Synthesis of Unnatural Poly(Amino Acid) - American Chemical Society

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“Green” Synthesis of Unnatural Poly(Amino Acid)s with Zwitterionic Character and pH-Responsive Solution Behavior, Mediated by Linear-Dendritic Laccase Complexes Ivan Gitsov, Lili Wang, Nikolay Vladimirov, Arsen Simonyan, David J Kiemle, and Andri Schütz Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm501126a • Publication Date (Web): 17 Oct 2014 Downloaded from http://pubs.acs.org on October 20, 2014

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“Green” Synthesis of Unnatural Poly(Amino Acid)s with Zwitterionic Character and pHresponsive Solution Behavior, Mediated by Linear-Dendritic Laccase Complexes

Ivan Gitsov,a,b* Lili Wang,b Nikolay Vladimirov,c Arsen Simonyan,b David J. Kiemle,b Andri Schutzd

a

b

The Michael M. Szwarc Polymer Research Institute;

Department of Chemistry, State University of New York - College of Environmental Science and Forestry, Syracuse, NY 13210; c

d

Ashland, Inc., Wilmington, DE 19808;

Department of Materials Science, Swiss Federal Institute of Technology, CH-8093 Zurich, Switzerland

*Corresponding author. E-mail address: [email protected]

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ACS Paragon Plus Environment

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ABSTRACT: This article describes the enzyme-catalyzed “green” synthesis of an unnatural poly(amino acid). DL-tyrosine was polymerized under environmentally friendly conditions using linear-dendritic laccase complexes as initiators and water as solvent. The influence of the dendron generation in the linear-dendritic copolymers, the monomer concentration, time and temperature on the polymers yields and molecular masses was investigated. Depending on the reaction conditions poly(tyrosine) with molecular mass (Mw) up to 82 kDa could be obtained in yields ranging between 45 % and 69 %. It was found that the linear-dendritic laccase complexes can induce further chain growth upon addition of fresh monomer to the preformed poly(tyrosine) in a fashion resembling the classic “living” polymerization. The structure of the poly(tyrosine) was investigated by NMR, FT-IR and MALDI-TOF and it was discovered that the polymer chains consist of phenol repeating units linked together by C-C and C-O bonds randomly distributed along the backbone of the polymers. The materials formed are completely watersoluble and behave as typical poly(zwitterions) changing charge and size with the medium pH. DLS measurements reveal that the zeta potential of the polymers can vary between +15 mV at pH 1.2 with hydrodynamic diameter (Dh) = 6.7 nm to -35 mV at pH 11.8 and Dh = 10 nm. The isoelectric point was found at pH = 2.3-2.6 where Dh of the polymer is at minimum (2.4 nm).

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1 1. Introduction Zwitterionic polymers (polyzwitterions)1 consistently attract substantial interest due to their specific nature and promising applications. The presence of opposite charges and its implications on the peculiar charge density distribution along the polymeric chain have proven most useful for applications in ion transport,2 ion chelation3 and emulsifying industries.3a The type of polyzwitterion, where both the positive and negative charge reside on the same repeating unit, are usually referred as polybetaines.1a,3a This class of polymers exhibit unusual aqueous solution behavior undergoing reversible conformational changes and phase transitions in response to external changes in environmental pH

4,5

and/or ionic strength.6 Promising features

of polybetaines are their biocompatibility and bioactivity associated with their highly hygroscopic nature.7 These properties make them very attractive polymers for specific bioapplications.3a,8,9 For example, zwitterionic polymers have been widely used as biomaterials for surface treatment to achieve pH- dependant adsorption/desorption of proteins.5,10 Amino acids are natural zwitterions due to the spontaneous protonation/deprotonation of the amino- and carboxylic groups with changes in pH. Therefore, the potential to produce corresponding stimuli-responsive polybetaines8,11 using multi-functional natural amino acids as zwitterionic monomers is particularly intriguing. Additional benefits arise from the sustainability of the monomers and their intrinsic biocompatibility. Tyrosine (Tyr) would represent a suitable candidate for the construction of such macromolecular nanoconstructs. It was shown in an elegant study by Kobayashi et al. that unnatural poly(tyrosine) could be prepared by horseradish peroxidase (HRP) catalyzed oxidation of tyrosinyl ester hydrochlorides and subsequent ester hydrolysis.12 3


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In our previous work, we have shown that laccase (EC 1.10.3.2, p-diphenol:dioxygen oxidoreductase) complexes with linear-dendritic copolymers are able to oxidize and polymerize a broad variety of phenolic and aromatic substrates in water.13 Therefore, this oxidative enzyme seems like another suitable natural catalyst for the polymerization of phenol-containing and water-insoluble Tyr. Previous articles, however, reported contradictory results on the ability of laccase to induce the oxidation or polymerization of Tyr.14 In this report, the first laccasemediated one-pot, efficient and environmentally benign oxidative polymerization of pure Tyr is reported. The synthesis is performed in water at temperatures close to ambient. The obtained polymer could be easily isolated from the reaction solutions and the biocatalytic complexes could be recycled and reused. The polymers formed have molecular masses (Mw) ranging from 4,000 Da to greater than 56,000 Da. Their solid-state and solution properties were investigated by a combination of spectroscopic-, thermal-, and mass-sensitive techniques. The zwitterionic poly(tyrosine) demonstrates interesting solution behavior over a wide range of the pH values.

