1 Highly ordered polypeptide with UCST phase-separation behavior

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Highly ordered polypeptide with UCST phase-separation behavior Sotaro Kuroyanagi, Naohiko Shimada, Shota Fujii, Tadaomi Furuta, Atsushi Harada, Kazuo Sakurai, and Atsushi Maruyama J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b10168 • Publication Date (Web): 31 Dec 2018 Downloaded from http://pubs.acs.org on December 31, 2018

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Journal of the American Chemical Society

Highly ordered polypeptide with UCST phase-separation behavior Sotaro Kuroyanagia, Naohiko Shimadaa, Shota Fujiib, Tadaomi Furutaa, Atsushi Haradac, Kazuo Sakuraib*, Atsushi Maruyamaa* aDepartment

of Life Science and Technology, Tokyo Institute of Technology, 4259 B-57, Nagatsuta,

Yokohama 226-8501, Japan E-mail: [email protected] bFaculty

of Environmental Engineering, The University of Kitakyushu, 1-1 Hibikino, Wakamatsu,

Kitakyushu City, Fukuoka, 808-0135 Japan cDepartment

of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1

Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan

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Abstract Manipulating phase separation structures of thermo-responsive polymers will enhance the usefulness of structure-controllable materials in the fields such as drug delivery and tissue engineering.

However,

behaviors of upper critical solution temperature (UCST) have been less investigated so far, despite of the importance of UCST.

Here, we examined two citrulline-based polypeptides, poly(D-ornithine-co-D-

citrulline) (PDOC) and poly(DL-ornithine-co-DL-citrulline) (PDLOC), to investigate how stereoregularity of the polypeptides influences UCST behavior, in addition with poly(L-ornithine-co-L-citrulline) (PLOC) previously studied.

Homochiral PLOC and PDOC showed phase separation temperatures (Tps) higher

than that of racemic PDLOC.

Moreover, PDLOC underwent liquid to coacervate phase separation at Tp,

whereas PLOC and PDOC underwent liquid to solid-like aggregation transitions.

From the structural

point of view, circular dichroism and small-angle X-ray scattering measurements revealed that homochiral PLOC and PDOC polypeptides formed α-helical structures, and assembled into a regular hexagonal lattice upon phase separation.

Interactions between the pendent ureido groups of homochiral POCs appear to

play pivotal roles in helical folding and assembly into the hexagonal structure. response to biodegradation was confirmed for both PLOC and PDLOC. considerably influenced by phase-separated structures.

In addition, Tp change in The biodegradability was

These findings of UCST-type POCs in this study

would provide important insights into structure-controllable and thermo-responsive biomaterials. 2 ACS Paragon Plus Environment

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1. Introduction Many thermo-responsive polymers have lower critical solution temperature (LCST) behavior under physiologically relevant pH and salt conditions.

The synthetic LCST-type polymer poly(N-

isopropylacrylamide) has been widely used in biomaterial applications such as biomolecule purification,1 thermoresponsive nanoparticles,2,3 and cell sheet engineering4,5 because it exhibits a soluble to insoluble transition between room temperature and body temperature.6,7

LCST-type thermoresponsive polymers

based on biocompatible and biodegradable polypeptides have also been synthesized.

Elastin like

peptides (ELPs), composed of repeats of the pentapeptide Val-Pro-Gly-X-Gly (the X stands for any amino acid except for proline), show LCST-type behavior accompanied by a conformational change from a random coil to a β-turn structure.8

Fusions of ELPs with proteins of interest prepared by genetic

engineering have potential for use in drug delivery9 and tissue engineering.10

Despite these broad uses

of thermo-responsive polypeptides, structural study of phase behavior is still few. There are few examples of synthetic polymers or polypeptides with upper critical solution temperature (UCST) behavior under physiologically relevant conditions.11–14

