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Ling and H. Tang, Polym. Chem., 2017, DOI: 10.1039/C7PY00170C.

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 Page 1 of 32 Polymer Chemistry
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DOI: 10.1039/C7PY00170C

Thermo and pH Dual Responsive Polypeptide Derived from “Clickable”

Poly(γ-3-methylthiopropyl-L-glutamate)
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Polymer Chemistry Accepted Manuscript

Chenglong Ge, Liang Zhao, Ying Ling, and Haoyu Tang*

Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry

of Education, Key Laboratory of Polymeric Materials and Application Technology of

Hunan Province, Key Laboratory of Advanced Functional Polymer Materials of

Colleges and Universities of Hunan Province, College of Chemistry, Xiangtan

University, Xiangtan, Hunan, 411105, China

Correspondence to: Haoyu Tang (Email: htang@[Link])

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ABSTRACT: Poly(γ-3-methylthiopropyl-L-glutamate) (PMTPLG) with “clickable”

thioether groups can be readily prepared from n-butylamine initiated ring-opening

polymerization of γ-3-methylthiopropyl-L-glutamic acid based N-carboxyanhydride

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(MTPLG-NCA). Polypeptides bearing sulfonium moieties and different alkyl (i.e.,

Polymer Chemistry Accepted Manuscript

methyl, n-butyl, and propargyl) pendants were prepared via the alkylation of

PMTPLG. They are able to show upper critical solution temperature (UCST)-type

thermoresponsive properties in methanol or ethanol depending on the alkyl pendants

and counter-anions. Polypeptides bearing sulfonium linkages, ammonium pendants

and tetrafluoroborate (BF4-) counter-anions (PPLG-MSEA-BF4) were prepared via the

copper-mediated [2+3] alkyne-azide 1,3-dipolar cycloaddition of polypeptides bearing

sulfonium moieties and propargyl pendants and subsequently ion-exchange reaction.

They showed UCST-type thermo and pH dual responsiveness in aqueous solutions.

Sharp solution phase separation of PPLG-MSEA-BF4 happened in a very small pH

range (∆pH = 0.05) when pH increased from 7.37 to 7.42 as suggested by dynamic

light scattering (DLS). The UCST-type phase transition temperature (Tpt) of

PPLG-MSEA-BF4 increased by 20 °C as the pH increased from 7.42 to 7.50 as

revealed by variable-temperature UV-vis spectroscopy. Increasing polymer or NaBF4

concentrations or decreasing NaCl concentration can further increase the Tpt.

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INTRODUCTION

Stimuli-responsive polymers have received much recent attention for their

interesting conformational or chemical changes in response to external triggers,1 such

as temperature,2-4 pH,5 light,6,7 and other stimuli. They have showed great potential in
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Polymer Chemistry Accepted Manuscript

various applications, including smart membranes,8 sensors,9,10 tunable catalysis,11

drug delivery,12,13 and tissue engineering.14,15 Among all external triggers, temperature

and pH are the most studied because that they are the most common environment

stimulation. Thermoresponsive polymers can be classified into two types, namely

polymers with a lower critical solution temperature (LCST) that undergo solution

phase separation at an elevated temperature,2 and polymers with an upper critical

solution temperature (UCST) that undergo solution phase separation at a reduced

temperature.3 The development of UCST-type thermoresponsive polymers and

understanding their structure-property relationship have currently gained increasing

attention.16,17 Moreover, thermo and pH dual responsive polymers have also been

developed toward realization of more sophisticated and useful materials.18-24

Generally, thermo and pH responsive moieties were incorporated in one polymer

chain by copolymerization method to achieve dual responsiveness. Nevertheless,

UCST-type thermo and pH dual responsive polymers are far less than their LCST

counterparts.

Polypeptides represent promising mimics of natural peptides or proteins with many

attractive properties, including biocompatibility, ordered conformations, and unique

self-assembly structures.25-28 Various stimuli-responsive polypeptides have been

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developed to simulate the intelligent biological systems and be readily used in various

biotechnological and biomedical applications.29,30 While they can be directly prepared

by ring-opening polymerization (ROP) of side-chain modified N-carboxyanhydride

(NCA) monomers,31 stimuli-responsive polypeptides with more complex functional

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Polymer Chemistry Accepted Manuscript

pendants still require postpolymerization of reactive polypeptides.32-35 Recently,

“clickable” polymers bearing thioether groups have gained increasing attention for

their unique ability to yield electropositive sulfonium moieties and versatile pendants

in one synthetic step.36-41 The thioether groups can be readily modified via oxidation

which endows polymers with redox responsive property42,43 and alkylation with alkyl

halides or epoxides.36-41 For example, Deming and co-workers have demonstrated that

poly(L-methionine) can be readily used for “click”-type side-chain modification via

alkylation with high efficiency and chemoselectivity.36,38 They have also developed

poly(S-alkyl-L-homocysteine)s with multiresponsive properties, such as thermo and

redox responsiveness.37 While sulfonium-based polymers have demonstrated

promising potentials as nonviral nucleic acid delivery vehicles,44 siRNA delivery

agents,45 cell penetrating materials,46 and so forth, stimuli-responsive polymers based

on sulfonium moieties have been less investigated.

In this contribution, we report a new “clickable” polypeptide, namely

poly(γ-3-methylthiopropyl-L-glutamate) (PMTPLG) that can undergo high efficient

alkylation to yield various polypeptides bearing sulfonium moieties and alkyl

pendants. It is worth to note that the term “clickable” used in this contribution is to

reflect a quantitative side-chain modification of thioether groups, which is different

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from the original definition of click reactions coined by Sharpless. UCST-type

thermoresponsive polypeptides bearing sulfonium moieties in alcoholic solvents have

been achieved by adjusting the alkyl pendants and counter-anions. Thermo and pH
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dual responsive polypeptides bearing sulfonium linkages and ammonium pendants

Polymer Chemistry Accepted Manuscript

have also been prepared from PMTPLG. The sulfonium linkages and ammonium

pendants endow the polymer with UCST-type thermo and pH responsiveness,

respectively, which is distinct from previously reported thermo and pH dual

responsive copolymers in terms of the polymer structures. The high efficient

alkylation of thioether groups provides more precise synthesis with regard to the

sequence of repeating units and higher density of functional groups which enable

sharp transitions as compared to copolymerization method. To the best of our

knowledge, this is the first example of sulfonium-based UCST-type thermoresponsive

or thermo and pH dual responsive polymers.