2. Experimental section 2.1. Materials. DL-Tyrosine (DL-Tyr) (99%), 2,2′-Azino-bis (3-ethylbenzo-thiazoline-6sulfonic acid) (ABTS), 4-Hydroxy-l-phenylglicine and Folin-Ciocalteau (FC) phenol reagent15 were purchased from Sigma-Aldirich Ltd (St. Louis, MO) and used as received. Deionized water (DI-H2O) (18.1 MΩ) was purified by a Barnstead Nanopure water system. Laccase was produced, isolated and purified from the basidiomycete white rot fungus Trametes versicolor (TvL) in a procedure described elsewhere.13a Linear-dendritic (LD) AB copolymer of [G3]PEO13k and asymmetric dendritic-linear-dendritic (DLD) ABA copolymers: [G3]-PEO13k-[G1] and [G3]-PEO13k-[G2] (n denotes the generation number) were synthesized via “living” ringopening polymerization of ethylene oxide initiated by third generation poly (benzyl ether) 4


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dendrons.16 Laccase modification was performed following procedure already described;17 the enzymatic copolymer complexes (ECC)s of TvL and LD or DLD copolymers (TvL/LD or TvL/DLD) were freshly prepared before each reaction. 15.0 ± 0.8 mg solid LD or DLD copolymers and 500 µL TvL were placed into 50 mL DI-H2O. The obtained solutions were stirred and equilibrated at room temperature for at least 4 hours prior to use. 2.2. Instrumentation and methods. 2.2.1. Size-Exclusion Chromatography (SEC). Aqueous SEC was carried out on Waters Alliance 2695 system and Waters 2414 differential refractive index detector (dRI). All sample solutions were filtered through a 0.45 µm filter before injection. The injected volume was 200 µL. The separation was achieved on a column set of Proteema guard, 1000 Å and 100 Å, 8 mm x 300 mm, 5µm (Polymer Standard Service, Mainz, Germany) in series in 70 % (0.08 M di- and 0.02 M mono-ammonium phosphate) / 30% acetonitrile at 40°C, and eluent flow rate of 0.8 mL/min. The calibration was performed with poly(styrene sulfonate) (PSS) standards (Polymer Standard Service, Mainz, Germany). Alternatively, absolute molar masses were determined using the same aqueous SEC line equipped with a multi-angle laser light scattering (MALLS) detector (DAWN-EOS, Wyatt Technology, Santa Barbara, CA), fitted with a helium-neon laser (λ=690 nm) and a K5 flow cell, and differential refractive index detector (dRI, Optilab rEX, Wyatt Technology). A 70% 0.2 M dibasic ammonium phosphate 30% acetonitrile (pH=8.5) mixture was used as eluent at a flow rate 0.7 mL/min. A dn/dc value of 0.212 mL/g was determined in the same solvent/temperature and used in the Mw calculations. ASTRA software (version 5.3.4) was utilized for data acquisition and analysis. 2.2.2. Fourier transform infrared spectroscopy (FT-IR). FT-IR spectra were collected on a Bruker Tensor 27 spectrometer (Bruker Optics, Inc., Billerica, MA) using attenuated total

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reflectance (ATR) technique on a ZnSe crystal. Spectra were acquired between 4000 and 600 cm-1 for 16 scans with a resolution of 4 cm-1. 2.2.3. NMR spectroscopy. All samples (normally 8–10 mg) were dissolved in D2O from Cambridge Isotope Laboratories Inc. NaOD (40% in D2O; from ABCR chemicals) was used to adjust the pH from neutral to basic conditions. 1H- and 1H-13C DEPT edited heteronuclear single quantum coherence (DEPT-HSQC) NMR spectra were run in deuterium oxide (D2O) at room temperature on 300 and 600 MHz Bruker AVANCE instrument. Some of the NMR spectra were recorded on a Bruker Avance 700 spectrometer, equipped with a BBI probe head, operated at 700 MHz and at 22°C. For 1H NMR at 700 MHz, a presaturation pulse was used to suppress the residual water peak at 4.72 ppm. 2D Correlation Spectroscopy, COSY (pulse program: cosygpqf. 2D homonuclear shift correlation using gradient pulses for selection) was performed without any water suppression by presaturation or watergate. For 2D Nuclear Overhauser Effect Spectroscopy, NOESY (pulse program: noesyphpr. 2D homonuclear correlation via dipolar coupling where dipolar coupling may be due to NOE or chemical exchange; phase sensitive and with presaturation during relaxation delay and mixing time), mixing times of 600 ms were applied. 2.2.4. UV-Vis spectroscopy. The UV-Vis measurements were conducted on Agilent 8453 UV-Vis spectroscopy system at room temperature. 2.2.5. Enzyme assay. Native TvL and enzymatic complexes of TvL (ECCs) were assayed for activities using ABTS as substrate at room temperature. 5 mM ABTS (in 10 mM sodium acetate buffer, pH 4.0) was freshly prepared. After blanking with 940 µL buffer and 50 µL of 5 mM ABTS solution, 10 µL of enzyme solution were added to the spectrometer cell. The solution was mixed by inversion and the absorbance at 414 nm (ε414 = 36000 M-1cm-1)18 was recorded 6


