Salt- and pH-Triggered Helix-Coil Transition of Ionic Polypeptides under Physiology Conditions

Article abstract of DOI:10.1021/acs.biomac.8b00204

Controlling the helix-coil transition of polypeptides under physiological conditions is an attractive way toward smart functional materials. Here, we report the synthesis of a series of tertiary amine-functionalized ethylene glycol (EG_x)-linked polypeptide electrolytes with their secondary structures tunable under physiological conditions. The resultant polymers, denoted as P(EG_xDMA-Glu) (x = 1, 2, and 3), show excellent aqueous solubility (>20 mg/mL) regardless of their charge states. Unlike poly-l-lysine that can form a helix only at pH above 10, P(EG_xDMA-Glu) undergo a pH-dependent helix-coil switch with their transition points within the physiological range (pH ～5.3-6.5). Meanwhile, P(EG_xDMA-Glu) exhibit an unusual salt-induced helical conformation presumably owing to the unique properties of EG_x linkers. Together, the current work highlights the importance of fine-tuning the linker chemistry in achieving conformation-switchable polypeptides and represents a facile approach toward stimuli-responsive biopolymers for advanced biological applications.

Full text of DOI:10.1021/acs.biomac.8b00204

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Article
Salt- and pH-Triggered Helix-Coil Transition of
Ionic Polypeptides under Physiology Conditions
Jingsong Yuan, Yi Zhang, Yue Sun, Zhicheng Cai, Lijiang Yang, and Hua Lu
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00204 • Publication Date (Web): 26 Mar 2018
Downloaded from http://pubs.acs.org on March 27, 2018
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Saltꢀ and pHꢀTriggered HelixꢀCoil Transition of Ionic Polypeptides
under Physiology Conditions
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Jingsong Yuan†, Yi Zhang†, Yue Sun†, Zhicheng Cai, ‡ Lijiang Yang,*, ‡ and Hua Lu*,†
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†Center for Soft Matter Science and Engineering, Key Laboratory of Polymer Chemistry and
Physics of Ministry of Education, Beijing National Laboratory for Molecular Sciences, College
of Chemistry and Molecular Engineering, Peking University, Beijing 100871, People’s Republic
of China
‡Institute of Theoretical and Computational Chemistry, Biodynamic Optical Imaging Center,
Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular
Engineering, Peking University, Beijing 100871, People’s Republic of China
ABSTRACT: Controlling the helixꢀcoil transition of polypeptides under physiological
conditions is an attractive way toward smart functional materials. Here we report the synthesis of
a series of tertiary amineꢀfunctionalized ethylene glycol (EGx)ꢀlinked polypeptide electrolytes
with their secondary structures tunable under physiological conditions. The resultant polymers,
denoted as P(EGxDMAꢀGlu) (x =1, 2, and 3), show excellent aqueous solubility (> 10 mg/mL)
regardless of their charge states. Unlike polyꢀLꢀlysine that can form helix only at pH above 10,
P(EGxDMAꢀGlu) undergo a pHꢀdependent helixꢀcoil switch with their transition points within
physiological range (~ pH 5.3ꢀ6.5). Meanwhile, P(EGxDMAꢀGlu) exhibit unusual saltꢀinduced
helical conformation presumably owing to the unique properties of EGx linkers. Together, the
current work highlights the importance of fineꢀtuning the linker chemistry in achieving
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conformationꢀswitchable polypeptides, and represents a facile approach towards stimuliꢀ
responsive biopolymers for advanced biological applications.
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INTRODUCTION
The αꢀhelical conformation of polypeptides/proteins is a pivotal structural motif that
governs a wide range of biological functions such as enhancing proteinꢀligand binding,
regulating proteinꢀprotein interaction, and enabling membrane activity of antimicrobial
peptides.1ꢀ7 Often, the onset of these biological functions is regulated by the coilꢀtoꢀhelix
transition of the peptides.8ꢀ10 One vivid example is perhaps the pHꢀdependent transmembrane
activity of pH low insertion peptides (pHLIPs), a family of peptides that undergo a sharp coilꢀtoꢀ
helix transition when the pH is lowered from 7.4 to 6.0.11ꢀ13 Inspired by this, it is intriguing to
mimic such natural behaviors in artificial systems under physiological conditions.14ꢀ16 For
synthetic polypeptides made by the ringꢀopening polymerization (ROP) of αꢀamino acid Nꢀ
carboxyanhydrides (NCAs),17ꢀ24 however, the transitions often occur under conditions far from
physiological environment. For example, polyꢀLꢀlysine (PLL) can form helix only at pH above
10 when its sideꢀchain ammoniums are deprotonated to neutral amines. This, unfortunately, also
leads to significantly decreased aqueous solubility. As such, charged PLL always adopts the less
interesting coil conformation under physiological conditions. In addition, the helicity of PLL is
vulnerable to various environmental stresses including high ionic strength and elevated
temperature.25 To address this dilemma, Lu and Cheng have previously constructed ultraꢀstable
helices by using ionic polypeptides with elongated hydrophobic linkers.26 They have concluded
that a hydrophobic linker separating the charge and the peptide backbone with a chargeꢀtoꢀ
backbone distance (CBD) of minimum 11 σ bonds is crucial to create stable helix resisting
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various environmental stresses. However, these helices lack the sometimes desired dynamics
such as the coilꢀtoꢀhelix transition. Moreover, the greasy hydrophobic linker can produce
considerable synthetic hurdles and lead to problems such as toxicity and poor water solubility.
