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Synthesis and Properties of Alternating Polypeptoids and Polyampholytes as Protein-Resistant Polymers Yue Tao, Shixue Wang, Xiaojie Zhang, Zhen Wang, Youhua Tao, and Xianhong Wang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01719 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018

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Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Synthesis and Properties of Alternating Polypeptoids and Polyampholytes as Protein-Resistant Polymers Yue Tao,[a,b] Shixue Wang,[a] Xiaojie Zhang,[c]Zhen Wang,[a] Youhua Tao*[a] and Xianhong Wang[a] a

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese

Academy of Sciences, 5625 Renmin Street, Changchun 130022, P. R. China b

c

University of Science and Technology of China, Hefei, 230036, P. R. China

Department of Polymer Science and Engineering, Hebei University of Technology, Tianjin

300130, P. R. China

ABSTRACT: Alternating polypeptoids are particularly appealing because alternating sequence may impart highly-ordered structure and special functions, while their simple synthesis still remains a key challenge. Herein, we describe that natural amino acid monomers can be polymerized via Ugi reaction in a step-growth fashion as an AA’ BB’ system, which leads to alternating polypeptoids with molecular weight up to 15 kg/mol. These alternating polypeptoids are thermally responsive and exhibit cloud points (Tcps) between 27 and 37 oC. Importantly, the marriage of high functionality of amino acids with Ugi reaction also enables the preparation of polypeptoids encoding both protected amino and carboxyl groups in the side chains with alternating arrangement. The cleavage of the protecting groups leads to alternating

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polyamholytes without any compositional drift. Such alternating polyamholytes not only exhibit high water solubility (> 100 mg/mL), but demonstrate the ability to resist aggregation with proteins. Moreover, the cell viability measurements reveal that these materials have minimal cytotoxicity to HeLa cells. Overall, this study offers us a simple way to prepare a variety of polypeptoids and polyamholytes as new protein-resistant materials for bioapplications.

INTRODUCTION Polypeptoids, including α-, β-, and γ-, as well as poly(ε-peptoid)s, are important peptidomimetic polymers that have attracted considerable interest because they combine advantages from both biopolymers and synthetic polymers.1-8 Among these, alternating polypeptoids are particularly interesting because alternating sequence may impart highly-ordered structure and special functions, which cannot be observed with random polypeptoids.9-10 Oligomeric peptoids with alternating sequence, which can be obtained by iterative solidphase submonomer synthesis method, have been extensively investigated in recent years.11 For example, Zuckermann et al. synthesized peptoid polymers with alternating sequence of hydrophobic and ionic monomers.12 These peptoids can self-assemble into highly-ordered, freefloating nanosheets. Blackwell and co-workers prepared a peptoid nonamer of alternating αchiral aromatic and achiral nitroaromatic monomer units and this alternating peptoid can adopt a helical structure in acetonitrile.13 Using alternating bulky α-chiral naphthyl monomers and achiral aromatic monomers, Blackwell and co-workers also delivered a unique peptoid ribbon secondary structure.14 Franzyk reported the synthesis of alternating peptoids that have both stability toward enzymatic degradation and activity against multidrug-resistant bacteria.15-16 Kirshenbaum developed cyclic peptoids bearing alternating methoxyethyl and phenylmethyl residues and demonstrated efficient formation of a reverse-turn conformation.17 Employing