EXPERIMENTAL SECTION

Materials. 3-Methylthiopropanol (98%), triphosgene (98%), iodomethane (99.5%),

1-bromobutane (98%), propargyl bromide (99%), 2-chloroethylamine hydrochloride

(98%), copper bromide (CuBr, 99%), deuterium oxide (D2O, D.99.9%),

N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA, 99%), NaI (98%), and

NaBF4 (98%) were purchased from Energy Chemical. Anhydrous tetrahydrofuran

(THF, 99%) and N,N-dimethylformamide (DMF, 99.9%) were dried over molecular

sieves before use. Chloroform-d (CDCl3, D.99.8%) + silver foil was purchased from

Cambridge Isotope Laboratories, Inc. Sodium azide (NaN3, 99.5%), dimethyl

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sulfoxide-d6 (DMSO-d6, 99.9 atom % D, contains 0.03% v/v TMS), and L-glutamic

acid (99%) were purchased from Sigma-Aldrich. Deionized water (DI-H2O) was

obtained from Aquapro AR1-100L-P11 water-purification system (Ever Young

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Enterprises Development Co., Ltd., P. R. China).

Polymer Chemistry Accepted Manuscript

Instrumentation. 1H nuclear magnetic resonance (1H NMR) spectra were recorded

on a Bruker ARX400 MHz spectrometer at room temperature. Chemical shifts (δ)

were reported in the units of ppm and referenced to the protonic impurities. The

polymer solution with concentrations of ~15 mg·mL-1 for 1H NMR test prepared by

directly mixing and shaking at room temperature. Gel permeation chromatography

(GPC) measurements were performed on a PL-GPC120 setup equipped with a column

set consisting of two PL gel 5 µm MIXED-D columns (7.5 mm × 300 mm, effective

molar mass range of 0.2-400.0 kg·mol-1) and PL-RI differential refractive index (DRI)

detector. DMF containing 0.01 M LiBr was used as the eluent at 80 °C at a flow rate

of 1.0 mL·min-1. Narrowly distributed polystyrene standards in the molar mass range

of (0.5-7.5) × 104 kg·mol-1 (PSS, Mainz, Germany) were utilized for calibration.

Polymer solutions for the GPC test with a concentration of 5 mg·mL-1 in 0.01 M

LiBr/DMF were prepared by directly mixing and shaking at room temperature. FTIR

spectra were recorded on a Thermo Scientific Nicolet 6700 FTIR spectrometer

equipped with an attenuated total reflection (ATR) sample holder. Solid samples were

placed on the diamond crystal window and pressed with a metal probe. Spectral

measurements were carried out in the transmittance mode (scan range = 4000-600

cm-1, resolution = 2 cm-1, number of scans = 2, 25 °C). Ultraviolet–visible (UV–vis)

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spectra were measured using an Agilent Cary 100 spectrometer. The polymer

solutions were prepared by stirring at temperatures above respective UCST-type phase

transition temperature (Tpt), and then placed in a quartz cell with a path length of 1.0
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cm. The transmittances of solutions were collected at the wavelength of 500 nm. The

Polymer Chemistry Accepted Manuscript

solutions were cooled from high temperatures to low temperatures for UCST-type

transitions with initial stabilization of 20 min. The transmittance at 500 nm was

recorded at every 2 °C decrement after a 5 min thermal equilibration at each

measurement. Tpt was determined at 50% of transmittance in the cooling cycle. For

ionic strength dependent studies, the salt concentration was adjusted by the addition

of NaX (X = Cl or BF4) and mixing at the temperature above respective Tpt. For pH

dependent studies, the solution pH was adjusted by the addition of aqueous HCl or

NaOH. The solutions pH values were determined by a PHS-3C pH meter (Shanghai

INESA Scientific Instrument Co., Ltd., P. R. China). Dynamic light scattering (DLS)

was conducted on a Zetasizer Nano ZS90 (Malvern Instruments, Ltd., UK) with a

He-Ne laser (λ = 633 nm) at a scattering angle of 90o (25 °C).

Scheme 1. Synthetic Route of PMTPLG and Polypeptides Bearing Various

Alkylmethylsulfonium Moieties

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Synthesis of γ-3-Methylthiopropyl-L-glutamate (MTPLG). L-Glutamic acid (2.0

g, 13.6 mmol) and 3-methylthiopropanol (3 mL) were mixed in a round-bottom flask

(100 mL) which was placed in an ice-water bath, followed by dropwise of sulfuric
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acid (0.8 mL) via a glass dropper (Scheme 1). The mixture was stirred at room

Polymer Chemistry Accepted Manuscript

temperature for 24 h. Milky white solution gradually became transparent. Then, the

solution was neutralized by triethylamine. Addition of isopropanol (50 mL) yielded a

white solid precipitate which was collected by filtration. Recrystallization in

ethanol/DI-H2O (V/V = 10/1) afforded the final product as a white flaky solid (2.6 g,

44% yield).

Synthesis of γ-3-Methylthiopropyl-L-glutamic Acid Based

N-Carboxyanhydride (MTPLG-NCA). MTPLG (0.75 g, 3.19 mmol), triphosgene

(0.38 g, 1.28 mmol), and anhydrous THF (10 mL) were mixed in a round-bottomed

flask (25 mL) under nitrogen (Scheme 1). The mixture was stirred at room

temperature for 24 h. Removal of the solvent under vacuum yielded a white solid

product which was then dissolved in ethyl acetate (50 mL) and washed with a cold

saturated NaHCO3/H2O solution and NaCl/H2O solution for 3 times. The organic

layer was separated and dried over anhydrous Na2SO4 at 0 °C. Filtration and

evaporation afforded a solid product which was then purified by silica gel

chromatography with ethyl acetate as the eluent. Finally, recrystallization in ethyl

acetate/hexane (V/V = 1/10) at -20 °C for 3 times afforded the final product as a white

crystalline solid (0.63 g, 76% yield). 1H NMR (CDCl3, δ, ppm): 6.68 (s, 1H, NH),

4.41 (t, 1H, -NHCHCH2-), 4.20 (t, 2H, -CH2CH2O-), 2.56 (t, 4H, -SCH2CH2- and

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-CH2CH2O-), 2.09-2.26 (m, 2H, -CH2CH2O-), 2.10 (s, 3H, -SCH3), 1.95 (m, 2H,

-CH2CH2CH2-).