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To manipulate the helixꢀcoil transition in neutral polypeptides, Deming and Li exploited the
redoxꢀresponsiveness of thioether group in ꢀcysteine derived polypeptides.27,28 Our group
L
previously showed that a photoꢀresponsive helixꢀcoil transition was realizable by introducing
azobenzene group in the side chain.29 Metallic coordination was also explored to achieve helixꢀ
coil transition.30 For ionic polypeptides, Schlaad et al. took advantage of the Columbic
interaction in glycosylated coꢀpoly(Lꢀglutamate)s.31,32 Bonduelle et al. investigated the charge
repulsion among side chain imidazole groups.33 Cheng and coworkers showed that such a
transition was achievable by imparting both hydrogen donor and acceptor in the side chain of
ionic homopolypeptides.34 Here, we hypothesize that ionic homopolypeptides with fineꢀtuned
amphiphilicity and linker length can realize switchable helixꢀcoil transition under physiological
conditions. For this purpose, we reason that oligo(ethylene glycol) (EGx) is an ideal linker
candidate. Although poly(ethylene glycol) (PEG) is widely considered as a hydrophilic polymer,
numerous studies have shown that the hydration status of PEG varied under different
physicochemical environments such as ionic strength and temperature.35ꢀ38 As such, we designed
a series of ionic poly(Lꢀglutamate) derivatives bearing EGx linker of varied lengths (x = 1ꢀ3),
which correspondingly give rise to CBDs ranging from 14 to 30 sigma bonds, respectively
(Scheme 1). We show that the secondary structure of the polypeptides can be switched by pH
and salt as demonstrated by circular dichroism (CD) spectroscopy, 2D nuclear overhauser effect
spectroscopy (NOESY), and molecular dynamics (MD).
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MATERIALS AND METHODS
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Materials. All chemicals were purchased from commercial sources and used as received
unless otherwise specified. Ultrapure water (15.0 Mꢁ, MilliꢀQ) was used in all experiments.
HMDS and anhydrous N,Nꢀdimethylformamide (DMF) were purchased from SigmaꢀAldrich (St.
Louis, MO, U.S.A.). Anhydrous dichloromethane (DCM), hexane, and tetrahydrofuran (THF)
were obtained by passing HPLC grade solvents through columns packed with neutral alumina or
activated 4 Å molecular sieves. 2,2ꢀDimethoxyꢀ2ꢀphenylacetophenone (DMPA) was purchased
from Aladdin Reagent Co. Ltd. (Shanghai, China). PolyꢀLꢀlysine was prepared by the ringꢀ
opening polymerization of εꢀcarboxybenzyl Lꢀlysine NCA (ZꢀLysNCA) and subsequently
deprotected following a previously reported procedure.39
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Characterizations. NMR spectra were recorded on a 400 MHz Bruker ARX400 or a 500
MHz AVANCE III FTꢀNMR spectrometer. Fourier transform infrared spectroscopy (FTꢀIR) was
recorded using a Bruker Tensor FTꢀIR spectrometer (Bruker, Bremen, Germany) in the range of
400–4000 cm−1 at a resolution of 2 cmꢀ1 and 32 scans. Tandem gel permeation chromatography
(GPC) experiments were performed on a system equipped with an isocratic pump (Model 1100,
Agilent Technology, Santa Clara, CA), a DAWN HELEOS 9ꢀangle laser light scattering detector
(also known as multiangle laser light scattering (MALLS) detector, Wyatt Technology, Santa
Barbara, CA) and an Optilab rEX refractive index detector (Wyatt Technology, Santa Barbara,
CA). The detection wavelength of MALLS was set at 658 nm. The temperature of both the
refractive index and the MALLS detectors was 25 °C. Separations were performed using serially
connected size exclusion columns (500, 103,104, and 105Å Phenogel columns, 5ꢂm, 7.8×300 mm,
Phenomenex, Torrance, CA) at 50°C using DMF containing 0.1 M LiBr as the mobile phase.
The molecular weights (MWs) of all polymers were determined based on the dn/dc values of all
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samples calculated offline by using the internal calibration system processed by the ASTRA V
software version 5.1.7.3 provided by Wyatt Technology. Circular dichroism (CD) spectroscopy
was carried out on a Bio–Logic MOS 450 CD spectrometer. The polymer solution was placed in
a quartz cell with a light path of 1.0 cm. The mean residue molar ellipticity was calculated by the
following formula: Ellipticity ([θ] in deg·cm2·dmolꢀ1) = (millidegrees × mean residue
weight)/(pathlength in millimeters × concentration of polypeptide in mg/mL). The αꢀhelix
contents of polypeptides were calculated using the following equation: % αꢀhelix = (ꢀ[θ222] +
3000)/39000.
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Experiment Sections.
General procedure for the preparation of HOꢀEGxene
Taking HOꢀEG3ene (2ꢀ(2ꢀ(2ꢀ(allyloxy)ethoxy)ethoxy)ethanol) as an example:
To an iceꢀbathed solution of triethyleneglycol (45.0 g, 300 mmol, 3.0 equiv) in THF (100 mL)
was added sodium hydride (3.8 g, 160 mmol, 1.6 equiv) in portion. The reaction was kept
stirring at room temperature for 15 min, after which 3ꢀbromopropene (12 g, 100 mmol, 1.0 equiv)
was added into the solution. After stirring for another 1 h, the solvent was removed by
evaporation. The residue was dissolved in DCM (200 mL) and the solid was removed by
filtration. The organic phase was washed with saturated NaCl and water (100 mL). The water
phase was extracted with DCM for another two times (200 mL). The organic phase was
combined and dried with Na2SO4. After evaporating the solvent, the crude product was purified
by chromatography (petroleum ether (PE): ethyl acetate (EA) = 7:3 ~ 1:1) to afford the pure
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product as a colorless oil (11.4 g, yield 60%). H NMR (400 MHz, CDCl3) δ 5.93 (ddt, J = 16.1,
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10.9, 5.7 Hz, 1H), 5.28 (dd, J = 17.2, 1.5 Hz, 1H), 5.19 (d, J = 10.4 Hz, 1H), 4.03 (d, J = 5.7 Hz,
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2H), 3.80 – 3.53 (m, 12H), 2.62 (s, 1H). C NMR (126 MHz, CDCl3) δ 134.79, 117.35, 72.65,
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72.39, 70.77, 70.73, 70.48, 69.51, 61.88. ESIꢀMS: calculated: m/z 190.1, found: m/z 213.2
[M+Na]+.