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iterative solution phase methods, Edwards and co-workers constructed a series of α, β alternating peptoids ranging from four to ten residues.18 Despite these remarkable efforts, the iterative synthesis is unsuitable for high-molecular weight polypeptoids because the yields must decrease with the successive reactions.19-20 Alternatively, the ring-opening polymerization (ROP) of N-substituted α-amino acid-N-carboxyanhydrides (NNCAs) offers an effective way to achieve high molecular weights polypeptoids,21-30 however, this approach is limited by the difficulties in sequence control. It has no doubt that design of a new method to obtain high molecular weight polypeptoids with alternating sequence is of great significance, but is a challenge. Polyampholytes bearing both positively and negatively charged repeat units on one polymer chain, are important zwitterionic polymers for their interesting properties, for example, stimuliresponsive properties, low protein adhesion, and so forth.31-36 Radical copolymerization of different unsaturated monomers bearing a positive or a negative charge is the representative strategy to synthesize polyampholytes.37 However, a major disadvantage of this approach is large compositional drift as well as the difficulties in the control over the charge distribution throughout the polymer chain, attributed to the difference in the reactivity of monomers.38 Actually, it is highly attractive to develop effective synthetic route toward polyampholytes with minimal drift in composition, as the solution performances (e.g., solubility, isoelectric point (pI)) of polyampholytes are closely related to the composition. Recently, Du Prez et al. prepared precisely alternating polyampholytes by employing thiolactone chemistry.39 In addition, proteinresistant or antifouling materials is an active area of research because of the vital role of nonspecific protein adsorption toward many biomedical applications.40-41 In recent years, increasing attention has been paid to zwitterionic polymers-based protein-resistant materials, mainly because of their ability to form a very stable hydration shell.42-43


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The Ugi reaction is a multicomponent reaction between acid, amine, isocyanide, and aldehyde.44-50 Recently, Meier et al. developed a very facile and modular process to synthesize diversely substituted polyamides through the Ugi four-component reaction for the first time.46 By the use of natural amino acid as an AB typed monomer, we have recently demonstrated that Ugi reaction can produce high molecular weight polypeptoids under mild conditions.51 Given the high functionality of amino acids,52-54 we anticipated that natural amino acid monomers may be polymerized via Ugi reaction in a step-growth fashion as an AA’ BB’ system, which is a polymerization method that leads to polymers with alternating sequences as already demonstrated by Carothers.55 For that to occur, the amino acid monomers should carry two amino or two carboxyl groups. Therefore, lysine and glutamic acid were selected as candidate monomers. In this contribution, we designed and synthesized a series of new alternating polypeptoids via the Ugi reaction of readily available natural amino acids (Scheme 1). At the same time, while many synthetic polypeptides have demonstrated remarkable thermoresponsiveness,56-57 very few studies have focused on the exploration of their analogues-thermoresponsive polypeptoids,58-59 probably owing to the limited synthetic method. In this context, we then reported that these alternating polypeptoids were thermally responsive and exhibited cloud points (Tcps) between 27 and 37 oC. Furthermore, the combination of high functionality of amino acids with Ugi reaction also allows us to obtain polypeptoids carrying both protected amino and carboxyl groups in the side chains with alternating arrangement. The cleavage of the protecting groups leads to alternating polyampholytes without any compositional drift (Scheme 1). The obtained polyampholytes contain zwitterions that endow the material with minimal cytotoxicity and good protein-resistant capability. The combination of biodegradability, cytocompatibility, and


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antifouling ability, suggests that alternating polypeptoids-based polyamholytes can be utilized as an ideal alternative protein-resistant material in biomedical science. To our knowledge, this is the first report of alternating polyamholytes through Ugi reaction, exhibiting appealing proteinresistant capability.

Scheme 1. Synthesis of alternating polypeptoids and alternating polyamholytes via Ugi reaction of natural amino acids

EXPERIMENTAL SECTION Materials and Methods. Tetrahydrofuran (THF) was refluxed with sodium, and distilled under argon atmosphere before use. Dichloromethane (DCM) was dried by refluxing with CaH2 followed by distillation. Benzaldehyde was stired with saturated sodium carbonate solution for 1h followed by separation from water, dried with Na2SO4 and subsequently distilled under reduced pressure. Methanol (MeOH) was refluxed with magnesium and iodine for 2 h, and distilled under N2. 1

H NMR spectra were recorded on a Bruker AV-300 or AV-600 spectrometer. Number-average

molecular weights (Mn) and polydispersity indexes (PDI) were determined by size-exclusion chromatography equipped with Waters 1515 HPLC pump, Waters Styragel HT3, HT4 columns


