(Nitrilotriacetic Acid)-End-Functionalized ... - ACS Publications

Chapter 20

Downloaded by CORNELL UNIV on May 29, 2012 | http://pubs.acs.org Publication Date (Web): March 20, 2012 | doi: 10.1021/bk-2012-1101.ch020

(Nitrilotriacetic Acid)-End-Functionalized Polystyrenes Synthesized by ATRP Mohammad Abdul Kadir,1 Hong Y. Cho,1 Bong-Soo Kim,1 Young-Rok Kim,2 Sun-Gu Lee,3 Unyong Jeong,4 and Hyun-jong Paik*,1 1Department

of Polymer Science & Engineering, Pusan National University, San 30 Jangjeon 2-dong Geumjeong-gu, Busan 609-735, Korea 2Department of Food Science, and Biotechnology & Institute of Life Sciences and Resources, College of Life Sciences, Kyung Hee University, Yongin, Korea 3Department of Chemical Engineering, Pusan National University, San 30 Jangjeon 2-dong Geumjeong-gu, Busan 609-735, Korea 4Department of Materials Science and Engineering, Yonsei University, 134 Shinchon-dong, Seoul, Korea *E-mail: [email protected]

The synthesis of nitrilotriacetic acid end-functionalized polystyrenes (NTA-PS) using t-butyl protected NTA initiators via atom transfer radial polymerization (ATRP) of styrene is described. The protected t-butyl group is subsequently removed at the α-chain end of polystyrene. After complexation of NTA chain ends of the polystyrenes with Ni2+ to produce Ni-NTA-PS, conjugation with (six histidine-tagged green fluorescent protein) His6-GFP via Ni2+/histidine (His) interaction and the self-assembly of the resulted bioconjugate are studied. Highly mesoporous NTA-PS fibers are also produced by taking advantage of interpenetrating phase separation between PS and poly(ethylene oxide) (PEO) during electrospinning. The specific interaction of Ni2+-complexed NTA-PS fibers with His enables to immobilize and purify only target proteins from highly heterogeneous protein mixtures.

© 2012 American Chemical Society In Progress in Controlled Radical Polymerization: Materials and Applications; Matyjaszewski, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Introduction

Bioconjugation of synthetic polymers with modified proteins have potential applications in biotechnology as improving the solubility, biocompatibility, and stability of hydrid materials (1). Generally polymer-protein bioconjugates are prepared by the reaction of pre-synthesized end-functional polymers with proteins (2). Recent advances in polymer synthesis, in particular, controlled radical polymerizations (CRPs), provided avenues to polymers with well-defined composition, molecular shape, chain length, and α,ω-functionality (3). The reactive chain-ends has been used to conjugate functional proteins. For example, using CRPs (4), well-defined polymers with N-succinimide ester (5, 6), aldehyde (7), thiol, maleimide (8, 9) or pyridyl disulfide (10) chain-ends have been used to conjugate with targeted proteins. Nitrilotriacetic acid (NTA) is a tetradentate ligand which occupies four of the six binding sites of Ni2+, leaving two free sites for histidine-tagged proteins (His-tagged proteins) to bind. The magnetism of the use of NTA and Ni2+ complexes is the selective binding due to the strong coordination with histidines. The NTA chelating chemistry with His-tagged proteins offers several advantages of biochemical recognition elements (11). First, fast and reversible reaction of Ni2+ with histidines, where competitor ligand, such as imidazole could relocate the histidine-tagged proteins. Second, Ni2+-NTA chemistry is a very well defined and efficient immobilization system in biology. NTA chelated with transition metals were successfully applied for protein purification (12) and detection (11, 13), as well as for surface immobilizations (14–18) and for tethering to lipid membranes (19–21). Conjugation of protein with polymers via NTA chemistry has been widly studied on selective protein binding. Kiessling et al. synthesized poly (N-methacryloxysuccinimide), where NTA derivatives were appended to the polymer backbone. After nickel complexation, histidine-tagged fibroblast growth factor 8b (His-tag FGF-8b) was clustered with NTA moiety through noncovalent interaction (22). Ober et al. polymerized acrylamide monomer with an NTA moiety, which was incorporated into 2-hydroxyethyl methacrylate (HEMA) hydrogels in a controlled fashion for the specific protein immobilization (23) Ramstedt et al. prepared NTA functionalized protein-resistant poly(oliogoethylene glycol methacrylate) (POEGMA) polymer brushes (24). POEGMA brush-coated surface prevent nonspecific interactions with original substrate. In this chapter, we will review the synthesis and characterization of NTA functionalized polystyrenes prepared by atom transfer radical polymerization (ATRP), one of CRPs technique, and their applications in bioconjugation and protein purification based on our recent publications (25, 26).