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HOꢀEG2ene (2ꢀ(2ꢀ(allyloxy)ethoxy)ethanol): 1H NMR (400 MHz, CDCl3) δ 5.91 (ddt, J = 16.1,
10.4, 5.8 Hz, 1H), 5.29 (dd, J = 17.2, 1.5 Hz, 1H), 5.20 (dd, J = 10.4, 1.0 Hz, 1H), 4.04 (d, J =
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5.7 Hz, 2H), 3.70 (m, 8H), 2.51 (s, 1H). C NMR (126 MHz, CDCl3) δ 134.65, 117.49, 72.64,
72.40, 70.61, 69.61, 61.94. ESIꢀMS: calculated: m/z 146.1, found: m/z 169.2 [M+Na]+.
HOꢀEG1ene (2ꢀ(allyloxy)ethanol): was purchased from Aladdin Reagent Co. Ltd. (Shanghai,
China) and used directly.
General procedure for the preparation of EGxeneꢀGlu
Taking EG3eneꢀGlu (γꢀ(2ꢀ(2ꢀ(2ꢀallyloxyethoxy)ethoxy)ethoxy)ꢀLꢀglutamate) as an example:
To a solution of Lꢀglutamate (3.0 g, 34 mmol, 1.0 equiv) suspended in 2ꢀ(2ꢀ(2ꢀ
o
(allyloxy)ethoxy)ethoxy)ethanol (11.4 g, 60 mmol, 2.94 equiv) at 0 C was added sulfuric acid
(1.8 g, 33.7 mmol, 1.65 equiv) dropꢀwise over 10 min. After stirring at room temperature
overnight, the viscous solution was poured slowly into mixed triethylamine (6.81 g, 67.4 mmol,
3.3 equiv) and isopropanol (250 ml) to yield a white precipitate that was collected by high speed
centrifugation and dried under vacuum. EG3eneGlu was purified by recrystallization in
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methanol/diethyl ether (v/v: 20/200) and completely dried (6.5 g, yield 46%). The amino acid
should be used within a few days, otherwise it should be kept under desiccators to protect
moisture. 1H NMR (400 MHz, D2O) δ 5.85 (ddt, J = 16.5, 10.6, 6.0 Hz, 1H), 5.24 (dd, J = 17.2,
1.5 Hz, 1H), 5.16 (d, J = 10.4 Hz, 1H), 4.18 (t, J = 6.0 Hz, 2H), 3.97 (d, J = 6.0 Hz, 2H), 3.71 –
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3.54 (m, 11H), 2.50 (t, J = 7.7, 2H), 2.06 (m,2H). C NMR (126 MHz, D2O) δ 174.51, 173.77,
133.67, 118.44, 71.74, 69.64, 69.60, 69.51, 68.70, 68.42, 64.08, 53.90, 29.79, 25.40. ESIꢀMS:
calculated: m/z 319.2, found: m/z 320.2 [M+H]+.
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EG2eneꢀGlu (γꢀ(2ꢀ(2ꢀallyloxyethoxy) ethoxy)ꢀLꢀglutamate): H NMR (400 MHz, D2O) δ 5.84
(ddt, J = 16.5, 10.7, 5.7 Hz, 1H), 5.23 (dd, J = 17.2, 1.5 Hz, 1H), 5.16 (dd, J = 10.4, 1.0 Hz, 1H),
4.19 (t, 2H), 3.96 (d, J = 5.9 Hz, 2H), 3.68 (m, 3H), 3.60 (m, 4H), 2.50 (t, J = 7.7, 2H), 2.07 (m,
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2H). C NMR (126 MHz, D2O) δ 174.53, 173.79, 133.68, 118.38, 71.72, 69.61, 68.60, 68.38,
64.05, 53.90, 29.78, 25.40. ESIꢀMS: calculated: m/z 275.1, found: m/z 276.1 [M+H]+.
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EG1eneꢀGlu (γꢀ(2ꢀallyloxyethoxy)ꢀLꢀglutamate): H NMR (400 MHz, D2O) δ 5.84 (ddt, J =
16.4, 10.5, 6.0 Hz, 1H), 5.23 (dd, J = 17.2, 1.5 Hz, 1H), 5.17 (dd, J = 10.4, 1.0 Hz, 1H), 4.20 (t,
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2H), 3.98 (d, J = 5.9 Hz, 2H), 3.67 (m, 3H), 2.50 (t, J = 7.9, 2H), 1.98 (m, 2H). C NMR (126
MHz, D2O) δ 174.53, 173.80, 133.48, 118.58, 71.71, 67.54, 64.10, 53.89, 29.77, 25.41. ESIꢀMS:
calculated: m/z 231.1, found: m/z 232.2 [M+H]+.
General procedure for the preparation of EGxeneꢀGluNCA
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Taking EG3eneꢀGluNCA (γꢀ(2ꢀ(2ꢀ(2ꢀallyloxyethoxy)ethoxy)ethoxy)ꢀLꢀglutamate Nꢀ
carboxyanhydride) as an example:
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To a mixture of EG3eneꢀGlu (1.0 g, 3.1 mmol, 1.0 equiv) and triphosgene (344 mg, 1.16 mmol,
0.37 equiv) under nitrogen was added anhydrous THF (30 ml) and αꢀpinene (641 mg, 4.7 mmol,
1.5 equiv). The solution was then heated to 50 oC for 3~5 h, after which the solvent was removed
under vacuum. The yellow oil was dissolved in a minimal amount of DCM and purified by
chromatography with the room humidity under 50%. The NCA was eluted with mixed PE and
EA (0.87 g, yield 80%). The pure NCA fractions were immediately combined and dried under
vacuum. The pure NCA was transferred into the glove box and stored in refrigerator under ꢀ20
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oC. H NMR (400 MHz, CDCl3) δ 7.32 (s, 1H), 5.90 (ddt, J = 16.1, 10.4, 5.8 Hz, 1H), 5.28 (dd, J
= 17.2, 1.5 Hz, 1H), 5.19 (d, J = 10.4 Hz, 1H), 4.47 (t, J = 5.8 Hz, 1H), 4.33 (m, 2H), 4.01 (t, J =
