 158−161. (26) Baker, P. J.; Patwardhan, S. V.; Numata, K. Macromol. Biosci. 2014, 14, 1619−1626. (27) Riekel, C.; Bränden, C.; Craig, C.; Ferrero, C.; Heidelbach, F.; Müller, M. Int. J. Biol. Macromol. 1999, 24, 179−186. (28) Miranda, R.-A.; Finocchio, E.; Llorca, J.; Medina, F.; Ramis, G.; Sueiras, J. E.; Segarra, A. M. Phys. Chem. Chem. Phys. 2013, 15, 15645− 15659. (29) Gosline, J. M.; Guerette, P. A.; Ortlepp, C. S.; Savage, K. N. J. Exp. Biol. 1999, 202, 3295−3303.

importantly, achieving a higher molecular weight by optimizing the condensation conditions will allow these spider-silk-like multiblock polypeptides to be used in bulk materials requiring high strength and toughness.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00006. Experimental details, schemes, 1H NMR spectra, MALDI-TOF mass spectra, GPC chromatograms, and AFM phase images (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge Drs. Hiroyasu Masunaga and Takaaki Hikima for the measurement of synchrotron WAXD at 45XU, SPring-8, Harima, Japan. This work was financially supported by Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT).

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DOI: 10.1021/acsmacrolett.7b00006 ACS Macro Lett. 2017, 6, 103−106