Synthesis of Poly(γ-3-methylthiopropyl-L-glutamate) (PMTPLG). A

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representative polymerization of MTPLG-NCA is as follows (Scheme 1).

Polymer Chemistry Accepted Manuscript

MTPLG-NCA (210 mg, 0.804 mmol) was dissolved in anhydrous DMF (2 mL) in a

vial under nitrogen. A measured volume of n-butylamine/DMF stock solution (CI =

0.1 mol·L-1, 161 µL, 0.016 mmol) was subsequently added with a syringe. The

reaction mixture was stirred at room temperature for 72 h. A white product was

precipitated from DI-H2O (20 mL) and collected by centrifugation and dried under

vacuum at room temperature (145 mg, 83% yield). 1H NMR (CDCl3, δ, ppm): 8.38 (s,

1H, NH), 4.18 (m, 2H, -CH2CH2O-), 3.96 (m, 1H, -CH2CHNH-), 2.65 (m, 2H,

-CH2CH2CH-), 2.55 (t, 2H, -CH2CH2S-), 2.29 (m, 2H, -CH2CH2CH-), 2.10 (s, 3H,

-SCH3-), 1.91 (m, 2H, -CH2CH2CH2-).

Synthesis of Poly(γ-3-propyl-L-glutamate) Dimethylsulfonium Iodide

Conjugate (PPLG-DMS-I). PMTPLG (20 mg, 0.092 mmol of sulfur) was dissolved

in DMF (3 mL), followed by addition of iodomethane (79 mg, 0.552 mmol, Scheme

1). The reaction was covered with aluminum foil and stirred at room temperature for

60 h. Then, the mixture was diluted with DI-H2O (6 mL) and dialysis against DI-H2O

in a dialysis bag with a 3000 molecular weight cut-off (MWCO) for three days.

Removing the solvent under vacuum afforded a glassy solid (28 mg, 86% yield). 1H

NMR (D2O, δ, ppm): 4.20 (m, 2H, -CH2CH2O-), 4.31 (m, 1H, -CH2CHNH-), 2.49 (m,

2H, -CH2CH2CH-), 3.37 (t, 2H, -CH2CH2S-), 2.17 (m, 2H, -CH2CH2CH-), 2.88 (d,

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6H, -SCH3-), 1.98 (m, 2H, -CH2CH2CH2-).

Synthesis of Poly(γ-3-propyl-L-glutamate) Methylbutylsulfonium Bromide

Conjugate (PPLG-MBS-Br). PMTPLG (40 mg, 0.184 mmol of sulfur) was

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dissolved in DMF (1 mL) under nitrogen, followed by addition of acetonitrile (1 mL)

Polymer Chemistry Accepted Manuscript

solution of NaI (83 mg, 0.553 mmol) and 1-bromobutane (252 mg, 1.84 mmol,

Scheme 1). The mixture was stirred at 80 °C for 72 h. Then, any precipitate was

removed by filtration. NaBr (10 eqv.) was added into the reaction solution, followed

by stirring at room temperature for 60 min. After that, the insoluble salts were

removed by filtration. The clear reaction solution was added into diethyl ether (20 mL)

to precipitate the product. The product was further purified by dissolving in acetone,

filtration to remove any salt residue, and dried under vacuum (51 mg, 78% yield). 1H

NMR (CDCl3, δ, ppm): 8.35 (s, 1H, NH), 4.15 (m, 2H, -CH2CH2O-), 3.95 (m, 1H,

-CH2CHNH-), 2.73 (m, 7H, -CH2CH2S- and -SCH3), 2.56 (m, 2H, -CH2CH2CH-),

2.25 (m, 2H, -CH2CH2CH-), 1.89 (m, 2H, -CH2CH2CH2-), 1.55 (m, 2H,

-CH2CH2CH3), 1.40 (m, 2H, -CH2CH2CH3), 0.91 (t, 3H, -CH2CH2CH3).

Synthesis of Poly(γ-3-propyl-L-glutamate) Methylpropargylsulfonium Bromide

Conjugate (PPLG-MPS-Br). PMTPLG (40 mg, 0.184 mmol of sulfur), propargyl

bromide (132 mg, 1.104 mmol) was dissolved in DMF (3 mL), followed by addition

of trifluoroacetic acid (126 mg, 1.104 mmol) to promote the reaction (Scheme 1). The

mixture was covered with aluminum foil and stirred at 60 °C for 60 h. Then,

trifluoroacetic acid and excess propargyl bromide were removed under vacuum at

room temperature, followed by addition of NaBr (10 eqv.) and stirred for 60 min.

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Filtration and precipitation from diethyl ether (40 mL) afforded a sticky solid which

was dissolved in acetone, followed by filtration and evaporation to afford a brown

solid (55 mg, 89% yield). 1H NMR (CDCl3, δ, ppm): 8.36 (s, 1H, NH), 4.18 (m, 2H,
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-CH2CH2O-), 3.95 (m, 1H, -CH2CHNH-), 2.64 (m, 2H, -CH2CH2CH-), 2.73 (s, 5H,

Polymer Chemistry Accepted Manuscript

-CH2CH2S- and -CH3), 2.16 (m, 2H, -CH2CH2CH-), 1.94 (m, 2H, -CH2CH2CH2-),

3.26 (s, 2H, -SCH2C-), 2.31 (s, 1H, -CH).