5.8 Hz, 2H), 3.77 – 3.49 (m, 10H), 2.64 – 2.42 (m, 2H), 2.32 – 2.42 (m, 1H), 2.16 – 2.04 (m, 1H).
13C NMR (126 MHz, CDCl3) δ 171.93, 170.41, 151.95, 134.55, 117.71, 72.32, 70.64, 70.56,
70.36, 69.44, 68.81, 63.64, 57.32, 30.51, 27.17. FTꢀIR (cmꢀ1): 2934 cmꢀ1, 2873 cmꢀ1, 2678 cmꢀ1,
1738 cmꢀ1, 1609 cmꢀ1, 1584 cmꢀ1, 1511 cmꢀ1, 1412 cmꢀ1, 1325 cmꢀ1, 1113 cmꢀ1, 996 cmꢀ1, 923 cmꢀ
1. ESIꢀMS: calculated: m/z 345.1, found: m/z 368.2 [M+Na]+.
EG2eneꢀGluNCA (γꢀ(2ꢀ(2ꢀallyloxyethoxy)ethoxy)ꢀLꢀglutamate Nꢀcarboxyanhydride): 1H
NMR (400 MHz, CDCl3) δ 7.09 (s, 1H), δ 5.90 (ddt, J = 16.5, 10.7, 5.7 Hz, 1H), 5.28 (dd, J =
17.2, 1.5 Hz, 1H), 5.20 (dd, J = 10.4, 1.0 Hz, 1H), 4.43 – 4.20 (m, 3H), 4.01 (t, J = 5.7 Hz, 2H),
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3.82 – 3.50 (m, 6H), 2.63 – 2.32 (m, 3H), 2.13 (m, 1H). C NMR (101 MHz, CDCl3) δ 171.85,
170.07, 151.73, 134.40, 117.56, 72.18, 70.22, 69.31, 68.71, 63.56, 57.34, 30.55, 27.03. ESIꢀMS:
calculated: m/z 301.1, found: m/z 324.2 [M+Na]+.
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EG1eneꢀGluNCA (γꢀ(2ꢀallyloxyethoxy)ꢀLꢀglutamate Nꢀcarboxyanhydride): H NMR (400
MHz, CDCl3) δ 6.84 (s, 1H), 5.92 (ddt, J = 16.2, 10.4, 5.8 Hz, 1H), 5.30 (dd, J = 17.2, 1.5 Hz,
1H), 5.24 (dd, J = 10.4, 1.0 Hz, 1H), 4.43 – 4.34 (m, 2H), 4.22 (m, 1H), 4.10 (m, 2H), 3.69 (m,
2H), 2.58 (t, J = 5.8 Hz, 2H), 2.38 (m, 1H), 2.12 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 172.05,
169.90, 151.45, 133.97, 118.47, 72.27, 67.41, 64.10, 57.67, 31.09, 27.46. ESIꢀMS: calculated:
m/z 257.1, found: m/z 280.1 [M+Na]+.
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General procedure for the polymerization of EGxeneꢀGluNCA
Taking EG3eneꢀGluNCA as an example:
In a glovebox, EG3eneꢀGluNCA (200 mg, 0.580 mmol, 50 equiv) dissolved in anhydrous DMF
(2 mL) was added to a HMDS stock solution in DMF (23.2 ꢂL×0.5 M, 1.0 equiv) and stirred for
15 h at room temperature. Upon complete consumption of the monomer as monitored by FTꢀIR
spectroscopy, an aliquot of the reaction mixture was diluted to 5 mg/mL in DMF containing 0.1
M LiBr and injected for GPC analysis. To obtain the purified P(EG3eneꢀGlu), the reaction
solution was poured into diethyl ether (80 mL), and the precipitate was collected by
centrifugation, washed extensively with diethyl ether (40 mL×2), and dried under vacuum (yields
∼70%). The polymer could be stored in a −20°C freezer for up to 2 months. 1H NMR (400 MHz,
CDCl3) δ 5.93 (m, 1H), 5.31 – 5.14 (m, 2H), 4.23 (br, 3H), 4.01 (br, 2H), 3.72 – 3.56 (br, 10H),
2.75 – 1.95 (br, 4H).
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P(EG2eneꢀGlu): 1H NMR (400 MHz, d6ꢀDMSO) δ 8.09 (br, 1H), 5.86 (m, 1H), 5.27 – 5.08 (m,
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2H), 4.24 (br, 1H), 4.12 (br, 2H), 3.92 (br, 2H), 3.54 (m, 6H), 2.35 (br, 2H), 2.10 – 1.70 (br, 2H).
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P(EG1eneꢀGlu): 1H NMR (400 MHz, d6ꢀDMSO) δ 8.20 (br, 1H), 5.84 (m, 1H), 5.27 – 5.09 (m,
2H), 4.30 – 4.15 (br, 3H), 3.94 (br, 2H), 3.54 (br, 2H), 2.37 (br, 2H), 2.10 – 1.70 (br, 2H).
General procedure for the synthesis of P(EGxDMAꢀGlu)50
Taking P(EG3DMAꢀGlu)50 as an example:
P(EG3eneꢀGlu)50 (20.0 mg, 0.0653 mmol “ene”, 1.0 equiv), N,Nꢀdimethyl mercaptoehtylamine
hydrochloride (37.0 mg, 0.0.261 mmol, 4.0 equiv) and dimethoxy phenyl acetophenone (DMPA,
1.8 mg, 0.007 mmol, 0.11 equiv) were dissolved in a DMF/water mixture (400/40 ꢂL) in a 5ꢀmL
vial. The vial was capped and the mixture was irradiated with a 365 nm UV lamp (1.0 W/cm2)
for 0.5 h. The reaction could be continued for another 0.5 h irradiation with the addition of
another 1.8 mg DMPA if necessary. The product was purified using a PDꢀ10 column and
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lyophilized to afford the product as a white gelꢀlike solid (20 mg, yield 75%). H NMR (400
MHz, D2O) δ 4.31 (br, 1H), 4.22 (br, 2H), 3.73 (br, 2H), 3.54 – 3.68 (m, 10H), 3.33 (t, J = 5.7
Hz, 2H), 2.90 – 2.84 (m, 8H), 2.62 (t, J = 5.7 Hz, 2H), 2.49 (br, 2H), 2.17 – 1.92 (m, 2H), 1.83 (q,
J = 5.7 Hz, 2H).