Recently, Deng et al. reported that -

helical polypeptides bearing 1-alkylimidazolium show UCST-type behavior driven by electrostatic interactions in water.15

Quiroz et al. reported resilin-like polypeptides that have UCST-type solution 3 ACS Paragon Plus Environment


behavior under physiologically relevant conditions.16

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Although the relationship between the secondary

structure and the UCST-type solution behavior has not been characterized, secondary structure has been shown to dramatically affect polymer self-assembly.17–20

For example, for amphiphilic polymers used

for drug delivery or as scaffolds, the rigid -helical structures are critical for self-assembly and function.21,22

The combination of peptides with well-defined assembling properties and thermo-

responsivity raises possibility to design novel biomaterials. We have reported that poly(allylurea)23–25 and poly(2-ureidoethyl methacrylate) copolymers exhibit UCST-type thermo-responsiveness driven by strong hydrogen bonding interactions of ureido groups under physiologically relevant conditions.26

We also reported that poly(ornithine-co-citrulline) (POC, Figure

1a) shows UCST-type behavior under physiological relevant conditions (Figure 1b).23 naturally occurring amino acid, citrulline, which has a ureido side chain.

POC contains a

The phase separation

temperature (Tp) of POC increased with increasing citrulline content and the molecular weight of main chain.

In our body, the conversion of arginine to citrulline dramatically affects the structure and function

of proteins, leading several human diseases, such as rheumatoid arthritis and Alzheimer’s disease.27–29 Citrulline residue were reported to act as a structural denaturant of natural proteins. study of POC having many citrulline residues is of interest.

Thus, structural

In this study, we prepared POCs with

homochiral or racemic backbones to explore the effect of the stereoregularity on thermoresponsiveness of 4 ACS Paragon Plus Environment

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the citrulline polypeptides.

Figure 1. (a) The structural formula of POC. (b) Photographs of L102k92.5 (1.0 mg/mL) in 10 mM HEPESNaOH (pH 7.5) containing 150 mM NaCl at 10 °C and 40 °C.

2. Experimental 2.1 Materials Poly(L-ornithine hydrobromide) (Mw 30,000~70,000), poly(DL-ornithine hydrobromide) (Mw >30,000), and Ni(COD)2 were purchased from Sigma-Aldrich. purchased from Watanabe Chemical Industries.

H-Orn(Z)-OH and H-D-Orn(Z)-OH were

Triphosgene, hydrogen bromide (30% in acetic acid),

and trifluoroacetic acid were purchased from Tokyo Chemical Industries. Tetrahydrofuran (dehydrated, stabilizer free), hexane (dehydrated), and 1,4-dioxane (dehydrated) were purchased from Kanto Chemical Companies.

2,2’-Bipyridyl, diethyl ether and potassium cyanate were purchased from Wako Pure



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Chemical Industries. 2.2 Syntheses of poly(ornithine-co-citrulline) Poly(L-ornithine) (PLO), poly(D-ornithine) (PDO), and poly(DL-ornithine) were synthesized by the ring opening polymerization of N-carboxyanhydride (NCA) amino acid derivatives initiated by a transition metal as previously described.30,31

In a 250-mL round-bottom flask H-Orn(Z)-OH (2.00 g, 7.51 mmol)

and triphosgene (1.11 g, 3.76 mmol) were stirred under vacuum for 2 h at room temperature. mL) was added under nitrogen, and the mixture was stirred at 50 °C for 3 h. turned clear, which indicated the completion of the reaction. hexane.

THF (20

The suspension gradually

The solution was precipitated with 60 mL

The crude NCA was purified by recrystallization two times using a mixture of hexane and THF

resulting in Orn(Z)-NCA in crystalline form (2.01 g, 91.6%). dissolved in 1,4-dioxane (15 mL) under nitrogen.

Orn(Z)-NCA (1.5 g, 5.13 mmol) was

The Orn(Z)-NCA solution was added to a

bpyNi(COD)/dioxane solution (1.13 mL, 0.1 or 0.2 mmol/mL).