Synthesis of Poly(γ-3-propyl-L-glutamate) Dimethylsulfonium

Tetrafluoroborate Conjugate (PPLG-DMS-BF4). PPLG-DMS-I (130 mg, 0.362

mmol of sulfonium) was dissolved in DI-H2O (5 mL), followed by addition of NaBF4

(398 mg, 3.62 mmol, Scheme 1). The solution was stirred at room temperature for 30

min. Then, it was dialysis against DI-H2O for 24 h in a 3000 MWCO dialysis bag.

The addition of NaBF4 and subsequent dialysis against DI-H2O were repeatedly for

three times. Removing the solvent under vacuum afforded a glassy solid (104 mg, 90%

1
yield). H NMR (D2O, δ, ppm): 4.25 (m, 2H, -CH2CH2O-), 4.36 (m, 1H,

-CH2CHNH-), 2.53 (m, 2H, -CH2CH2CH-), 3.40 (t, 2H, -CH2CH2S-), 2.20 (m, 2H,

-CH2CH2CH-), 2.92 (d, 6H, -SCH3-), 2.05 (m, 2H, -CH2CH2CH2-).

Synthesis of Poly(γ-3-propyl-L-glutamate) Methylbutylsulfonium Iodide

Conjugate (PPLG-MBS-I) and Poly(γ-3-propyl-L-glutamate)

Methylbutylsulfonium Tetrafluoroborate Conjugate (PPLG-MBS-BF4).

PPLG-MBS-Br (50 mg, 0.141 mmol of sulfonium) was dissolved in DMF (2 mL),

followed by addition of NaBF4 (163 mg, 1.49 mmol) or NaI (223 mg, 1.49 mmol).

The reaction mixture was stirred at 60 °C for 30 min. After cooling down to room

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temperature, inorganic salts were precipitated and removed by filtration. Then, the

solution was added into diethyl ether (40 mL) to afford a solid product. The product

was repeatedly reacted with NaI or NaBF4 in DMF (5 mL) and precipitated from
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diethyl ether (40 mL). The product was further purified by dissolving in chloroform,

Polymer Chemistry Accepted Manuscript

filtration to remove any salt residue, and dried under vacuum (PPLG-MBS-I: 47 mg,

84% yield; PPLG-MBS-BF4: 41 mg, 81% yield). 1H NMR (CDCl3, δ, ppm) of

PPLG-MBS-I: 8.42 (s, 1H, NH), 4.17 (m, 2H, -CH2CH2O-), 3.95 (m, 1H,

-CH2CHNH-), 2.77 (m, 7H, -CH2CH2S- and –SCH3), 2.55 (m, 2H, -CH2CH2CH-),

2.25 (m, 2H, -CH2CH2CH-), 1.91 (m, 2H, -CH2CH2CH2-), 1.55 (m, 2H,

-CH2CH2CH3), 1.40 (m, 2H, -CH2CH2CH3), 0.92 (t, 3H, -CH2CH2CH3). 1H NMR

(CDCl3, δ, ppm) of PPLG-MBS-BF4: 8.37 (s, 1H, NH), 4.18 (m, 2H, -CH2CH2O-),

3.96 (m, 1H, -CH2CHNH-), 2.80 (m, 7H, -CH2CH2S- and -SCH3), 2.56 (m, 2H,

-CH2CH2CH-), 2.22 (m, 2H, -CH2CH2CH-), 1.92 (m, 2H, -CH2CH2CH2-), 1.56 (m,

2H, -CH2CH2CH3), 1.41 (m, 2H, -CH2CH2CH3), 0.92 (t, 3H, -CH2CH2CH3).

Synthesis of Poly(γ-3-propyl-L-glutamate) Methylpropargylsulfonium Iodide

Conjugate (PPLG-MPS-I) and Poly(γ-3-propyl-L-glutamate)

Methylpropargylsulfonium Tetrafluoroborate Conjugate (PPLG-MPS-BF4).

PPLG-MPS-I and PPLG-MPS-BF4 were prepared using the same procedure as

PPLG-MBS-X (X = I or BF4). 1H NMR (CDCl3, δ, ppm) of PPLG-MPS-I: 8.35 (s, 1H,

NH), 4.21 (m, 2H, -CH2CH2O-), 3.97 (m, 1H, -CH2CHNH-), 2.62 (m, 2H,

-CH2CH2CH-), 2.76 (s, 5H, -CH2CH2S- and -CH3), 2.18 (m, 2H, -CH2CH2CH-), 1.97

(m, 2H, -CH2CH2CH2-), 3.27 (s, 2H, -SCH2C-), 2.31 (s, 1H, -CH). 1H NMR (CDCl3,

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δ, ppm) of PPLG-MPS-BF4: 8.37 (s, 1H, NH), 4.19 (m, 2H, -CH2CH2O-), 3.97 (m,

1H, -CH2CHNH-), 2.65 (m, 2H, -CH2CH2CH-), 2.74 (s, 5H, -CH2CH2S- and -CH3),

2.17 (m, 2H, -CH2CH2CH-), 1.95 (m, 2H, -CH2CH2CH2-), 3.26 (s, 2H, -SCH2C-),
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2.33 (s, 1H, -CH).

Polymer Chemistry Accepted Manuscript

Synthesis of 2-Azidoethylamine. 2-Chloroethylamine hydrochloride (2.00 g, 17.0

mmol) and sodium azide (1.68 g, 25.5 mmol) were dissolved in DI-H2O (10 mL). The

reaction solution was stirred at 75 °C for 96 h. After cooling down to room

temperature, the pH value of the aqueous solution was adjusted to 10-11 with sodium

hydroxide. Then, the solution was extracted with diethyl ether (5 × 30 mL). The

organic layer was combined and dried over anhydrous Na2SO4 at 0 °C. Filtration and

evaporation afforded a clear liquid product (0.96 g, 65% yield). 1H NMR (CDCl3, δ,

ppm): 2.87 (t, 2H, -CH2CH2N3), 3.36 (t, 2H, -CH2CH2N3).