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P(EG2DMAꢀGlu)50: 1H NMR (400 MHz, D2O) δ 4.31 (br, 1H), 4.20 (br, 2H), 3.70 (br, 2H), 3.50
– 3.65 (m, 6H), 3.31 (t, J = 5.7 Hz, 2H), 2.84 (m, 8H), 2.60 (t, J = 5.7 Hz, 2H), 2.46 (br, 2H),
2.20 – 1.90 (m, 2H), 1.81 (q, J = 5.7 Hz, 2H).
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P(EG1DMAꢀGlu)50: 1H NMR (500 MHz, D2O) δ 4.33 (br, 1H), 4.26 (br, 2H), 3.68 (br, 2H), 3.58
(t, J = 7.2 Hz, 2H), 3.29 (t, J = 5.7 Hz, 2H), 2.87 – 2.80 (m, 8H), 2.61 (t, J = 5.7 Hz, 2H), 2.48
(br, 2H), 2.15 – 1.90 (m, 2H), 1.82 (q, J = 5.7 Hz, 2H).
CD spectroscopy
Polypeptides were dissolved in DI water at a concentration of 0.05 mg/ml. The pH value of the
solution was adjusted by adding a specific volume of concentrated HCl or NaOH, and the
solution pH was measured by a pH meter (Oakton Instruments, Vernon Hills, IL, USA). After
the pH was adjusted to the desired value, the polypeptide solution was transferred to a quartz
o
cuvette (pathlength=1 or 10 mm) for CD tests at 25 C. For the temperatureꢀdependent CD
measurement, the temperature of the sample chamber where the quartz cuvette was placed was
controlled by a water bathe with programmed heating ramp. The samples were equilibrated at
each temperature for at least 10 min before measurement.
pH titration and apparent pKa measurement
The pH titration experiments were conducted by following a previously reported
procedure.40Briefly, P(EGxDMAꢀGlu)50 or PLL50 (5 mg) was dissolved in HCl (0.1 M × 5 ml) to
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adjust the initial pH of the polymer solution below 4.0. Then NaCl (8.8 mg) was added to the
solution to adjust the salt concentration to ~150 mM. pH titration was carried out by adding
small aliquots (5 ꢂL increment) of NaOH solution (0.1 M for P(EGxDMAꢀGlu)50) or 1 M for
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PLL50) at 25 C. The solution pH, measured by a pH meter, was plotted as a function of total
added volume of NaOH.
NOESY experiments
P(EGxDMAꢀGlu)50 for NOESY were dissolved in D2O at 10 mg/mL. 2D NOESY experiments
were performed on a 500 MHz AVANCE III FTꢀNMR spectrometer with a Cyroꢀprobe. The
pulse sequence was noesygpph19 with acquisition time of 107 ms and 26.7 ms. The
π/2 pulse width was 8.85ꢂs. The recycle delay was 2.0 s, and τm was 100 ms.
Simulation of P(EG3DMAꢀGlu)20
We used the Amber 14 Molecular Dynamics Package to perform the allꢀatom simulation of the
polypeptide. To build the initial polypeptide configuration, we used Gaussview to generate sideꢀ
chain coordinates, and Gaussian 09 to optimize the sideꢀchain configuration at a HF/6ꢀ31G*
level of theory. Then the sideꢀchain was grafted to a helical backbone template by Amber14. The
polypeptide was modeled with the Amber force field, augmented with some nonꢀbonded
interactions computed using the RESP ESP charge Derive Server (R.E.D.Server,
http://upjv.q4mdꢀforcefieldtools.org/REDServer). The backbone was simulated using force field
parm10.dat and the sideꢀchain was simulated using force field gaff.dat. Partial charges were
assigned from quantum mechanical predictions using Firefly_7.1.G on the R.E.D.Server. The
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polypeptide has a net charge of +21e due to the positively charged terminal group on each sideꢀ
chain and on the Nꢀterminal of backbone. The net charge was neutralized by adding Na+ and Clꢀ
ions. The whole system was placed in a box with periodic boundary condition and solvated by
TIP3P water molecules at 298K and 1 bar.
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Cytotoxicity experiments
In a 5% CO2 containing humidified atmosphere, HeLa cells were grown in Dulbecco's Modified
Eagle Medium (DMEM, Corning, Manassas, US) supplemented with 10% Fetal Bovine Serum
(FBS) and 4T1 cells were maintained in RPMI 1640 (Corning, US) supplemented with 10% FBS
at 37 °C. Cells were seeded at a density of 5000 cells/well for 24 h in a 96ꢀwell plate prior to the
experiment to allow the complete cell attachment to the plate. The cells were incubated with
P(EGxDMAꢀGlu)50 at appropriate concentrations for 24 h with fresh medium added (n=3). The
cell viabilities relative to the untreated group (100%) were determined by 3ꢀ(4,5ꢀdimethylthiazolꢀ
2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) assay following the manufacturer’s procedure.
Membrane activity experiments
The LUV was received as a kindly gift from Dr. Yuting Sun and used directly, which was
prepared following a general procedure described below: The thin lipid film was prepared by
evaporating a solution of 25 mg lipid mixture (DOPC:Cholesterol:DSPE ꢀPEG2000 = 50:50:5)
in 1 mL mixted solvent of MeOH/CHCl3 (volume ratio = 1:1) on a rotary evaporator at room
temperature. After which the thin lipid film was further dried under vacuo overnight. The
resulting film was dissolved in 1.0 ml buffer containing 50 mM 5(6)ꢀcarboxyfluorescein (CF), 10
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mM Tris and 10 mM NaCl (pH 7.4) for more than 30 min, and then subjected to several times
(usually more than 5) of freezeꢀthaw cycles. The LUV was then extruded through a
polycarbonate membrane with pore size of 200 nm for more than 20 times. Extravesicular
components were removed by passing a Sephadex Gꢀ50 filled conlumn with 10 mM Tris, 107
mM NaCl, pH 7.4 buffer as eluent.