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and a Waters 2414 Refractive index Detector (eluent: DMF containing 0.01M LiBr; flow rate: 1 mL/min; temperature: 50 oC; standard: PS). MALDI-TOF/MS was performed on a Bruker atuoflex III mass spectrometer in linear, positive ion mode equipped with 355nm smart beam laser. The matrix was DCTB, and solvent was methanol. The cloud point temperature was determined as the temperature corresponding to that of solutions reaching 50% of the initial transmittance, and was measured as described elsewhere.53 The aggregation studies of polyampholytes in the presence of lysozyme were evaluated using DLS as described elsewhere.41 The isoelectric points (pI) test was measured on a PHS-3C pH meter. Typical Procedure for the Synthesis of Alternating Polypeptoids. Taking the synthesis of P1 as an example, lysine methyl ester (480.6 mg, 3 mmol), N-Boc-glutamic acid (741.7 mg, 3 mmol) and isobutyl aldehyde (475.9 mg, 6.6 mmol) were dissolved in MeOH (4 mL) first, and stirred for 2h. Then tert-butyl isocyanide (548.6 mg, 6.6 mmol) was added to proceed the Ugi polymerization at 25 oC for 4 days. After evaporation of all volatiles, the residue was dissolved in DCM (2 mL) and precipitated twice in petroleum ether. The product polymer was then centrifuged and dried under vacuum to yield white powder (1.86 g, 91 %). Typical Procedure for the Synthesis of Alternating Polyampholytes. Taking the synthesis of polyampholyte P10 as an example, LiOH (aq, 4.5 mL, 1 M) was added to a solution of P1 (1.0 g) in THF(6 mL) and the mixture was stirred for 24 h at 25 oC. Then, diluted aqueous HCl solution was added until the pH reached 2, after evaporation of the solvent, the solution of P1 in CH2Cl2 (6 mL) was treated with TFA (2 mL) at 0 oC and was stirred for 3 h at 25 oC. Then, the solution was concentrated in vacuo and subsequently dialyzed (MWCO 1000) to yield the final polymer (0.67 g, 80 %).


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The Isoelectric Points (pI) Test. The isoelectric points (pI) of the polyampholytes were tested by PHS-3C pH meter. The solution of P13 in distilled water at concentration of 1 mg/mL was treated with 0.1 M NaOH until pH~13. And then the aqueous solution was titrated with 0.2 M HCl until pH~2.

Cell Viability. HeLa cells were seeded in 96-well plates at a density of 105 cells/well. The cells were incubated in DMEM at 37 °C. After 24 h, fresh medium containing polypeptoids diluted to the desired concentrations was added to the corresponding well, respectively. After incubation for 48 h at 37 °C, cell viability was measured by MTT method.

RESULTS AND DISCUSSION Monomer Synthesis. The key monomer in this contribution, lysine methyl ester 1, was prepared from the esterfication reaction of commercially available lysine monohydrochloride, followed by treating with 1 M Na2CO3 and extracting with chloroform (Scheme 2). The 1H NMR and

13

C

NMR spectra confirm the monomer structure (Figure S2). The oligo-ethylene-glycol (OEG) functionalized isocyanides used in this work were synthesized upon dehydration from their corresponding formamide precursors which were prepared from primary amines (Scheme 2). The structures of OEG3-isocyanide 4b and OEG4-isocyanide 4c were confirmed by 1H NMR and 13C NMR (Figure S6, and S4).


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Scheme 2. Synthesis of lysine methyl ester and the functional isocyanides (OEG4-isocyanide) a

a

Reagents and conditions: (i) SOCl2, CH3OH, ice bath, 1 h; reflux, 6 h; (ii) Na2CO3, CHCl3/H2O,

2 h; (iii) phthalimide, DIAD, PPh3, THF; 12 h; (iv) NH2NH2.H2O, EtOH, overnight; (v) ethyl formate, reflux, 24 h; (vi) POCl3, NEt3, CH2Cl2, 0 oC to rt, 24 h.