Scheme 2. Synthetic Route of Polypeptides Bearing Methylsulfonium Linkages and

Ethylammonium Pendants

Synthesis of Poly(γ-3-propyl-L-glutamate) Bearing Methylsulfonium Chloride

and Ethylammonium Chloride (PPLG-MSEA-Cl). PPLG-MPS-Br (100 mg, 0.298

mmol of propargyl groups), 2-azidoethylamine (70 mg, 0.596 mmol), and

N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA, 36 µL, 0.149 mmol) were

dissolved in DMF (5 mL) in a glass vial (10 mL) under nitrogen, followed by addition

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of CuBr (22 mg, 0.149 mmol, Scheme 2). The reaction solution was stirred at room

temperature for 24 h and quenched by exposure to air. The product was purified by

dialysis against NaCl aqueous solution (0.31 mol·L-1) in a 3000 MWCO dialysis bag
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for 48 h (HClaq was added to protonate amino groups and remove copper ions, pH =

Polymer Chemistry Accepted Manuscript

5-6) and subsequently dialysis against DI-H2O for 24 h. Removing the solvent under

vacuum afforded a glassy solid (126 mg, 95% yield). 1H NMR (DMSO-d6, δ, ppm):

8.35 (s, 1H, NH), 8.10 (s, 1H, -NCH=C-), 4.64 (s, 2H, -NCH2CH2-), 4.04 (s, 3H,

-SCH2C- and -NHCHCH2-), 3.78 (s, 2H, -CH2CH2O-), 3.34 (s, 2H, -NCH2CH2-), 2.48

(s, 5H, -CH3 and -SCH2CH2-), 2.32 (s, 2H, -CHCH2CH2-), 1.97 (m, 2H,

-CHCH2CH2-), 1.83 (m, 2H, -CH2CH2CH2-).

Synthesis of Poly(γ-3-propyl-L-glutamate) Bearing Methylsulfonium

Tetrafluoroborate and Ethylammonium Tetrafluoroborate (PPLG-MSEA-BF4).

PPLG-MSEA-Cl (132 mg, 0.637 mmol of chloride ions) was dissolved in DI-H2O (5

mL), followed by addition of NaBF4 (653 mg, 5.94 mmol, Scheme 2). The product

precipitated immediately after addition of NaBF4. The reaction mixture was stirred at

room temperature for 30 min, followed by dialysis against DI-H2O in a 3000 MWCO

dialysis bag for 24 h. The product was repeatedly stirred in the aqueous solution of

NaBF4 and dialysis against DI-H2O. Removing the solvent under vacuum afforded a

glassy solid (157 mg, 95% yield). 1H NMR (DMSO-d6, δ, ppm): 7.93 (s, 1H, NH),

8.02 (s, 1H, -NCH=C-), 4.55 (t, 2H, -NCH2CH2-), 4.05-4.21 (m, 3H, -SCH2C- and

-NHCHCH2-), 3.77 (s, 2H, -CH2CH2O-), 3.31 (t, 2H, -NCH2CH2-), 2.48 (s, 5H, -CH3

and –SCH2CH2-), 2.32 (s, 2H, -CHCH2CH2-), 1.97 (m, 2H, -CHCH2CH2-), 1.83 (m,

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2H, -CH2CH2CH2-).

RESULTS AND DISCUSSION

γ-3-Methylthiopropyl-L-glutamic acid based N-carboxyanhydride (MTPLG-NCA)

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was synthesized in two steps from L-glutamic acid (Scheme 1). Specifically,

Polymer Chemistry Accepted Manuscript

3-methylthiopropanol reacted with L-glutamic acid in the presence of sulfuric acid via

a monoesterification to afford γ-3-methylthiopropyl-L-glutamate (MTPLG).

MTPLG-NCA was prepared via a cyclization of MTPLG with triphosgene at room

temperature in anhydrous THF. White crystalline MTPLG-NCA (Figure S1a of

Supporting Information) can be readily obtained by recrystallization in ethyl

acetate/hexane solvent mixture. The molecular structure of MTPLG-NCA was

verified by 1H NMR (Figure 1a) and FTIR (Figure S1b of Supporting Information).

Figure 1. 1H NMR spectra of (a) MTPLG-NCA and (b) PMTPLG in CDCl3, and (c)
PPLG-DMS-BF4 in D2O (asterisks signify DMF signals).
Ring-opening polymerization (ROP) of MTPLG-NCA was conducted at room

temperature in anhydrous DMF using n-butylamine as the initiator to yield

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poly(γ-3-methylthiopropyl-L-glutamate) (PMTPLG, Scheme 1). An aliquot of the

polymerization solution was taken for analysis of monomer conversion by FTIR. The

conversion can be calculated according to the intensity variations with reaction time

of vC=O bands at around 1863 and 1776 cm-1 (Figure S1b of Supporting Information).
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Polymer Chemistry Accepted Manuscript

PMTPLG was precipitated in DI-H2O when the monomer conversion was 100%.

PMTPLG samples with various number average molar masses (Mns) and narrow

molar mass distribution (Mw/Mn) can be readily obtained by adjusting the initial

[M]0/[I]0 ratios (Table 1). Both Mns and degree of polymerizations (DPs) were

determined by GPC (Figure 2) and 1H NMR analysis based on the end-group method

(Figure S2 of Supporting Information). Specifically, DP (NMR) was calculated from

the proton integration ratios of methylene groups from the side-chains (e.g., He) and

from the n-butyl end-groups (e.g., Hc’). Deviation of the DP (NMR) from the

theoretical values (Table 1) likely due to the activated monomer mechanism (AMM)

of ROP47 and/or the presence of residual impurities (e.g., water) in the monomers.