The reaction mixture was stirred for 24

h at 25 °C and then precipitated with 75 mL diethyl ether and dried in vacuo.

In order to remove the

protecting group, polyOrn(Z) was dissolved in trifluoroacetic acid (15 mL), to which HBr (30% in acetic acid, 0.87 mL) was added.

After stirring for 3 h at room temperature, the final product was isolated by

precipitating in excess diethyl ether, redissolving in distilled water, dialyzing against distilled water, and freeze-drying. 6 ACS Paragon Plus Environment

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Molecular weights of the polymers containing poly(L-ornithine hydrobromide) and poly(DL-ornithine hydrobromide), purchased from Sigma-Aldrich, were determined by gel permeation chromatography (GPC) using OHpak SB-804 HQ and 806 M HQ columns (Shodex) on a LC-2000Plus series (Jasco) system equipped with a multi-angle static light scattering (MALS) detector (DAWN-HELEOS II, Wyatt). Polymer samples were dissolved in deionized water to a concentration of 10 mg/mL.

The polymers were

eluted with 0.5 M acetic acid and 0.2 M sodium nitrate (0.8 mL/min) at 25 °C. Polymerization of H-D-Orn(Z)-OH and H-DL-Orn(Z)-OH and characterization of the resulting poly(D-ornithine hydrobromide) and poly(DL-ornithine hydrobromide) were carried out using similar procedures. To obtain POC various amounts of potassium cyanate were added to poly(ornithine hydrobromide) in 1 M imidazole-HCl buffer (pH 6.5), and then the mixtures were incubated at 50 °C for 48 h. polymers were purified by dialysis against water and then lyophilized.

The

The polymers (2.5 mg/mL in D2O

containing 1% NaOD) were characterized by 1H NMR (Bruker Avance 400) at 60 °C. 2.3 Transmittance measurements POC solutions in pure water and D2O were diluted with buffer to result in solutions of 10 mM HEPES/NaCl (pH 7.5), 150 mM NaCl.

Transmittance at 500 nm of POC solutions at various

concentrations at temperatures from 50 to 5 °C was measured on a Shimadzu UV-1650PC UV−visible 7 ACS Paragon Plus Environment


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spectrophotometer equipped with a Peltier temperature controller at scanning rate of 1 °C/min.

Tp was

defined as an initial point of temperature where the transmittance first began to drop. 2.4 Optical microscopic observation POC solutions in D2O were diluted with buffer to result in solutions of 10 mM HEPES/NaCl (pH 7.5), 150 mM NaCl.

Optical images of 1.0 mg/mL copolymer solutions were recorded below the UCSTs

using a Biozero microscope (Keyence). 2.5 Transmission electron microscopic observation Transmission electron microscopy (TEM) images were taken using a JEOL JEM-1400Plus electron microscope with an accelerating voltage of 100 kV.

A drop of each POC solutions (5.0 mg/mL in 10

mM HEPES-NaOH buffer (pH 7.5) containing 150 mM NaCl) was mounted on a carbon-coated Cu grid and negatively stained with 2% methylamine tungstate (Nanoprobes, Stony Brook, N.Y.) twice, followed by suctioning off the excess fluid using a filter paper. 2.6 Circular dichroism measurements Circular dichroism (CD) spectra of polypeptides (0.5 mg/mL) in 10 mM Tris/HCl (pH 7.5) containing 150 mM NaCl were measured on a J-820 CD spectropolarimeter (Jasco) equipped with a Peltier temperature controller.

Samples were placed in 10-mm quartz cells, and each spectrum was collected

from 200 to 250 nm at a scan rate of 100 nm/min with a response time of 2 s. 8 ACS Paragon Plus Environment

Data were averaged over

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5 accumulations.