Synthesis of Alternating Polypeptoids via Ugi Reaction. Employing the Ugi polymerization of the AA’ BB’-type monomers lysine methyl ester 1, N-Boc-glutamic acid 2, and isobutyraldehyde 3a, and tert-butyl isocyanide 4a, we can easily prepare alternating polypeptoids in a one-pot “AA’ + BB’ + C + D” process (Scheme 1). All polymerizations were performed in MeOH at room temperature. Employing a small excess (10% excess) of monofunctional components guaranteed lysine methyl ester and N-Boc-glutamic acid to complete consumption and get polypeptoids. Initially, we evaluated different polymerizations varying the monomer concentration (Table 1, P1 and P5-P7). As expected, higher monomer concentration favored both polypeptoid yields and the molecular weights. However, if the concentration was too high, the resultant Mn decreased again; this can be attributed to high viscosity of the reaction mixture, which might result in low mobility of polymer chains. A similar trend in isocyanide-based multicomponent polymerization was also observed by Meier and Li et al.46, 60 In addition, though performing the polymerization for 120 h produced polymers with a slightly higher molecular weight (Table 1, P9), we have ultimately established the protocol of


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Ugi reaction of natural amino acids to be at 0.75 M for 96 h in MeOH. Under optimized polymerization conditions, we were pleased to observe alternating polypeptoid P1 (91 % yield) with a Mn = 15.1 kg/mol and a molecular weight distribution Đ = 2.2 was obtained.

Table 1. Results of Ugi reaction of amino acids. a

a

Polymer

Monomers

[1] (mol/L)

t (day)

Mn (kg/mol)b

Ð (Mw/Mn)b

Yield (%)c

P1

1+2+3a+4a

0.75

4

15.1

2.2

91

P2

1+2+3b+4a

0.75

4

10.5

2.3

86

P3

1+2+3a+4b

0.75

4

11.2

1.9

57

P4

1+2+3a+4c

0.75

4

9.0

1.8

54

P5

1+2+3a+4a

0.25

4

13.7

2.1

76

P6

1+2+3a+4a

0.5

4

12.8

2.0

82

P7

1+2+3a+4a

1

4

13.9

1.8

82

P8

1+2+3a+4a

0.75

3

13.7

1.7

80

P9

1+2+3a+4a

0.75

5

15.9

2.4

92

Polymerizations are performed in CH3OH at room temperature. The monofunctional

components are fed 2.2 equiv to 1 or 2. bMeasured by SEC with polystyrene as standard and DMF as eluent. cCalculated based on polymers recovered after precipitation. The structure of P1 was characterized by 1H, 13C, and COSY NMR spectroscopy (Figure 1B, Figure S7-S8). All the signals attributable to the repeating unit of P1 that should be produced by the Ugi reaction are visible. COSY NMR spectroscopy (Figure S8, black box) showed the correlation of h and j, suggesting the formation of side-chain amide group. The alternating structure of P1 was further supported by the MALDI-TOF-MS spectrometry (Figure 1D). The


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MALDI-TOF-MS spectrum of P1 displays three series of peaks, each separated by intervals of 679.8 Da which corresponds to the molar masses of the repeating unit. Unfortunately, the end groups cannot be determined from the MALDI-TOF-MS spectrum of P1.49, 51 Additionally, it should be noted that the obtained polypeptoid theoretically had three kinds of regio-structures, head-to-head (H-H), tail-to-tail (T-T), and head-to-tail (H-T) structure (Scheme S1). We have thus demonstrated that a simple Ugi reaction provides a relatively facile method toward alternating polypeptoids.

Figure 1. (A) Deprotection of alternating polypeptoid P1. (B) 1H NMR spectra of P1 (upper panel, Table 1, entry 1) and deprotected P10 from polypeptoid P1 (lower panel) in CDCl3. (C) Overlaid SEC traces (DMF at 50 oC) of the alternating polypeptoids P1-P4 obtained by the Ugi reaction in MeOH at 25 oC. (D) The MALDI-TOF-MS spectrum of P1; the spacing between the peaks corresponds to the molar masses of the repeating unit. To expand the scope of this polymerization and get more alternating polypeptoids with different side groups, we conducted the Ugi reaction of lysine methyl ester and N-Boc-glutamic acid with different aldehyde 3b, or isocyanides 4b-c under similar conditions (Table 1, P2-P4).