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immediately every 7 s for 2 min. Enzyme activities were expressed in microkatals (µkat), the amount of enzyme that catalyzes the transformation of 1 µmol of substrate at room temperature. 2.2.6. Phenolic content of poly(tyrosine). A calibration curve was made where the absorbance was plotted against concentration. 4-hydroxy-L-phenylglicine was chosen as a standard for calibration purpose because it is water-soluble and structural analog of DL-Tyr. A stock solution of 6.05 mM was diluted to series of concentrations. The content of phenolic hydroxyl groups was quantified by measuring the absorbance at 760 nm. In a typical procedure,19 1.0 mg poly(tyrosine) was placed into a 50 mL volumetric flask followed by adding 3 mL of FC reagent and 30 mL DI-H2O. The solution was swirled to mix and incubated for 5~8 min. Then 10 mL sodium carbonate (20 wt %) was added and H2O was finally added to the 50 mL line. The mixture was further incubated for 2 h. The absorbance at 760 nm was recorded. Blank solution was prepared following the same procedure but without poly(tyrosine) added. 2.2.7. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The MALDI-TOF spectra were acquired on Bruker Autoflex III system equipped with Smartbeam II laser source (Nd-YAG laser, 266 or 355 nm). All spectra were collected in a linear positive mode. The laser attenuation was set to the lowest value possible to obtain high resolution spectra. The matrix solution was prepared by dissolving 2,5-dihydroxy benzoic acid (DHB) in 70% acetonitrile and 30% TFA (0.1%) at a concentration of 80 mg/mL. Sample solutions were prepared in 0.1% TFA at a concentration of 1 mg/mL. Samples were spotted following the dried-droplet method, where 1 µL droplet of premixed solutions (sample/matrix mixing ratio of 1:7) was spotted on MTP 384 target plate (polished steel, Bruker Daltonics). 7


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2.2.8. Thermogravimetric analysis (TGA). TGA measurements were conducted on a TGA Q5000IR (TA Instruments). The temperature was programmed to increase from room temperature to 700 °C at a rate of 20 °C min-1 in the presence of nitrogen gas (10 mL×min-1). 2.2.9. Differential scanning calorimetry (DSC). DSC analyses were performed on a DSC Q200 instrument (TA Instruments). The analyses were carried out in a heating/cooling/heating cycle, where polymer samples were heated to 175°C at 10°C min-1, cooled to −50°C at 5°C/min, and then heated back to 175°C at 10°C/min. 2.2.10. Dynamic Light Scattering (DLS). DLS studies were conducted on a Malvern Zetasizer Nano Series instrument at a fixed detecting angle of 173°. Size was calculated using a CONTIN program. Zeta potential measurements were performed on the same instrument using a Malvern U-shaped folded capillary cuvette. An aqueous solution was prepared by dissolving 10.45 mg of poly(tyrosine) into 6 mL DI-H2O. The initial solution pH was then adjusted to 11.8 by adding 1 mL of ~0.05 M NaOH. The solution was then titrated by continuous addition of ~0.05 M HCl. 800 µL of solution at each desired pH was withdrawn and set aside for Dh measurement. A total volume of 2.71 mL HCl solution was consumed to give a final pH at 1.2. For the zeta potential measurements aqueous solutions of poly(tyrosine) at a concentration of 1.0 mg/mL were prepared. The same solutions were adjusted to the desired pH using HCl and NaOH and sequentially injected into the cuvette for zeta potential determination. Each apparent Dh or zeta potential value reported is an average of multiple measurements (minimum 3). The error bar represents a standard deviation. 2.2.11. In vitro cytotoxicity assay.The MTS assay was used to evaluate the cytotoxicity of poly(tyrosine) on the cell viability against normal healthy cell line (Chinese hamster ovary, CHO) and cancer cell line (Raji). CHO and Raji cells were seeded in 96-well plate at the cell 8


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densities of 4 × 103 and 8 × 103 cells/well respectively. Raji cell was directly used after seeding to treat with different concentrations of poly(tyrosine) polymer, while the CHO cell was incubated overnight before adding the polymer solution. After 72 h incubation, CellTiter 96® Aqueous Cell Proliferation Reagent, which is composed of MTS and an electron coupling reagent PMS, was added to each well. The cell viability was determined by measuring the absorbance at 490 nm using a microplate reader (SpectraMax M2, Molecular Devices, USA). Untreated cells served as a control. Results were shown as the average cell viability [(ODtreat−ODblank)/(ODcontrol−ODblank)×100%] of triplicate wells. 2.3. Syntheses. 2.3.1. ECCs-catalyzed polymerization of DL-Tyr. In a typical reaction, to a 10 mL freshly prepared TvL/[G3]-PEO13k-[G2] complex, 20 mg finely ground DL-Tyr was added. The reaction solution was stirred vigorously at room temperature (22°C) or 45°C. After 24 or 72 h, the unreacted insoluble DL-Tyr was centrifuged off and washed twice with 5 mL DI-H2O. The poly(tyrosine) was isolated and precipitated from the decanted supernatant by adjusting the solution pH to 2.6 - 3.0 using HCl and NaOH, and then separated by centrifugation. The purification was further conducted by adding the isolated crude poly(tyrosine) in 1 mL DI-H2O, followed by slowly adjusting pH until the polymer dissolved. The aqueous solution was then precipitated into 10 mL THF to obtain a dark-brown solid product (yield at 45°C/24h: 57%, Mn= 4800, Mw/Mn= 4.60, see Table 1). The product yield reported herein is calculated from the mass of purified poly(tyrosine) divided by the initial total mass of DL-Tyr monomer.