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The membrane activity experiments were conducted by following a previously reported
procedure.41 LUV stock solution (10 µL) was diluted 10000 fold with a buffer containing 10 mM
Tris, 107 mM NaCl (pH 7.4) with a final lipid concentration 0.5 ꢂM. The CF efflux was
monitored at ꢀem 517 nm after mixing the diluted LUV solution (10 µL ), Tris buffer (80 µL with
10 mM Tris, 107 mM NaCl, pH 7.4) and polypeptide solutions with different concentration (10
µL, 10 mM Tris, 107 mM NaCl, pH 7.4) for 2 min. The 100% CF efflux was determined by
using 1.0% aqueous triton Xꢀ100 (10 µL, 10 mM Tris, 107 mM NaCl, pH 7.4) instead of
polypeptide solution. Effective concentration for polypeptide EC50 and Hill coefficient n were
determined by plotting the fractional activity as a function of amine concentration c and fitting
them to the Hill equation:
Y= Y0+ (YMAX − Y0) / {1 + (EC50/ c)n}
where Y0 is Y without polypeptide, YMAX is Y with 1.0% triton Xꢀ100 instead of polypeptides, i.e.
100% CF efflux, EC50 is the concentration of polypeptide required to reach 50% activity and n is
the Hill coefficient.
RESULTS AND DISCUSSION
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To generate ionic polypeptides with varied EGx linkers, we prepared three ꢀglutamateꢀ
L
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based monomers denoted as EGxeneꢀGluNCA (x = 1ꢀ3). All monomers were synthesized in a
concise and straightforward manner within 3 steps. Briefly, bishydroxyꢀterminated
oligo(ethylene glycol) (HOꢀEGxꢀOH) were reacted with 3ꢀbromopropene to etherize one
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hydroxyl group to generate HOꢀEGxene, which were then esterified by reacting with ꢀglutamic
L
acid to afford EGxꢀylated ꢀglutamate derivatives EGxeneꢀGlu. The EGxeneꢀGlu were converted
L
to the corresponding EGxeneꢀGluNCA in dry THF using triphosgene, and purified by column
chromatography in ambient atmosphere with ~60%ꢀ80% yield. All precursors and monomers
were comprehensively characterized (Figure S1ꢀS16). Next, we polymerized EGxeneꢀGluNCA
by using hexamethyldisilazane (HMDS) as the initiator (Scheme 1). At the feeding
monomer/initiator ratio (M/I) of 20/1, 50/1 and 100/1, gel permeation chromatography (GPC)
gave monomodal peaks for all resultant P(EGxeneꢀGlu) polymers with controlled molecular
weights (MW) and low polydispersity indices (Ð) (Table 1 and Figure S17). 1H NMR spectra of
P(EGxeneꢀGlu) exhibited characteristic allyl peaks (Figure S18ꢀS20). Next, we functionalized
P(EGxeneꢀGlu)50 via the thiolꢀene chemistry by using N,Nꢀdimethyl mercaptoehtylamine
hydrochloride to afford the highly waterꢀsoluble P(EGxDMAꢀGlu)50. The tertiary amine was
selected here because Gao et al. have previously demonstrated that tertiary amines have tunable
1
apparent pKa near the physiology range.40 The alkene peaks disappeared completely in the H
NMR spectra of the purified P(EGxDMAꢀGlu)50, which suggested a nearly 100% grafting
efficiency (Figure S21ꢀS23).
Scheme 1. Synthesis of the EGxeneꢀGluNCA monomers and the ionic P(EGxDMAꢀGlu)
polypeptides.
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HOOC NH2
, NaH
THF, r.t. 1 h, 60%-70%
C3Cl6O3
Br
H-Glu-OH
O
O
HO
H
HO
THF, 50 oC, 4 h, 60%-80%
H2SO4, r.t. 12 h, 46%-50%
x
x
O
x
O
O
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HO-EGxene
H
EGxene-Glu
O
O
O
O
H
NH
•
HCl
N
N
∗
N
O
HMDS
∗
HS
∗
∗
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n
n
DMF, r.t. 70%-80%
O
x
DMF, H2O, UV, 1 h, 60%-75%
O
x
O
S
N
O
O
O
O
O
O
x
x = 1, 2, 3
EGxene-GluNCA
P(EGxene-Glu)n
P(EGxDMA-Glu)n
Table 1. HMDSꢀmediated ROP of EGxeneꢀGluNCAsa.
b
c
Entry
1
Monomer
DPexp.
50
DPobt.
52
MWobt. (g·molꢀ1)d
11200
Ðe
EG1eneꢀGluNCA
1.12
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EG2eneꢀGluNCA
EG3eneꢀGluNCA
EG3eneꢀGluNCA
EG3eneꢀGluNCA
50
20
54
25
14000
7700
1.13
1.04
1.09
1.05
50
55
16720
31900
100
105
aAll polymerizations were analyzed at more than 95% monomer conversion, which was
determined by monitoring the NCA anhydride peak at 1853 cmꢀ1 in FTꢀIR spectroscopy. DPexp.
b
c
= expected degree of polymerization (feeding M/I ratio). DPobt. = obtained degree of
polymerization, the dn/dc values of P(EGxeneꢀGlu) were determined to be 0.0465 (x = 1), 0.0547
(x = 2), and 0.0590 (x = 3) mL/g, respectively. MWobt. = MW obtained. Ð = polydispersity
index, determined by GPC.
d
e
To study the pHꢀinduced helixꢀcoil transition of P(EGxDMAꢀGlu)50, all samples were
dissolved in ultrapure water at 0.05 mg/mL. The pH values of all solutions were carefully
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adjusted by either 1M HCl or NaOH, and measured by a pH meter. CD spectra of the polymers
depicted a rapid and characteristic coilꢀtoꢀhelix transition as the pH raised from ~3 to 9 (Figure
1A and S24). The maximum helicities of the polymers were measured ~ 80ꢀ84%, which were
fairly high for 50ꢀmer polypeptides. Next, the helicity was plotted as a function of pH to compare
the pHꢀinduced coilꢀtoꢀhelix transition of all polymers (Figure 1B). For clarity and simplicity
purposes, we defined the pH as the halfway transition point (H50ꢀpH) when a polymer adopts 50%
of its maximum helicity. Interestingly, the H50ꢀpH of P(EGxDMAꢀGlu)50 was measured as ~ 5.3
(x = 1), 5.5 (x = 2), and 6.8 (x = 3), respectively. In contrast, the H50ꢀpH of PLL was ~10.6.