Table 1. Ring-opening polymerization of MTPLG-NCA in DMFa

Mnb c DPd
entry [M]0/[I]0 Mw /M n
Calcde GPCf NMRg GPCf NMRg
1 20 4350 5050 4350 1.19 23 20
2 50 10860 9700 6520 1.13 45 30
3 80 17380 15200 11950 1.07 70 55
4 100 21730 21700 13250 1.08 100 61
a
Polymerizations were conducted in DMF at room temperature for 72 h using
n-butylamine as the initiator. b Number-average molar mass. c Molar mass distribution
determined by GPC. d Degree of polymerization. e Theoretical polymer molar masses
were calculated from the [M]0/[I]0 and the conversion which was 100% as monitored
by FTIR. f Mn or DP determined by GPC. g Mn or DP determined by 1H NMR.
The molecular structure of PMTPLG was characterized by 1H NMR (Figure 1b)

and FTIR (Figure 3a). The chemical shifts in the 1H NMR spectrum were consistent
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with PMTPLG structure and similar to MTPLG-NCA, except that the proton signal of

–NH groups (6.69 ppm, Figure 1a) for MTPLG-NCA disappeared due to the ROP

while the proton signal of amide bonds for PMTPLG appeared at 8.37 ppm (Figure
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1b). In the FTIR spectrum, PMTPLG showed characteristic bands at 3280, 2934, 1728,

Polymer Chemistry Accepted Manuscript

1652, and 1547 cm-1, corresponding to vN-H, vC-H, vC=O, amide I, and amide II,

respectively.

Figure 2. GPC chromatographs of PMTPLG samples prepared from ring-opening

polymerizations with different initial monomer to initiator ratios ([M]0/[I]0).

Figure 3. FTIR spectra of (a) PMTPLG and polypeptides bearing various sulfonium
moieties and alkyl pendants, and (b) polypeptides bearing sulfonium linkages and
ammonium pendants in the solid state.
The “click”-type side-chain modification of PMTPLG was based on alkylation of

thioether groups with alkyl iodides or bromides under mild conditions (Scheme 1).

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Specifically, PMTPLG reacted with iodomethane at room temperature in DMF to

yield poly(γ-3-propyl-L-glutamate) dimethylsulfonium iodide conjugate

(PPLG-DMS-I). It can also react with 1-bromobutane or propargyl bromide in the

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presence of NaI or trifluoroacetic acid to yield poly(γ-3-propyl-L-glutamate)

Polymer Chemistry Accepted Manuscript

methylbutylsulfonium bromide conjugate (PPLG-MBS-Br) or

poly(γ-3-propyl-L-glutamate) methylpropargylsulfonium bromide conjugate

(PPLG-MPS-Br), respectively. In order to study the effect of counter-anions on their

solution properties, polypeptides bearing sulfonium pendants and various

counter-anions, namely poly(γ-3-propyl-L-glutamate) dimethylsulfonium

tetrafluoroborate conjugate (PPLG-DMS-BF4), poly(γ-3-propyl-L-glutamate)

methylbutylsulfonium iodide conjugate (PPLG-MBS-I), poly(γ-3-propyl-L-glutamate)

methylbutylsulfonium tetrafluoroborate conjugate (PPLG-MBS-BF4),

poly(γ-3-propyl-L-glutamate) methylpropargylsulfonium iodide conjugate

(PPLG-MPS-I), and poly(γ-3-propyl-L-glutamate) methylpropargylsulfonium

tetrafluoroborate conjugate (PPLG-MPS-BF4) were prepared via ion-exchange

reactions (Scheme 1). The reactions were conducted in DI-H2O or DMF depending on

polymer solubility and with the presence of corresponding salts, such as NaI and

NaBF4. To achieve a quantitative conversion, the resulting polypeptides were

repeatedly reacted with the corresponding salts.

The molecular structures of the resulting polypeptides bearing sulfonium and

various alkyl pendants were also characterized by 1H NMR (Figure 1c, Figure 4a,

Figure S3-S4 of Supporting Information, and Experimental Section) and FTIR (Figure

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3). A significant peak shift of Hg on methyl groups indicated the success of alkylation

of thioether groups. Specifically, the Hgs for PMTPLG (Figure 1b), PPLG-DMS-I

(Figure S3 of Supporting Information), PPLG-MBS-Br (Figure S4 of Supporting

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Information), and PPLG-MPS-Br (Figure 4a) were 2.10, 2.77, 2.72, and 2.73 ppm,

Polymer Chemistry Accepted Manuscript

respectively. 1H NMR analysis also suggested high grafting efficiency of methyl,

n-butyl, and propargyl groups. For example, the grafting efficiency of methyl or

propargyl groups were 95% and 98%, respectively, which can be calculated according

to the integrations of Hg (Figure S3 of Supporting Information) or Hg + Hh (Figure 4a)

and Hf on methylene groups next to sulfonium moieties. Moreover, the peak shift of

Hg on methyl groups from 2.77 ppm for PPLG-DMS-I to 2.84 ppm for

PPLG-DMS-BF4 indicated the success of ion-exchange reaction. In the FTIR spectra

(Figure 3), the appearance of B-F vibration mode (vB-F) at around 1020 cm-1 for

PPLG-DMS-BF4, PPLG-MBS-BF4, and PPLG-MPS-BF4 further confirmed the

success of ion-exchange reactions.

Figure 4. 1H NMR spectra of (a) PPLG-MPS-Br in CDCl3, (b) PPLG-MSEA-Cl and

(c) PPLG-MSEA-BF4 in DMSO-d6.

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PPLG-MPS-Br with propargyl pendant can be further modified via a

copper-mediated [2+3] alkyne-azide 1,3-dipolar cycloaddition. 2-Azidoethylamine

was used to react with PPLG-MPS-Br under mild condition in DMF with the presence
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of CuBr and N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA) under nitrogen

Polymer Chemistry Accepted Manuscript

(Scheme 2). The intermediate was further treated with NaCl and HCl aqueous

solutions to ion-exchange and protonate the amino pendants (Scheme 2). The product,

namely poly(γ-3-propyl-L-glutamate) bearing methylsulfonium chloride and

ethylammonium chloride (PPLG-MSEA-Cl) was subsequently treated with NaBF4 via

the ion-exchange reaction to yield poly(γ-3-propyl-L-glutamate) bearing

methylsulfonium tetrafluoroborate and ethylammonium tetrafluoroborate

(PPLG-MSEA-BF4). The molecular structures of PPLG-MSEA-X (X = Cl, BF4) were

confirmed by 1H NMR (Figure 4b-4c) and FTIR (Figure 3b). In the 1H NMR

spectrum of PPLG-MSEA-Cl (Figure 4b), the chemical shifts at 8.34, 8.11, 4.64, and

3.78 ppm corresponded to the proton signals of ammonium chloride, Hi on triazole

groups, Hj and Hk on ethidene groups, respectively. The 1:2 integration ratio of Hi and

Hf (Figure 4b) indicated a quantitative grafting efficiency. In the 1H NMR spectrum of

PPLG-MSEA-BF4 (Figure 4c), noticeable peak shifts of ammonium groups (-NH3+)

and water residue as compared to PPLG-MSEA-Cl indicated the success of

ion-exchange reaction.