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3. Results and Discussion 3.1. Polymerization of tyrosine catalyzed by linear-dendritic copolymer/enzyme complex. Laccases are members the lignolytic oxidative enzymes family. They are glycoproteins with copper cluster containing active sites, which predominantly act on a broad range of substrates of phenolic and aniline compounds. At the structural level, they have a high content of N-glycans residues which could provide the suitable anchoring units for enzyme modifiers. Our previous studies have shown that the facile physical modification of laccase using amphiphilic LD block copolymers composed of poly(ethylene glycol) and poly(benzyl ether) dendrons, could selectively adsorb on the N-glycan fragments through a hydrophobic, π-H interactions. A schematic illustration of the formation of supramolecular ECC from enzyme TvL and LD copolymer of [G3]-PEO13k-[G2] is shown in Scheme 1. The ECC has shown enhanced catalytic activity on oxidizing hydrophobic substrates of interest, such as the oxidation of fullerene,17 and homopolymerization and copolymerization of bisphenol A and diethylstilbestrol.13b In this study, a series of LD copolymers varying in their hydrophobic dendrons fractions are used to modify TvL. The activities of native TvL and the obtained ECCs were determined against ABTS to be 71.4±0.3, 72.0±1.2, 70.8±1.5 and 70.9±0.9 µkat/L for TvL, TvL/[G3]-PEO13k, TvL/[G3]PEO13k-[G1] and TvL/[G3]-PEO13k-[G2] respectively (Figure 1). It shows that the modification has no significant and negative effect on the enzyme activity.

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Scheme 1. Formaiton of a TvL/[G3]-PEO13k-[G2] enzymatic complex.

74.00 73.00

Laccase activity, µkat/L

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72.00 71.00

71.96 71.42 70.84

70.89

70.00 69.00 68.00 67.00

Figure 1. Activity toward ABTS for the native TvL and its LD or DLD copolymer complexes. The activities are mean values from 3 replicate assays with error bars representing a standard deviation.

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The poor aqueous solubility of Tyr presents a serious limitation to its enzymatic polymerization. The solubility of Tyr in water in the enzyme active range (pH = 4-7) is very low,20 which dramatically affects the efficiency of the reaction in aqueous media in the presence of native laccase. Additionally, the typical protocol of using water miscible organic co-solvent at the expense of enzyme activity is not applicable since Tyr is not soluble in most organic solvents, as well. Kobayashi pioneered the enzymatic synthesis of unnatural poly(tyrosine) in aqueous solution by an indirect approach,12 where tyrosine ester hydrochlorides were used as substrates in an HRP-catalyzed polymerization to afford water-insoluble products. Subsequent alkaline hydrolysis yielded poly(tyrosine). In this report, the prepared ECCs are able to induce directly the polymerization of Tyr in water without conducting additional protecting/deprotecting procedures. The bulky hydrophobic dendrons of LD copolymers physically adhere to the Nglycan residues of laccase, effectively serving as hydrophobic pockets for accumulating the water-insoluble monomer via hydrophobic-hydrophobic interactions. These supramolecular complexes could facilitate the mass transfer from the binding domain to the active site of enzyme, as shown in our previous work.17 The enhanced performance provided by ECCs in the biopolymerization of Tyr was evaluated in terms of polymer yield and molecular mass. Both TvL and the TvL/[G3]-PEO13k-[G2] were employed to investigate the characteristic reaction optima in terms of temperature (T) and reaction time (t) with an initial substrate concentration ([Tyr]) at 2 mg/mL. Compared to the native TvL, increase in poly (tyrosine) yields is observed for ECCs at both 22 and 45°C (Table 1). It indicates that the polymerization occurs more efficiently at 45°C probably due to the maximum in catalytic activity and increased mobility of the solid Tyr leading to increased diffusion into the complex and active site. 12


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The effect of two reaction times, 24 and 72 h, on the product yield is shown in Table 1. For each case, the reaction was conducted at 45°C with an initial [Tyr] of 2 mg/mL. With prolonged reaction time (72 h), the yield of obtained poly(tyrosine) increases from 20 to 30 % for TvL (entries 3 and 5, Table 1) and from 57 to 63 % for TvL/[G3]-PEO13k-[G2] (entries 4 and 6, Table 1), respectively. Notable molecular mass increases are also observed, an indication for slow and continuous chain growth over the extended reaction time. The molecular mass increase is not exponential, which might hint to absence of step-growth propagation mechanism. Figure 2 shows the overlaid SEC chromatograms for the poly(tyrosine)s of entries 3-6 in Table 1. Entry 3 is a polymer obtained at 45°C for 24 h with TvL as catalyst and displays similar SEC profile to the polymer produced by the TvL/[G3]-PEO13k-[G2] (entry 4) the latter having slightly higher molecular mass. Identical trends are obtained for the polymers obtained at 72 h (entries 5 and 6). The results suggest that TvL and ECCs polymerize the Tyr under the same mechanism while the ECCs functions more efficiently to promote the polymer production. Table 1. Poly(tyrosine) yield obtained with TvL and TvL/[G3]-PEO13k-[G2] catalysts at different temperature (T) and time (t) with initial [Tyr] = 2 mg/mL. T t Yield MW Characterization by SECa °C h % Mn Mw Mp Mw/Mn 1 TvL 22 24 8.1 1920 5390 2516 3.7 2 TvL /[G3]-PEO13k-[G2] 22 24 22.6 1060 2900 2047 2.7 3 TvL 45 24 19.5 4250 13000 7700 3.1 4 TvL/[G3]-PEO13k-[G2] 45 24 57.2 4810 22100 13010 4.6 5 TvL 45 72 29.9 6500 27220 34500 4.2 6 TvL/[G3]-PEO13k-[G2] 45 72 62.7 7900 56030 33330 7.1 a Determined by conventional calibration with PSS standards, see experimental for details. Entry

Biocatalyst

13


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Molecular mass

Figure 2. Aqueous SEC dRI traces of poly(tyrosine)s: entries 3-6 in Table 1. Entries 3 and 5 are polymers catalyzed by TvL at 45°C with reaction times of 24 and 72 h, respectively; entries 4 and 6 are polymers catalyzed by TvL/[G3]-PEO13k-[G2] at 45°C with reaction times of 24 and 72 h, respectively. See experimental for analysis details. The reaction optima (45°C, 72 h) were then used for the polymerization of Tyr at three distinct concentrations ([Tyr] = 2 mg/mL, 3 mg/mL and 5 mg/mL) to understand the influence of substrate loading (i.e., availability) on the product yield. The results are summarized in Figure 3 and Table 2 and imply that the optimal loading capacity of all ECC catalysts is 2 mg/mL.