304 In Progress in Controlled Radical Polymerization: Materials and Applications; Matyjaszewski, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Synthesis and Characterization of NTA End-Functionalized Polystyrenes


The NTA end-functionalized polystyrenes can be synthesized using initiators (2a or 2b) by ATRP in the presence of CuCl catalyst system (Scheme 1). The use of 4,4′-di(5-nonyl)-2,2′-bipyridine (dNbpy) instead of N,N,N′,N′,N′′-pentamethyldiethylenetriamine (PMDETA) and employing halogen exchange technique (27) provide better control on polymerizations (Table I).

Scheme 1. Preparation of Ni2+-complexed-NTA end-functionalized polystyrene (Ni-NTA-PS). (Reproduced with permission from reference (25). Copyright 2011.)

Polymerization with initiator 2b together with dNbpy yields well-defined polystyrene, 5 (entries 4-6 in Table I) as 2b initiator shows fast activation rate. In addition, fast deactivation rate with bpy based catalyst also contributes to the improvement of initiation efficiency (28). The polymerization of styrene with 2b along with CuCl/dNbpy catalyst system shows typical characteristics of living polymerization. The first order kinetic plot is linear and the rate of radical generation is faster than that of 2a. The molecular weight increases linearly with conversion, which shows good agreement with theoretical molecular weight. The molecular weight distribution is as narrow as 1.09 (Figure 1 and Figure 2).



Table I. ATRP of styrene using NTA derivative initiators at 110 °C. (Reproduced with permission from reference (25). Copyright 2011.) Mw /Mn

Mn, theorya [103]

154

1.45

24.7

26

170

1.67

18.7

5.75

27

11.0

1.26

6.1

CuCl/2dNbpy

14

55

13.5

1.21

11.8

2b

CuCl/2dNbpy

14

72

4.9

1.09

4.2

2b

CuCl/2dNbpy

20

70

3.9

1.08

2.7

No.

[M]0 /[I] 0

[I]0

Catalyst

Time [hr]

Conv. [%]

1

672

2a

CuBr/PMDETA

17

34

2

672

2a

CuCl/PMDETA

7

3

196

2a

CuCl/2dNbpy

4

196

2b

5

48

6

29

Mn, GPC [103]

1: [M]0=7.9 M, [I]0=[CuBr/PMDETA]0=1.2 × 10-2 M, anisole=7.8 × 10-1 mL. 2: [M]0=4.3 M, 2[I]0=[CuCl/PMDETA]0 = 1.3 × 10-2 M, anisole = 7.8 mL. 3: [M]0=4.3 M, 2[I]0=[CuCl/2dNbpy]0 = 4.5 × 10-2 M, anisole = 1.5 mL. 4: [M]0=4.3 M, 2[I]0=[CuCl/2dNbpy]0 = 4.5 × 10-2 M, anisole = 1.5 mL. 5: [M]0=4.3 M, 2[I]0=[CuCl/2dNbpy]0 = 1.8 × 10-1 M, anisole = 1.0 mL. 6: [M]0=4.3 M, [I]0=[CuCl/2dNbpy]0 = 1.5 × 10-1 M, anisole = 3.0 mL. a Mn, theory = conversion × ([M]0/[I]0) × Mmonomer + Minitiator.

Figure 1. Results of styrene ATRP using 2b (entry 5, Table I); (A) first-order kinetic plots, (B) Mn and Mw/Mn evolution on monomer conversion. (Reproduced with permission from reference (25). Copyright 2011.)