The solubility of PMTPLG and polypeptides bearing various sulfonium moieties

was tested in various solvents (Table S1 of Supporting Information). All polymers

showed good solubility in polar aprotic solvents (e.g., DMF and DMSO). Nonionic

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PMTPLG and polypeptides bearing sulfonium moieties and long alkyl pendants (e.g.,

n-butyl and propargyl) showed good solubility in less polar solvents (e.g., chloroform

and THF), yet poor solubility in water. To the contrary, polypeptides bearing
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sulfonium moieties and methyl pendants or ammonium salts showed poor solubility in

Polymer Chemistry Accepted Manuscript

chloroform and THF, but water-solubility. Polypeptides bearing sulfonium moieties

and alkyl pendants showed upper critical solution temperature (UCST)-type

thermoresponsive properties in methanol (MeOH), except PPLG-MBS-BF4 and

PPLG-MPS-BF4 bearing long alkyl pendants and more hydrophobic counter-anions

(i.e., BF4-). Additionally, PPLG-MBS-Br and PPLG-MPS-Br bearing more

hydrophilic counter-anions (i.e., Br-) showed UCST-type thermoresponsive properties

in ethanol (EtOH). It is believed that the mechanism of UCST-type phase transitions

in alcoholic solvents mainly originated from the H-bonding interactions between

hydroxyl groups of the solvents and polar moieties and linkages of the polymers (e.g.,

ester bonds).4,48 Herein, we assumed that the electrostatic interactions between

sulfonium and counter-anions and hydrophilic interactions between alkyl groups of

both polymer and solvent molecules should have also played important roles in

polymer solubility and thermoresponsiveness.

Variable-temperature UV-vis spectroscopy was used to study the UCST-type

solution phase behaviors in alcoholic solvents (Figure 5). The UCST-type phase

transition temperature (Tpt) was determined from 50% of the transmittance in the

cooling cycle. In MeOH, the Tpts of the thermoresponsive polypeptides bearing

sulfonium moieties were in the temperature range of 13.3-34.5 °C. It increased with

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decreasing the alkyl chain length or hydrophilicity of the counter-anions. For example,

PPLG-MBS-Br bearing the longest alkyl pendants and most hydrophilic

counter-anions (i.e., Br-) showed the lowest Tpt in MeOH. While the effect of alkyl
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chain length on the Tpts in EtOH was similar to that in MeOH, the Tpts of

Polymer Chemistry Accepted Manuscript

PPLG-MBS-Br and PPLG-MPS-Br in EtOH were 27.4 °C and 33.7 °C, respectively,

which were higher than respective Tpts in MeOH likely due to the increase of

hydrophobicity and decrease of polarity of the solvent.48 Moreover, PPLG-MBS-Br

and PPLG-MPS-Br showed slightly sharper solution phase transition in MeOH than

in EtOH (Figure 5). However, more research efforts are demanded to understand this

phenomenon.

Figure 5. Plots of transmittance at λ = 500 nm versus temperature for (a) methanol

solutions of PPLG-DMS-X (X = I, BF4), PPLG-MPS-X (X = Br, I), and
PPLG-MBS-X (X = Br, I), and (b) ethanol solutions of PPLG-MBS-Br and
PPLG-MPS-Br (polymer concentration = 1 mg·mL-1).
It is known that poly(L-glutamic acid) (PLGA) and poly(L-lysine) (PLL) are

pH-responsive polypeptides.29,30 Their pH-induced coil-to-helix transitions arise from

the protonation and deprotonation of carboxyl or amino groups. Therefore, we

reasoned that polypeptides bearing sulfonium linkages and ammonium pendants (e.g.,

PPLG-MSEA-X, X = Cl, BF4) may also show pH-responsiveness. While

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PPLG-MSEA-Cl with Cl- counter-anions showed no solution phase separation in a

wide pH-range (pH = 1-14). It is interesting to observe that PPLG-MSEA-BF4 with

more hydrophobic counter-anions showed noticeable pH-induced solution phase

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separation after a slightly pH variation (∆pH = 7.42-7.37 = 0.05, Figure 6a). The

Polymer Chemistry Accepted Manuscript

solution phase separation should have resulted from the decreased hydrophilicity and

charge repulsion between ammonium and sulfonium moieties by formation of amino

groups and consequently forming intermolecular electrostatic interactions between

sulfonium and counter-anions. This has been suggested by DLS analysis (Figure 6b).

At pH ≤ 7.37, the aggregation size of PPLG-MSEA-BF4 was no more than 15.1 nm.

However, it significantly increased as the pH slightly increased (∆pH = 0.05) and

gradually tended to a constant value (Figure 6c).