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90 80

2mg/mL

3mg/mL

5mg/mL

70 60

Yield, %

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50 40 30 20 10 0

Figure 3. Poly(tyrosine) yield dependence of the initial [Tyr]. The product yields are mean values from 4 replicate reactions with error bars representing a standard deviation.

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Table 2. Molecular mass and yield of poly(tyrosine) obtained by enzymatic polymerization at 45°C, 72 h using ECC with different linear-dendritic copolymers.

Biocatalyst

TvL/[G3]-PEO13k

TvL/[G3]-PEO13k-[G1]


a

[Tyr] mg/mL

Yield %

2

MW Characterization by SECa Mn

Mw

Mp

Mw/Mn

69.8

3500

13930

6500

4.0

3

65.2

2700

11500

11310

4.2

5

41.6

2500

13800

12400

5.5

2

66.7

3400

21400

25800

6.3

3

49.7

2400

13100

9400

5.5

5

40.0

3500

41400

27900

11.8

2

62.7

7900

56000

33300

7.1

3

63.1

2600

13800

11200

5.3

5

47.7

4600

19300

17900

4.2

Determined by conventional calibration with PSS standards, see experimental for details.

Two sets of samples were further characterized under the same conditions (see the experimental section) using an online multi-angle laser light scattering (MALLS) detector in aqueous SEC to give the apparent absolute molecular mass (Figure 4 and Table 3). The relative molar mass of poly(tyrosine) determined using PSS as standard is much lower than the absolute molar mass measured by SEC-MALLS (see Table 3). This noticeable discrepancy could be attributed to a considerably smaller hydrodynamic volume of poly(tyrosine) in water at pH 8.5 compared to the values of PSS standards. Polyanionic PSS adopts an extended conformation at pH 8.5 due to the electronstatic repulsion between the repeating units, while the zwitterionic 16


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poly(tyrosine) has a more compact conformation because the repulsion would be minimized by the presence of positive charge on the same monomer. Another possible explanation for the observed differences in the molecular mass data could be that the poly(tyrosine) has a hyperbranched architecture, leading to small hydrodynamic diameter compared to the linear standards. This hypothesis of a densely branched topology is not supported by DLS measurements, which show that the hydrodynamic volume of poly(tyrosine) is freely tunable (continuously expands and contracts) with pH adjustment. Thus, the polymer predominantly has a linear backbone bearing amino acid zwitterions as the side groups.

Table 3. Comparison of poly(tyrosine) molecular masses determined by conventional SEC versus PSS standards and SEC−MALLS measurements. SEC-PSSb Entrya

Biocatalyst

Mn

Mw

×10-3 (Da)

SEC-MALLSc

Mw/Mn

Mn

Mw

Mp

×10-3 (Da)

Mw/Mn

1

TvL

4.25

13.0

3.1

42.9

62.2

48.8

1.4

2


4.80

22.1

4.6

32.0

61.1

54.9

1.9

3


4.32

23.8

5.5

41.0

82.4

68.4

2.0

4


6.03

45.3

7.5

32.6

80.1

83.1

2.5

a

Entries 1 and 2 are polymers obtained at 45°C for 24 h in the presence of TvL and TvL/[G3]-PEO13k-[G2] respectively; Entries 3 and 4 are polymers isolated from the same reaction solution in the presence of TvL/[G3]-PEO13k-[G2] at two time interval of 24 and 72 h respectively. b Molecular mass obtained by aqueous SEC measurements (PSS standards). c Molecular mass obtained by aqueous SEC−MALLS measurements with refractive index increment dn/dc = 0.212 mL/g.

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Figure 4. Aqueous SEC-MALLS profiles of poly(tyrosine)s (entries 1-4) in Table 3. Traces produced by the 90° detector of the Wyatt MALLS instrument, see experimental part for details. The lines above the chromatography traces show the molecular mass range (left Y-axis) across the elution volume of the peaks.

The

role

of

laccase

in

vivo

is

well

accepted

to

catalyze

the

polymerization/depolymerisation of lignin and/or lignin precursors.21 Herein, additional postpolymerization experiments were designed as depicted in Scheme 2 in an attempt to gain insight into the chain propagation mechanism. Either poly(tyrosine) or a mixture of poly(tyrosine) and Tyr were used as the starting substrates for both TvL and ECC. In the case of TvL (Scheme 2a), the initial poly(tyrosine)-A (produced by the enzyme complex at 45°C for 72h, entry 3 in Table 1 and Figure 2) shows signs of decomposition after incubation as indicated by a appearance of a significant lower molecular mass shoulder in the recovered poly(tyrosine)-B (Figure 5a, green trace) and Mp decreases from 7700 to 3700 with Mw/Mn = 4.5 vs the initial value of 3.1. The addition of Tyr monomer to poly(tyrosine)-A generates a product, poly(tyrosine)-C, with a 18