Titration of P(EGxDMAꢀGlu)50 and PLL by NaOH (Figure S25) implied that the apparent pKa
values (defined as the pH when 50% of all the ionizable amines are protonated) of the polymers
correlated with their H50ꢀpHs fairly well (Table S1).40 Thus, it appeared that the coilꢀtoꢀhelix
transition of P(EGxDMAꢀGlu)50 was dependent on the protonation status of the tertiary amines,
similar to PLL.
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Figure 1. The pHꢀdependent coilꢀtoꢀhelix transition. (A) Overlay of CD spectra of P(EG3DMAꢀ
Glu)50 at varied pHs. (B) The pHꢀdependent helicity of P(EGxDMAꢀGlu)50 and PLL50. The
helicity was calculated using the following equation: helicity = (ꢀ[θ222] + 3000)/39000 ×100%.42
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We then examined the salt effect by gradually increasing the salt concentration in
P(EGxDMAꢀGlu)50 solutions. As shown in Figure 2A and S26, all P(EGxDMAꢀGlu)50 exhibited
an unexpected but clear saltꢀinduced coilꢀtoꢀhelix transition, when the polymers were initially in
the unfolded state at an acidic pH (e.g. 4.5). Maximum helicities were reached with the addition
of ~120 mM NaCl. In sharp contrast, PLL did not show any sign of helical conformation even at
500 mM NaCl, the highest salt concentration we tested. When all polymers initially adopted high
helical contents (e.g. at basic pHs), P(EGxDMAꢀGlu)50 were found to have slightly enhanced
helicity following the addition of NaCl, whereas PLL showed a helixꢀtoꢀcoil conformational
switch under the same condition (Figure 2B and S26). Thus, it was clear that P(EGxDMAꢀGlu)50
and PLL had completely opposite conformational switches with respect to salt. To examine the
conformational transition in different lyotropic salts, we also measured the CD spectra of
P(EG3DMAꢀGlu)50 in (NH4)2SO4, Na2SO4, and NH4Cl at pH 4.5. To facilitate the direct
comparison between different salts, the helicity of the polymer was plotted as a function of the
logarithm of ionic strength (logI). Upon the increase of ionic strength, interestingly, P(EG3DMAꢀ
Glu)50 adopted a more sensitive coilꢀtoꢀhelix transition in the kosmotropic (NH4)2SO4 and
Na2SO4 than in NaCl and NH4Cl (Figure 2C and S27). Thus, it appeared that anion had a greater
saltingꢀout effect than cation, which was also observed in proteins.43 Overall, the results
indicated that the synthetic polypeptides followed a similar trend with proteins as far as the
interaction with different Hofmeister salts was concerned. To verify the stable αꢀhelix of the
ionic polypeptides at low pH with salt, we performed allꢀatom MD simulation, a method that has
been widely used for protein conformation analysis.44 P(EG3DMAꢀGlu)20 was selected as a
model polypeptide and simulated using AMBER12 suite of programs. All of the terminal amine
groups were protonated to simulate the low pH environment. During the 50 ns simulation, the
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fully charged P(EG3DMAꢀGlu)20 remained intact αꢀhelix conformation in NaCl solution,
highlighting the robustness of our ionic helical polypeptides (Figure 2D and Supporting Movie).
Moreover, the helicities of P(EGxDMAꢀGlu)50 decreased only slightly even at a temperature up
to 70 oC, further underscoring their excellent helical thermostability (Figure S28).
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Figure 2. The saltꢀinduced helix formation. (AꢀB) The helicity of P(EGxDMAꢀGlu)50 and PLL50
as a function of NaCl concentration (A) when all polymers initially adopted a coiled
conformation (pH 4.5); (B) at conditions when all polymers initially adopted a helical
conformation (pH 7.4 for P(EGxDMAꢀGlu)50 and 11.1 for PLL50). (C) The helicity of
P(EG3DMAꢀGlu)50 as a function of the logarithm of ionic strength in four different salts: NaCl,
NH4Cl, Na2SO4, and (NH4)2SO4. (D) MD Simulation of P(EG3DMAꢀGlu)20 in NaCl solution
with fully charged side chains (50 ns).
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Next, we conducted 2D NOESY experiments to investigate the chemical environment of
P(EGxDMAꢀGlu)50 at different pH and ionic strength (Figure 3).45 Taking P(EG3DMAꢀGlu)50 in
D2O as an example, no cross peaks belonging to NOE signals was observed in the spectrum at
pH 7 (Figure 3B). Interestingly, when the solution was acidified to pH 3, several cross peaks
attributable to the NOE signals were clearly seen (Figure 3C). For example, the spectrum
suggested that the EG3 linker protons (He and Hf) and the glutamate protons (Hb and Hc) were
spatially in close contact with each other (< 0.5 nm) as evidenced by the NOE cross peaks. The
protons of the inner methylene adjacent to the thioether (Hi) also showed considerable crosstalk
with protons structurally far away from them (e.g. Ha, Hd, He, and Hf). Together, the spectra
implied the occurrence of intramolecular entanglement of side chains when the backbone of
P(EGxDMAꢀGlu)50 underwent a helixꢀtoꢀcoil transition from pH 7 to 3. Moreover, when the
polymer restored its helicity at pH 3 by the aid of 80 mM NaCl, the NOESY spectrum once
again gave no obvious NOE peaks and appeared very similar to the spectrum at pH 7 without salt
(Figure 3B and 3D). It was worth mention that no signal was detected for all solutions in
dynamic light scattering (DLS), suggesting there was no aggregation under the condition tested,
and thus ruling out the possibility of intermolecular interactionꢀinduced NOE effect (data not
shown). Similar NOESY results were obtained for both P(EG1DMAꢀGlu)50 and P(EG2DMAꢀ
Glu)50 (Figure S29ꢀS30).