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Hydrophobic polypeptoid with rigid phenyl side groups P2 was prepared in 86 % yield. Watersoluble polypeptoids P3-P4 were also prepared by Ugi reaction with OEG3-isocyanide 4b and OEG4-isocyanide 4c, respectively. All the 1H NMR spectra are consistent with the expected structures of P2-P4 (Figures S9, S10 and S12). As indicated in SEC traces (Figure 1C), all these polypeptoids are with moderate molecular weights. For example, OEG-functionalized P3 and P4 showed Mn about 11.2 kg/mol, and 9.0 kg/mol, respectively. It has already been demonstrated that the Ugi polymerization existed “ring-closing reaction”, which may be responsible for the relative low molecular weight of the resultant alternating polypeptoids.46, 60

Thermoresponsive Behavior of the Polypeptoids. Thermoresponsive polymers that can react to

temperature

stimuli

have

received

considerable

interest.

Many

biodegradable

thermoresponsive polymers, such as polyesters, and polypeptides, have been extensively investigated toward biomedical applications. In this context, thermoresponsive polypeptoids is of great interest. The resultant polypeptoids P3 and P4 were soluble in cold water at neutral pH. The thermoresponsiveness of these alternating polypeptoids was investigated. Heating of the aqueous solution of P4 caused a transition from clear solution to turbid emulsion (Figure 2A). The thermoresponsive behavior in water was investigated at varying temperatures by measuring the light transmittance of the polypeptoid solutions at a wavelength of 500 nm (Figure 2B). Alternating polypeptoid with OEG4 side chains delivered much higher cloud point than that with OEG3 side chains (e.g., 37 oC for P4 and 27 oC for P3, respectively.), which was reasonable due to the higher hydrophilicity of alternating polypeptoids with longer OEG side chains. Additionally, Figure 2B also demonstrated that the phase transition of P3 and P4 was reversible, that is, heating and cooling cycles were comparable with minimal hysteresis.


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Figure 2. (A) Visual turbidity change of P4 upon heating the aqueous solution. (B) Temperature dependence of transmittance for the aqueous solutions (2 mg/mL) of P3 and P4 (500 nm, heating or cooling at a rate of 1 °C min-1). To further sight the phase transition of these alternating polypeptoids, the temperaturevariable 1H NMR analysis (Figure 3) of the phase transition of the 2 mg/mL polypeptoids solution was performed. As shown in Figure 3A, with temperature rising from 30 to 50 oC the peaks weaken, revealing thermoresponsive dehydration of P4. The dehydration of the polymer chains is in general due to the weakened hydrogen bonding between the polymer chains and water when temperature is above cloud points.61 To further analyze dehydration of the polypeptoids in water, two typical characteristic signals, i.e, CH2OCH2 (k, δ = 3.6 ppm), and OCH3 (m, δ = 3.3 ppm) were chosen, since these two signals were more visible and they could be affected by hydrogen bonding. As indicated, at 30 °C, the two signals were distinctly observed for P4 (Figure 3A). Starting from 35 °C, with the increase of temperature, characteristic peaks of P4 decreased substantially, accompanied by a broadening in width. This result is in agreement with the Tcps values measured by UV-vis. Meanwhile, it was deemed that


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the dehydration of alternating polypeptoids upon heating is mainly ascribed to the weakened hydrogen bonding between the OEG moieties with H2O, although the polypeptoid backbone can also take part in formation of hydrogen bonding.58 The temperature-dependent 1H NMR spectra of P3 were also measured, and the similar dehydration of the P3 with the increasing temperature is detected (Figure 3B).

Figure 3. 1H NMR spectra of P4 (A) and P3 (B) in D2O at various temperatures. The signals are normalized by the solvent peak at δ = 4.79 ppm.