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similar molecular mass distribution as poly(tyrosine)-B (Figure 5a, blue trace) and the same trend of decreasing molecular mass. The results indicate that the enzyme could primarily induce a radical group formation in the soluble poly(tyrosine)-A, which undergoes a reversible additionfragmentation process with two competing reactions running simultaneously: chain growth with the freshly added Tyr and chain degradation of the incubated polymer. A parallel experiment was carried out in the presence of ECC as catalyst and the same poly(tyrosine) A (Scheme 2b). A similar decomposition process is occurring, but at a much slower rate as evidenced by the small changes in the molecular mass distributions of the isolated product - poly(tyrosine)-B’ vs the initial polymer - Mw/Mn = 3.3 vs the initial value of 3.1 (Figure 5b, red trace and blue trace, respectively). Impressively, when both Tyr and poly(tyrosine)-A were added to the lineardendritic laccase complex, a polymer with profound molecular mass increase and broadened molecular mass distribution was isolated - poly(tyrosine)-C’ Mp = 12200 with Mw/Mn = 6.8 (Scheme 2b and Figure 5b, green trace). The chain extension of the preformed poly(tyrosine) indicates that with the assistance of LD copolymers sufficiently large amounts Tyr radicals are produced to couple with the accessible chain ends of the water-soluble preformed polymer. This “quasi-living” character of the polymer-supported enzymatic polymerization might open new avenues in the construction of multi-block copolymers.

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Scheme 2. Post-polymerization reactions of poly(tyrosine)-A with and without monomer (Tyr) addition at 45°C for 72 h: (a) in the presence of TvL; (b) in the presence of ECC.

Molecular mass

Molecular mass

Figure 5. SEC studies of post-polymerization reactions as illustrated in Scheme 2. dRI detector signals are shown in color corresponding to the color of the products in Scheme 2. Poly(tyrosine)-A: preformed polymer isolated, purified and added to a new enzymatic solution; poly(tyrosine)-B and B’: polymers isolated after incubation of poly(tyrosine)-A with TvL and ECC, respectively; poly(tyrosine)-C and C’: polymers isolated after incubation of poly(tyrosine)A and new portion of Tyr with TvL and ECC, respectively, see Scheme 2. 20


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Scheme 3. Postulated initial radicals and their coupling products from the Tyr oxidative polymerization, catalyzed by TvL/LD copolymer complex.

3.2. Characterization of poly(tyrosine). Typically, the catalytic cycle of laccaseinduced polymerization involves a one-electron mechanism. Initially, the enzyme oxidizes the substrate to generate free radicals (Scheme 3), which undergo successive oxidative couplings to form a polymer. The enzyme shows a high chemoselectivity to phenol groups, but lacks regioselectivity by producing either C-C or C-O coupling products as elucidated in Scheme 3 and Figure 6a,b. The chemical composition of poly(tyrosine) is investigated using NMR analysis. As illustrated in Figure 7, the 1H NMR spectrum reveals characteristic broad peaks, which may 21


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result from the combined effects of polydispersity and backbone stiffness (low mobility of the polymer repeating units). The signals ranging from 2.2–3.7 ppm, 3.4–4.0 ppm and 6.1–8.0 ppm are assigned to H7(Hβ), H8(Hα) and aromatic protons, respectively(structures shown in Figure 7a,b). In particular, the sharp peaks embedded in the aromatic region (Figure 7a) could be assigned either to the corresponding a, b, c and/or a’, b’ protons of the end groups (Figure 6c,d) due to their increased degree of mobility or to the aromatic protons of an unreacted monomer, which might have been co-precipitated during the isolation of the polymerization products. In order to avoid further ambiguity poly(tyrosine)s from different polymerization experiments were subjected to dialysis over 3 days (membrane molecular mass cut-off = 3 kDa). While two of the sharp doublets were not observed, the other signals persistently appeared in the NMR spectra (Figure 7b). Their assignment as signals from tyrosine monomer is excluded for two reasons: (1) a complete purification has been carried out to remove any residual monomer and (2) the 1,4substituted tyrosine monomer would give rise to only two inequivalent resonances due to the 2 ortho- and 2 meta protons instead of the three peaks displayed in the spectrum. The assignments are shown in Figure 6c,d. The chemical shifts of the aromatic carbon atoms, which are closely related to π-electron densities22 might be able to point toward the coupling manner of two adjacent Tyr monomers leading either to C-C or C-O linkages. For a C-C coupling block (Figure 6a), both ortho positions (C2,6) participate in the covalent bond formation of the polymer backbone. As for the case of C-O coupling (Figure 6b), the ortho position corresponding to the phenoxy moiety (C-O bond) (C3, 6’) remains free of substitution and should have a high πelectron density due to the inductive effect. As a result, the

13

C carbon shifts of C3 and C6’

(Figure 8b) could be utilized as a characteristic carbon shift to distinguish C-O coupling from CC coupling. Therefore, a 2D 1H-13C DEPT-HSQC analysis (Figure 8) enhanced with solvent 22


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1 13 (D2O) suppression was performed to extract more information from the H- C correlations to

identify C-C and C-O coupling. The aromatic protons correlate to two dominant carbons shifts at (7.14, 130.19) ppm and (6.93, 117.04) ppm. The signal located at 130.19 ppm is assigned to all the meta carbon atoms, while the correlation at 117.04 ppm is the typical resonance from ortho atoms, which exclusively results from C-O coupling.