Temperature dependence of mean molar residue ellipticity at 222 nm and 208 nm

([θ]222 and [θ]208, respectively) were measured in the step scan mode of the J-820 spectropolarimeter at a scan rate of 1 °C/min with a response time of 2 s. 2.7 Molecular dynamics simulations All the molecular dynamics (MD) simulations were conducted using AMBER 2016 MD software.32 -Helical structures of 24-residue PLO and poly(L-citrulline) (PLC) were constructed initially, and force field parameters of each residue were determined by employing the general AMBER force field (GAFF) with the AM1-BCC partial charges.

Each system was fully solvated with explicit solvent (TIP3P water).

The total numbers of atoms were 12,699 and 12,249 for PLO and PLC systems, respectively.

Each system

was energetically minimized for 300 steepest descent steps and equilibrated for 200 ps with positional restraints on the backbone residues under NPT conditions at 300 K and 1 bar.

Finally, a 100-ns

production run was performed for each system. 2.8 Small-angle X-ray scattering measurements Small-angle X-ray scattering measurements were carried out on the BL40B2 beamline at SPring-8, Japan.

A 30 cm × 30 cm imaging plate (Rigaku R-AXIS VII) detector was used and placed 0.7 m away

from the sample.

The wavelengths of the incident beam (λ) were 0.10 and 0.071 nm.

This setup

provided a q range of 1 – 10 nm−1, where q is the magnitude of the scattering vector defined by q = 4π/λ 9 ACS Paragon Plus Environment


sinθ with a scattering angle of 2θ.

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The X-ray transmittance of 10 mg/mL POC samples in 10 mM

HEPES/NaCl (pH 7.5), 150 mM NaCl at various temperatures was measured using ion chambers located before and behind the samples. 2.9 Biodegradation of POCs in vitro Reaction solutions containing 1.0 mg/mL of POCs in 10 mM HEPES/NaOH (pH 7.5), 150 mM NaCl, in the presence or absence of 5 or 50 g/mL Proteinase K from Tritirachium album (Sigma-Aldrich) were incubated at 37 °C or 17 °C for different time intervals. 5 min resulting in denaturation of Proteinase K.

Reactions were stopped by heating at 90 °C for

Transmittance of the samples were analyzed as above.

Samples were also evaluated by 15% SDS-PAGE stained with SimplyBlue SafeStain (Invitrogen).

3. Results & Discussion 3.1 UCST-type behavior of chiral or racemic POCs We previously reported that L isomers of homochiral POC, PLOC, show UCST-type solution behavior.23

Here, homochiral and racemic POCs with various citrulline contents and molecular weights

were prepared (Table 1).

The DL racemic polypeptide, PDLOC, and D homochiral polypeptide, PDOC,

exhibited UCST-type solution behavior under physiologically relevant conditions as well as PLOC.

Tps

of PLOC having similar citrulline content increased with increasing molecular weight (Figure 2a) and 10 ACS Paragon Plus Environment

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reached a plateau around 69 kDa.

PDLOC showed similar molecular weight dependence.

also increased with increasing polymer concentration (Figure S1).

The Tps

However, the concentration

dependence on Tp of POCs is small as compared with other UCST polymers.11-13,15,16

The phase

separation behavior was reversible and had a small hysteresis (approximately 5 °C) between cooling and heating measurements (Figure S2).

We previously reported that the Tps of poly(allylamine-co-allylurea)

polymers in D2O were about 10 °C higher than those in H2O because deuterium substitution of ureido groups enhances hydrogen bonding.23

This deuterium effect was also observed for PDOC and PDLOC

(Figure S3 and Table S1).



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Table 1. Properties of Citrulline Polypeptides Used in This Study Optical activity

Code

M w (kDa)a

In feed b

Citrulline content c (mol %)

T p d (°C) 0.50 mg/mL 1.00 mg/mL

L30k 0

L

30.1

0.0

1 Highly ordered polypeptide with UCST phase-separation behavior

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