Figure 2. Gel permeation chromatogram traces of NTA-PS (entry 5, Table I). (Reproduced with permission from reference (25). Copyright 2011.)

The presence of NTA moiety in PS (entry 6 in Table I) can be verified by 1H NMR (Figure 3). Peaks at 1.33 (a, (CH3)3-) and 1.35 ppm (b, (CH3)3-) are assigned to t-butyl protons and peaks at 2.71 (h, -CH2-), 3.15 (d, -CH-), 3.38 (c, -CH2-) 4.25 (n, -CH-Cl) and 5.17 (i, -NH-) ppm are clearly assigned to NTA moiety in Figure 3 (A). Based on the integral ratio of peak h (2.6-2.8 ppm), to the phenyl ring proton (6.2-7.4 ppm), the Mn of 3b is calculated to be 4,750 g/mol. Mn calculated from 1H NMR agrees well with those from GPC (Mn, GPC = 4,900).



Figure 3. (A) 1H NMR spectra of α–(p-NTA)-polystyrene, 3b, (Mn, GPC = 3,900; Mw/Mn = 1.08,), CDCl3 100%, and (B) α-(NTA)-polystyrene, 4b, (Mn, GPC = 3,500; Mw/Mn = 1.18), mixed solvent system (CDCl3/MeOH-d4 = 99/1 by vol. %). (Reproduced with permission from reference (25). Copyright 2011.)

Applications of NTA End-Functionalized Polystyrenes NTA-modified polystyrene and its chemistry to complex with Ni2+; which was explained in introduction part, enable NTA-PS to become a functional material. Accordingly it has applications in bioconjugation and bioseparation. 308 In Progress in Controlled Radical Polymerization: Materials and Applications; Matyjaszewski, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Formation of Micellar Aggregates and Conjugation with Protein


NTA-PS has an amphiphilic property due to hydrophilic NTA moiety and hydrophobic PS chain. Therefore, it produces micellar aggregates when water is added to THF solution containing 4b (without Ni2+) and 5b (with Ni2+), respectively. SEM and TEM studies show that the size of aggregates is ~500 nm (Figure 4). Aggregates are spherical and relatively uniform in size. Energy dispersive x-ray (EDX) fluorescence analysis confirms the presence of Ni2+ in the aggregates.

Figure 4. SEM and TEM images of the aggregates from the α-(NTA)-polystyrene; A-1 and A-2 are without nickel, B-1 and B-2 are with nickel. (Reproduced with permission from reference (25). Copyright 2011.)

His6-GFP binding property on the spherical aggregates produced from 4b (without Ni2+) and 5b (with Ni2+) is observed with fluorescence microscopy (Figure 5). The fluorescence intensity of 4b (A-1) is weaker than that of 5b (B-1), which is due to the absence of nickel for chelating between His6-GFP and NTA on the surface of spherical aggregates specifically (4b). According to Figure 5, the adsorption of His6-GFP on the surface of spherical aggregates of 4b is irreversible and non-specific, because the fluorescence intensity is rarely reduced even after treating with excess imidazole. However, in the case of 5b, the fluorescence intensity decreases from 100 (before rinsing with imidazole, Figure 5, B-1) to 14 (after rinsing with imidazole, Figure 5, B-2) due to the specific and reversible binding of nickel on the surface of 5b with His6-GFP.



Figure 5. Fluorescence microscope images of the aggregates from the α-(NTA)-polystyrene; A-1; non-specific binding of His6-GFP with 4b, A-2; after rinse with excess imidazole, B-1; Specific binding of His6-GFP to NTA of 5b in presence of Ni, B-2; after rinse with excess imidazole. (Reproduced with permission from reference (25). Copyright 2011.)