Figure 6. Structures of poly(tyrosine) with C-C coupling (a) and C-O coupling (b); end group of C-C coupling (c), and C-O coupling (d).

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(a)

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

(b)

Figure 7. (a) 700 MHz 1H NMR spectrum of poly(tyrosine) recorded at 22°C in D2O/NaOD with presaturation of the water signal at 4.72 ppm. Impurity peaks are marked with (×); (b) 600 MHz 1H NMR spectrum of purified poly(tyrosine) recorded in D2O at 50°C. Proton assignments are shown in Figure 6c,d. 24


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(a)

6

HDO

1

2

6

2’ 3’

1

2”

1’

H7(Hβ)-C7(Cβ)

8 4’ 7

5

3 4

3

5

6’

4

7(β)

5’

H8(Hα)-C8(Cα)

7(β) 8(α)

C-C coupling

8(α)

C-O coupling

Figure 8. 2D 1H-13C DEPT-HSQC NMR spectrum of poly(tyrosine), recorded in D2O and the assignment of the proton and carbon atoms. (a) full range 1H-13C correlation, (b) 1H-13C correlation of the aromatic region. 25


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Both 2D COSY and NOESY spectra (an example is shown in Figure 9) show weak coupling among the adjacent protons in the Tyr repeating units [a, b to H7(Hβ); and H7(Hβ) to H8(Hα), see Figure 6 for proton assignments]. These are expected interactions, however, and no conclusions concerning the structural folding (tertiary structure) can be drawn from these measurements.

Figure 9. 2D NOESY spectrum of poly(tyrosine) recorded at room temperature in D2O/NaOD at 700 MHz, with a mixing time of 0.6 s (see figure 7a for the corresponding 1HNMR spectrum). 26


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Explicit evidence for the formation and further polymerization of the possible resonance structures (A, B and C) in Scheme 3 could be also obtained via MALDI-TOF mass spectrometry. The spectrum in Figure 10 shows the presence of polymerization products with degree of polymerization (DP) between 3 and 11, which are separated from each other by 179 m/z [M 2H]+. Further confirmation of the isolated polymers is provided by FT-IR spectra which indicate that the monomer addition proceeds randomly through the formation of C-C and/or C-O linkages in the phenol groups as shown in Figure 11. Quantitative information for the relative content of these linkages in the polymer backbone could not be achieved at this time. Titration with FC reagent15 shows the presence of 21-24 % phenol groups, an amount only partially supported by the FT-IR analyses. For example, as shown in Figure 11, the broad absorption in the 3600-2300 cm-1 region results from superimposed O-H and NH3+ stretching bands. The aliphatic C-H stretching results in a strong peak at 2882 cm-1 while the aromatic C-H stretching in the region of 3045-3013 cm-1 becomes relatively weak as a result of polymerization. The sharp and broad peak at 1601 cm-1 presents a superimposing of (C=O)-O and aromatic C=C stretching. The O-H and C-O(H) stretching vibration of the tyrosine phenol group at 3201 and 1242 cm-1 are barely visible in the polymer, where the C-O-C stretching vibration at 1099 cm-1 is dominant.

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Figure 10. Representative MALDI-TOF MS spectrum of poly(tyrosine).

Figure 11. FT-IR-ATR spectra of monomer DL-Tyr (bottom) and poly(tyrosine) (top). 28


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TGA of poly(tyrosine) under nitrogen shows a total weight loss of 60 % up to 700°C accompanied by three distinct weight loss stages (Figure 12). The result implies that the unnatural poly(tyrosine) displays thermal stability up to 675°C without the breakdown of its polyaromatic backbone. At the first weight loss stage (T< 100°C) TGA registers a 7 % weight loss resulting from the loss of either free or H-bonded water. This is followed by a 16 % weight loss (theoretical, 25 wt %) at 221°C (the temperature reported was taken as midpoint of the weight loss at the inflection point of the TGA derivative curve), which might be associated with decomposition of the carboxylic acid group on the side chain. The next two consecutive weight losses at 384 and 507°C correspond to the breakdowns of amine and phenol group respectively (21 and 15 wt% theoretically). However, the apparent lower weight losses compared to the theoretical ones indicate incomplete decompositions. It could be primarily attributed to the heat consumption/dissipation needed to break the inter- and intramolecular electrostatic interactions when a constant heating rate is used in TGA analysis. No melting or glass transitions were detected by DSC measurements (Figure 13), which suggests that strong electrostatic interactions between the zwitteronic polymer chains strongly hamper the segmental chain movement and consequently suppress any visible thermal transitions.

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Figure 12. TGA thermogram of poly(tyrosine) samples analyzed from room temperature to 700°C at 20°C/min under nitrogen flow.

Figure 13. DSC cooling and heating runs for the poly(tyrosine).

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3.3. Aqueous solution properties of poly(tyrosine). As a characteristic feature, polyzwitterions composed of acidic and basic species possess an isoelectric point (IEP), at which the negative and positive charges are fully balanced and the polymer is electrically neutral. Aqueous electrophoresis study via zeta potential measurement (Figure 14) indicates that the net charge on the poly(tyrosine) (Entry 6 in Table 1) is close to zero at pH 2.6. Either side of this IEP, the polymer has either positive zeta potential because of amine protonation or negative zeta potential as a result of the formation of carboxylate anions at high pH values. As pH is adjusted from the IEP below, both amine and carboxylate groups get protonated along the polymer chain, converting the polybetaine into a cationic polyelectrolyte. While at pH greater than 2.6, positively charged amine and carboxylic acid groups undergo a gradual increase of the degree of deprotonation, which affords the poly(tyrosine) predominant negative charge to give a anionic polyelectrolyte. Thus, the poly(tyrosine) with intact amino acid moieties displays pH-responsive and reversible transition from polycation, polyzwitterion to polyanion in a salt-free aqueous solution.