Figure 6. (a) Optical images of PPLG-MSEA-BF4 aqueous solutions with different

pH values at room temperature (polymer concentration = 5 mg·mL-1). (b) DLS size
distribution plots of PPLG-MSEA-BF4 aqueous solutions with different pH values at
25 °C (polymer concentration = 1 mg·mL-1). (c) Plots of aggregation size versus pH
value for PPLG-MSEA-BF4 aqueous solutions with increasing the pH values (The
error bar was calculated by performing DLS experiments in triplicate).
Previously, we have demonstrated that polypeptides bearing ionic liquid pendants

were able to show reversible UCST-type solution behaviors in DI-H2O due to a

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mainly electrostatic interaction mechanism.32,34 We reasoned that PPLG-MSEA-BF4

at basic conditions (e.g., pH ≥ 7.42) may also exhibit a reversible UCST-type

thermoresponsiveness in a heating/cooling cycle (Figure S5 of Supporting

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Information). The Tpt of PPLG-MSEA-BF4 aqueous solution was significantly

Polymer Chemistry Accepted Manuscript

affected by pH variation (Figure 7, Figure S6 of Supporting Information). It increased

by 20 °C as the pH increased from 7.42 to 7.50 while the increment of Tpt was around

10 °C per 0.1 pH value as the pH increased from 7.50 to 7.80. The UCST-type phase

behavior of PPLG-MSEA-BF4 also showed a good reversibility in terms of pH

variation (Figure 7b). The difference value of Tpt (∆Tpt) in a pH increasing/decreasing

cycle was below 4 °C.

Figure 7. (a) Plots of transmittance at λ = 500 nm versus temperature for

PPLG-MSEA-BF4 aqueous solutions with increasing the pH values. (b) Plots of Tpt or
∆Tpt versus pH value for PPLG-MSEA-BF4 aqueous solutions with increasing and
decreasing the pH values (polymer concentration = 5 mg·mL-1).
Given that the electrostatic interactions could be affected by the concentration of

ions and ionic strength, we further investigated the polymer and salt concentration

dependence studies on Tpts of PPLG-MSEA-BF4 aqueous solution at constant pH

value (i.e., pH = 7.50) by variable-temperature UV-vis spectroscopy (Figure S7-S8 of

Supporting Information). The Tpt increased with increasing of polymer concentrations

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due to the increased polymer-polymer interactions per volume, which was similar to

our previous report.34 The increment of Tpt resulted from the change of polymer

concentration was about 4 °C per 1 mg·mL-1 and it was more pronounced at low
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polymer concentrations (Figure 8a). In the salt concentration dependent study, the Tpt

Polymer Chemistry Accepted Manuscript

increased as NaBF4 concentration increased or as NaCl concentration decreased.

Similar results have been reported and the authors have suggested that the Tpt was

affected by ion-exchange reaction between ionic liquid pendants and salts.49 The Salt

concentration dependent study can be further explained by the Hofmeister effect as

follows. Cl- has a salting in effect, namely, the Tpt decreased with increasing NaCl salt

concentration. To the contrary, BF4- has a salting out effect, namely, the Tpt increased

with increasing NaBF4 salt concentration, which was likely caused by destabilizing

the H-bonds between water and the polar groups of the polymer and consequently

resulted in higher Tpt to hydration.50

Figure 8. Plots of Tpt versus (a) polymer concentration or (b) salt concentration for
PPLG-MSEA-BF4 in DI-H2O or in NaX (X = Cl or BF4) aqueous solutions,
respectively (polymer concentration = 5 mg·mL-1).

CONCLUSIONS

We have demonstrated that “clickable” poly(γ-3-methylthiopropyl-L-glutamate)

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(PMTPLG) with thioether groups can be readily prepared from n-butylamine initiated

ring-opening polymerization of γ-3-methylthiopropyl-L-glutamic acid based

N-carboxyanhydride (MTPLG-NCA). Alkylation of PMTPLG with iodomethane,

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1-bromobutane, or propargyl bromide can yield polypeptides bearing sulfonium

Polymer Chemistry Accepted Manuscript

moieties and various alkyl pendants with high grafting efficiency (≥95%). The

presence of sulfonium moieties enables the resulting polypeptides with UCST-type

thermoresponsiveness in alcoholic solvents. The UCST-type phase transition

temperature (Tpt) can be tuned by adjusting the counter-anions via the ion-exchange

reactions. Polypeptides bearing sulfonium linkages, ammonium pendants and

tetrafluoroborate (BF4-) counter-anions (PPLG-MSEA-BF4) can be readily prepared

via the copper-mediated [2+3] alkyne-azide 1,3-dipolar cycloaddition and

ion-exchange reaction. It showed good water-solubility at pH ≤ 7.37 and became

insoluble at pH ≥ 7.42 due to the deprotonation of ammonium pendants and formation

of intermolecular electrostatic interactions between sulfonium and counter-anions. At

pH ≥ 7.42, PPLG-MSEA-BF4 showed reversible UCST-type phase behaviors mainly

due to the electrostatic interaction mechanism. The Tpt was significantly affected by

pH, polymer and salt concentrations. It increased with the increasing of pH values,

polymer or NaBF4 concentrations. Nevertheless, it decreased with the increasing of

NaCl concentration. In summary, the high efficient postpolymerizations including

alkylation of thioether groups of PMTPLG, copper-mediated [2+3] alkyne-azide

1,3-dipolar cycloaddition, and ion-exchange reaction enable a facile preparation of

sulfonium-based stimuli-responsive polypeptides with tunable properties. Our results

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provide a new platform to develop functional polymeric materials and the

structure-property relationship shall shed some light on future molecular design of

stimuli-responsive polymers.
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ASSOCIATED CONTENT

Polymer Chemistry Accepted Manuscript

Supporting Information Available. Optical image and FTIR spectrum of

MTPLG-NCA (Figure S1), 1H NMR spectra of PMTPLG samples prepared from

ring-opening polymerizations with different initial monomer to initiator ratios (Figure

S2), PPLG-DMS-I (Figure S3), and PPLG-MBS-Br (Figure S4), solubility of

PMTPLG and polypeptides bearing various sulfonium moieties (Table S1), and

variable-temperature UV-vis analyses of pH-dependent study (Figure S5),

reversibility of UCST-type phase behavior (Figure S6), polymer concentration study

(Figure S7), and salt concentration study (Figure S8) for PPLG-MSEA-BF4.

ACKNOWLEDGEMENTS

This work is supported by Hunan Provincial Natural Science Foundation of China

(2016JJ3110) and Xiangtan University start-up fund (12QDZ06).

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Graphical Abstract

Thermo and pH dual responsive polypeptides bearing sulfonium linkages and

ammonium pendants were prepared from “clickable” thioether-containing
polypeptides.
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Polymer Chemistry Accepted Manuscript