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Synthesis of Polyampholytes with Alternating Amino and Carboxyl groups. After the successful preparation of alternating polypeptoids, the corresponding polyampholytes encoding both amino and carboxyl groups in the side chains were recovered by the cleavage of protecting groups (Figure 1A). 1H NMR analysis of the polyampholyte P10, prepared from alternating polypeptoid P1, displayed the disappearance of all of the tert-butyl protons around 1.38 ppm, and methyl ester protons around 3.68 ppm (Figure 1B), suggesting the complete deprotection. We have thus demonstrated that a simple Ugi reaction provides a relatively facile method toward alternating polyamholytes without any compositional drift. The polyampholyte P13 from OEG4-functionalized polypeptoid P4 is a highly water-soluble polymer encoding alternating sequence of -COOH and -NH2 pendants. It remained fully dissolved in the range of pH = 2-12. The high solubility of alternating polyampholytes over a broad pH range is characteristic of the interactions between the adjacent ionic groups, which inhibit complete charge compensation within the polyampholyte that is typically associated with insolubility.39 The isoelectric points (pI) was 6.5 for P13 and 5.0 for P12, respectively, as determined by potentiometric titration (Figure 4A, and S16).


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Figure 4. (A) Potentiometric titration curve and first differential of polyampholyte P13, prepared from alternating polypeptoid P4. (B) Particle size measured by DLS as a function of time for polyampholytes P12 (from the corresponding polypeptoid P3) and P13 dissolved in 1 wt % lysozyme in PBS, the concentration of polyampholytes is 1 wt %. The error bars are the standard deviations of three measurements.

Protein-Resistant Behavior and Cytotoxicity. Another remarkable feature of the resultant polyampholyte that was obtained by Ugi reaction is the presence of zwitterions, which have been demonstrated for their appealing protein-resistant behavior.62-63 Protein-resistant materials have been used as implant coatings to improve biocompatibility and as the shell of drug delivery


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nanocarriers for prolonged blood circulation. P12 and P13 were selected as the model polymers to study the antifouling characteristics. As literatures pointed out, dynamic light scattering (DLS) is a valuable tool for protein resistant characterization.41, 64-66 To determine the nonfouling nature of P12 and P13, we measured the hydrodynamic size of P12 and P13 as a function of time in a lysozyme-containing medium. Lysozyme was selected as the model protein.41 Both P12 and P13 were found to be relatively stable in PBS containing 1 wt % lysozyme (Figure 4B). These results demonstrate the potential utility of these hydrophilic zwitterionic polypeptoids as proteinresistant materials for bioapplications. The cytotoxicity of the alternating polypeptoid P4 and the corresponding polyampholyte P13 was assessed using HeLa cells and an MTT assay. PEG (5000 g/mol), a benchmark proteinresistant materials, was used for comparison. Both P4 and P13 showed minimal cytotoxicity toward HeLa cells, with greater than 87% cell viability in the 0.125-2.0 mg/mL polymer concentration range (Figure 5).


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Figure 5. Cell viability of alternating polypeptoid P4 and polyampholyte P13 compared to PEG (5000 g/mol), as evaluated by MTT method following treatment with polymers for 48 h. The error bars are the standard deviations of three measurements. CONCLUSION In conclusion, Ugi chemistry has opened up a new route for the preparation of a variety of polypeptoids with alternating structure in a simple way. The resultant alternating polypeptoids are thermally responsive, with cloud points (Tcps) between 27 and 37 oC. Moreover, the combination of high functionality of amino acids with Ugi reaction also enables the preparation of alternating polyampholytes with high solubility (> 100 mg/mL), and appealing antifouling behavior in a mild, and feasible fashion. Indeed, the alternating structure and diverse polymer properties described here offer a new direction for the synthesis of polypeptoids and polyampholytes as new protein-resistant materials.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Experimental details and characterization data. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant Nos. 21474101 and 51673192) and “The Hundred Talents Program” from the Chinese Academy of Sciences. REFERENCES (1)

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