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Figure 14. The variation of zeta potential as a function of pH for a 1.0 mg/mL aqueous solution of the poly(tyrosine) at 25°C. The conformational behavior of the zwitterionic poly(tyrosine) is strongly influenced by the nature and net charge of the polymer chain. As to the pH-responsive poly(tyrosine), the conformational change is detected by measuring the hydrodynamic diameter (Dh) variation as a function of solution pH (Figure 15). All analyte solutions were prepared from a stock solution by adjusting to the desired pH using NaOH and HCl at low ionic strength (~0.01 M). At around the IEP point, the polymer shows both intra- and intermolecular associations driven by the electrostatic attractions. Upon dilution with NaCl (aq.), intramolecular interaction dominates and the polymer is in its most compact conformation as reflected by the minimum in the hydrodynamic diameter, measured to be 2.4 nm (Figure 15 and Figure 16, green/left peak). In contrast, as polymer concentration increases in the IEP region, a dramatic increase in Dh is measured to be the onset of precipitation due to the strong intermolecular associations as shown 32


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in Figure 16 red/right peak. When the value of pH drops to 1.2, the protonated polymer chain adopts an extended conformation caused by the electrostatic repulsion between the repeat units, giving an increased Dh of 6.7 nm. Adjusting pH above the IEP, the columbic repulsion between the negatively charged repeating units similarly causes the polymers to adopt extended conformations in solution. The Dh undergoes a gradual increase to 10 nm at pH =11.8. The greater size of the anionic poly(tyrosine) is attributed to the further deprotonation of phenol group (pKa=10.07) into a phenoxide anion resulting in a doubly negatively charged repeating unit. It should also be noted that the poly(tyrosine) shows an anti-polyeletrolyte behavior. At the IEP, the diluted polymer shows no expansion of Dh upon addition of salt, the only result being an increased solubility due to the reduced intermolecular interactions caused by the salt screening effect.

Figure 15. Variation of Dh as a function of solution pH for aqueous solutions of poly(tyrosine) at 25°C. Polymer concentrations are 1.1-1.5 mg/mL with exception at pH 2.6 where concentration of ~0.3 mg/mL was used. 33


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Figure 16. Hydrodynamic diameter, Dh, of poly(tyrosine) at IEP (pH=2.3-2.6) for a diluted (green) and concentrated (red) aqueous solution at 25°C. The pH-responsive and polyelectrolyte-polyzwitterion tunable properties make poly(tyrosine) a promising candidate in developing anti-fouling materials.23 In addition, it could be potentially used as non-viral vector for gene delivery.24 As to this application, the ammonium cation of poly(tyrosine) has potential to complex DNA,24,25 while its carboxylate anion permits the shielding of inter-complex coacervation, which remains a major problem for positively charged polyelectrolyte/DNA complexes. In order to assess the applicability of poly(tyrosine) for biomedical use the toxicity of the polymer towards normal and tumor cell lines is briefly investigated in pilot experiments.

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Figure 17. Cytotoxicity of poly(tyrosine) against the Raji and CHO cell lines. The cytotoxicity assessment of poly(tyrosine) in culture was evaluated via the MTT assays on two cell lines (Raji and CHO) with concentrations ranging from 0.3 to 500 µg/mL (Figure 17). In all cases, these preliminary results confirm that the poly(tyrosine) presented here shows no significant cytotoxicity over relatively broad concentration range. Therefore, this polybetaine should be suitable for biomedical applications. Detailed biological evaluation of this new class of polymer is now underway.

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4. Conclusions The results obtained confirm that both laccase and its complexes with LD copolymers are able to catalyze the polymerization of Tyr. Only low polymer yields are achieved with native TvL, due to the limited “availability” of the substrate, which is not soluble in aqueous media. The LD copolymer assisted biocatalysis, on the other hand, allowed for efficient and direct synthesis of zwitterionic pseudo-poly(amino acid)s without the need for additional protecting group chemistry and subsequent deprotection procedures. The resulting polymers have high molecular masses and water solubility in salt-free aqueous media over a wide pH range, exhibiting a rich solution behavior. The nature of the surface charge, the charge density of polymers, solution conformation and their Dh can be tuned simply by adjustment of solution pH. Of particular importance is the observed “quasi-living” behavior of the polymerization system with linear-dendritic laccase complexes as initiators. This phenomenon is currently further explored for the one-pot formation of block copolymers.

5. Acknowledgments The authors wish to thank Prof. Mathew M. Maye (Syracuse University) for provided access to DLS instrumentation, Michael Rooney (REU student from Northwestern University) for his assistance with the DLS measurements and Prof. Juntao Luo (SUNY Upstate Medical University) for the toxicity evaluation. Partial funding for this study, provided by NSF (CBET 0853454 and EEC-1156942) is gratefully acknowledged.

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ToC Graphic

“Green” Synthesis of Unnatural Poly(Amino Acid)s with Zwitterionic Character and pHresponsive Solution Behavior, Mediated by Linear-Dendritic Laccase Complexes Ivan Gitsov, Lili Wang, Nikolay Vladimirov, Arsen Simonyan, David J. Kiemle, Andri Schutz

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Synthesis of Unnatural Poly(Amino Acid) - American Chemical Society

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