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Figure 3. (A) 1H NMR spectra of P(EG3DMAꢀGlu)50. (BꢀD) 2D NOESY spectra of P(EG3DMAꢀ
Glu)50 at pH 7 and no salt (B); at pH 3 and no salt (C); and at pH 3 with 80 mM NaCl (D). The
blue circles represent additional NOE signals when P(EG3DMAꢀGlu)50 is in coiled conformation.
Based on the experimental results and the simulation, we proposed our prototype model for
the pHꢀ and saltꢀinduced coilꢀtoꢀhelix transition of P(EGxDMAꢀGlu) (Figure 4). At a relatively
basic pH in water (e.g. >7.0), it was not surprising that the neutral polymers all adopted the αꢀ
helical conformation. Notably, P(EGxDMAꢀGlu) were highly soluble in water because of the
hydrophilic EGx linkers and the terminal amines, even at the electronic neutral form. When the
tertiary amines were protonated at an acidic pH, the charge repulsion became predominate and
led to the disruption of the helix, similar to PLL. Nevertheless, the transition pH shifted to 5.5ꢀ
6.5 due to the presence of the EGx and the thioether linkers. With the addition of salt, the helix
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restored owing to possibly two reasons. One potential reason was the screen effect of the salt that
could shield the side chain charge repulsion. Although this screen effect was also applicable to
PLL, it was worth mention that the charge repulsion in PLL was appreciably greater than that in
P(EGxDMAꢀGlu) because the CBD of PLL (5 σ bonds) was significantly shorter relative to
P(EGxDMAꢀGlu) (14, 22, and 30 σ bonds for EG1, EG2, and EG3, respectively). Another reason
was attributable to the amphiphilicity of the EGx linker. It was postulated that the salt in solution
could compete for water molecules with the EGx linker, which altered the hydration state of side
chain and ultimately led to the enhanced helicity of the polypeptides.37,46 With more rigorous
works undergoing with our collaborators, we expect to report the more detailed biophysical
mechanism and the theoretical prediction in a separated work later.
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Figure 4. Cartoon illustration of the pHꢀ and saltꢀinduced conformation transition of
P(EGxDMAꢀGlu).
Ionic helical polypeptides are promising biomaterials for their helicityꢀinduced
transmembrane activity.47ꢀ49 However, the linker chemistry has not yet be fully explored to
achieve balanced toxicity and activity. To this end, we evaluated the toxicity and the membrane
activity of P(EGxDMAꢀGlu)50. MTT assay results using 4T1 cells indicated that the IC50 of
P(EGxDMAꢀGlu)50 were 96.9 (x =1), 264.5 (x =2), and >500 (x =3) µg/mL, respectively (Figure
5A and Table S2). We also studied the membrane activity of the polymers by using LUVs⊃CF,
i.e., large unilamellar vesicles (LUVs) composed of the 1, 2ꢀdioleoylꢀsnꢀglyceroꢀ3ꢀ
phosphocholine (DOPC), cholesterol and distearoyl phosphoethanolamineꢀPEG2000 (DSPEꢀ
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PEG2000) and loaded with 5(6)ꢀcarboxyfluorescein (CF) (Figure 5B and Table S2).41
Interestingly, the membrane disruption EC50 of P(EG3DMAꢀGlu)50 was measured to be 0.0108
ꢂg/mL, which is ~6ꢀ10 fold more potent than P(EG2DMAꢀGlu)50 (EC50 ~ 0.123 ꢂg/mL) and
P(EG1DMAꢀGlu)50 (EC50 ~ 0.0657 ꢂg/mL). Together, the data suggested that by elongating the
repeating unit of EG linker, the polymer could gain higher membrane activity with lower
cytotoxicity.
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Figure 5. (A) Cytotoxicity of P(EGxDMAꢀGlu)50 toward 4T1 cancer cell measured by the MTT
assay. (B) Membrane activity of P(EGxDMAꢀGlu)50 assessed using CFꢀloaded LUVs.
CONCLUSIONS
In conclusion, we successfully synthesized a series of amineꢀfunctionalized EGxꢀlinkered
polypeptide electrolytes. The resultant P(EGxDMAꢀGlu) polymers showed excellent aqueous
solubility regardless of their charge status. Unlike PLL that can form helix only at pH above 10,
CD spectroscopy indicated that P(EGxDMAꢀGlu) underwent a pHꢀdependent coilꢀtoꢀhelix switch
right within the physiological range. More interestingly, P(EGxDMAꢀGlu) exhibited unusual
saltꢀinduced helical conformation owing to the unique properties of EGx linkers. Preliminary
studies indicated that P(EG3DMAꢀGlu) was considerably less toxic, but in the meantime
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preserved an appreciable higher membrane activity, compared to its analogues P(EG1DMAꢀGlu)
and P(EG2DMAꢀGlu). The current work thus highlights the importance of fineꢀtuning the linker
chemistry in achieving conformationꢀswitchable polypeptides and represents a facile approach
towards stimuliꢀresponsive biopolymers for advanced biological applications.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
Characterization data (1H NMR, 13C NMR, MS), GPC curves, CD spectra, pH titration
curves, NOESY spectra
A supporting movie
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: chemhualu@pku.edu.cn
*Eꢀmail: lijiangy@pku.edu.cn
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGEMENTS
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This work is supported by National Natural Science Foundation of China (21722401, 21474004,
21434008 and U1430237). H.L. thanks the support from the Youth ThousandꢀTalents Program
of China. We gratefully acknowledge Dr. Yuting Sun and Prof. Xing Chen for the help in
membrane activity experiments and Hailin Fu and Prof. Yao Lin for the discussion of the
physical model.
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