On the other hand, slow addition of Ni-NTA-PS (Mn=21,800) dissolved in DMF (4 vol. % of water) to deionized water containing His6-GFP produces well defined micelles due to specific interaction between polymer and protein via NTANi2+/His6-tag interaction and amphiphilic nature of the resulted bioconjugate. The size of micelles decreases when competitor ligand (e.g.; imidazole) is added to the polymer-protein micellar solution due to the release of GFP by imidazole as is evidenced by TEM and DLS studies (Figure 6). We also studied the effect of solvents (DMF and THF) on self-assembly structures of resulted bioconjugate following the slow addition of polymer (Ni-NTA-PS, Mn=21,800) dissolved in respective solvents to deoinized water containing His6-GFP under stirring. We observed that H2O/DMF system produced well defined micellar aggregates, while H2O/THF produced ill defined and larger aggregates (Figure 7). This might be due to poor solubility of His6-GFP in THF.



Figure 6. TEM and DLS data of Ni-NTA-PS (Mn=21,800) with His6-GFP in water/DMF: (A) after conjugation (B) after addition of excess imidazole. Scale bar of TEM images = 500 nm. (Reproduced with permission from reference (25). Copyright 2011.)

Figure 7. TEM images of Ni-NTA-PS (Mn=21,800) with His6-GFP: (A) in water/DMF (B) in water/THF.

Formation of Mesoporous Fibres for Protein Purification Highly mesoporous NTA-PS fibers could be produced by employing the phase separation between NTA-PS and polyethyleneoxide (PEO) during electrospinning. A mixture solution of PEO and NTA-PS was electrospun on a collector substrate. The polymer solution in chloroform (9 wt % polymer in total solution) was accelerated through a metal needle at 12 kV. Diameter of the as-spun fibers was about 1 μm (Figure 8(A)). Due to the poor miscibility between the two polymers, the polymers phase separated as the concentration increased during electrospinning. The equivalent mixture of the two polymers (1:1, w/w) forms a bicontinuous network structure inside the as-spun fibers. The porous feature on the fiber surfaces is shown in Figure 8(B). The pore size on the 311 In Progress in Controlled Radical Polymerization: Materials and Applications; Matyjaszewski, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


fiber surfaces ranged from 50 to 300 nm. The fractured cross-sectional image in Figure 8(C) displays the interpenetrating mesopores. The TEM image in Figure 8(D) clearly visualizes the mesoporous structure of the fibers. The mesoporous structure creates a lot of surfaces and helps the diffusion of proteins, which is effective for a rapid binding-and-elution response.

Figure 8. SEM and TEM images showing the mesoporous structure of NTA-PS fibers. (A,B) Low and high magnification of mesopores on the fiber surfaces, (C) cross-sectional SEM image of a fractured fiber, and (D) TEM image showing 3D interpenetrating pores. (Reproduced with permission from reference (26). Copyright 2010.)

These highly mesoporous NTA-PS fibers could be utilized for the functionalized microfluidic channels. The PDMS molds were directly placed on mesoporous fiber mats. The top and bottom were covered with slide glasses and tightly pressed (Scheme 2). The pressurized nanofibers by the molds were densified and did not allow water penetration. Meanwhile, the unpressurized area maintained the loose structure of the as-spun fibers so that water absorption was immediate. The unpressurized region could play as the microfludic channels. The specific affinity of Ni to imidazole moieties in histidines enabled large amount of specific binding to the recombinant proteins. Supplying pure imidazole replaced the binding and released the proteins. 312 In Progress in Controlled Radical Polymerization: Materials and Applications; Matyjaszewski, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Scheme 2. Layout of the microfluidic device and illustration of purification of histidine-tagged proteins using the mesoporous NTA-PS fibers. (Reproduced with permission from reference (26). Copyright 2010.)

Conclusions The synthesis of nitrilotriacetic acid end-functionalized polystyrenes (NTAPS) using t-butyl protected NTA initiators via atom transfer radial polymerization (ATRP) of styrene is described. Due to presence of NTA moiety, NTA-PS has potential applications in bioconjugation and protein purification.

Acknowledgments This work was supported by the Active Polymer Center for Pattern Integration (R11-2007-050-02002-0) and WCU (World Class University) program (R33-10035-0) through the National Research Foundation (NRF) grant funded by the Korea government (MEST).

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(Nitrilotriacetic Acid)-End-Functionalized ... - ACS Publications

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