VBA dodecyl methacrylate. DMA. The preparation of these new polymers was investigated via both free radical polymerization (RP) and atom transfer radical ...
SOLUTION SELF-ASSEMBLY OF SYNTHETIC COPOLYMERS BEARING COMPLEMENTARY NUCLEIC ACID FUNCTIONALITIES Jean-François Lutz,*1 Rainer Nehring1 and Andreas F. Thünemann2 1
Fraunhofer Institute for Applied Polymer Research Geiselbergstrasse 69, Golm 14476, Germany 2 Federal Institute for Materials Research and Testing Richard-Willstaetter-Strasse 11, 12489 Berlin, Germany
Introduction The self-assembly of low molecular weight or macromolecular buildingblocks are straightforward routes for constructing organized nanostructures.1 Such processes are typically driven by various types of supramolecular interactions such as van der Waals forces, π-π interactions, electrostatic interactions or H-bonding.2 Hence, for macromolecular self-assembly, polymeric building-blocks capable of creating such intermolecular interactions are needed: polymeric amphiphiles, polyelectrolytes or polymers bearing Hbonding moieties. In the latter case, natural macromolecules such as DNA or RNA are very influential, since they are known to undergo complex Hbonding directed self-assembly. Thus, the preparation of synthetic polymers, which similarly to DNA or RNA bear nucleic acid moieties (thymine, uracil, adenine, cytosine or guanine) was always an important challenge of polymer chemistry.3 In that context, the present work reports the synthesis and characterization of a new class of nucleic acids substituted polymers prepared via the radical copolymerization of dodecyl methacrylate (DMA) with styrenic derivatives containing either thymine or adenine moieties (Scheme 1). Scheme 1. Molecular structure of the monomers used in the present study.
O
N
N
O
O 1-(4-vinylbenzyl)thymine VBT
NH2
N
Results and Discussion Synthesis of nucleic acid containing copolymers. The radical polymerization of nucleic acid substituted monomers VBT and VBA was studied. Both monomers could be homopolymerized at 60°C, using AIBN as an initiator. However, the resulting homopolymers were found to be only soluble in a few polar aprotic solvents. In order to prepare more practical polymers bearing nucleic acids functionalities, we studied the radical copolymerization of dodecyl methacrylate (DMA) with either VBT or VBA. DMA (Scheme 1) was chosen as a comonomer for two main reasons. First, this monomer possesses long alkyl chains, which allow solubility in a large number of organic solvents. Moreover, the reactivity ratios of methacrylates and styrene derivatives are usually below unity, which suggest a tendency towards alternation of the comonomers during copolymerization. The latter was targeted since it would lead in the present case to the formation of polymers with a an homogeneous chain to chain composition (i.e. no composition drift) and a quite regular distribution of nucleic acids along each chain. Table 1. Preparation of Nucleic Acids Substituted Polymers Via RP or ATRP.a M1b
N
O HN
0.02 mmol) was added via a syringe. The mixture was heated at 90°C in an oil bath. After 20 hours, the experiment was stopped by opening the flask and exposing the catalyst to air. The final polymer was purified by precipitation in methanol, filtrated and dried under vacuum. Analytical measurements. Molecular weights and molecular weight distributions were determined by SEC performed in either THF or NMP as eluent. Monomer conversions were calculated by 1H NMR (spectra were recorded in DMSO-d6 on a Bruker DPX-400 operating at 400.1 MHz). For UV-Vis, FT-IR and optical microscopy, the studied polymers were directly added in CHCl3 with a concentration in the range 1-10 mg.mL-1 depending on the measurements. Before each measurement, the solutions were heated for five minutes at 60°C in a close tube. UV spectra were recorded on a Cary 5000 UV-VIS-NIR spectrometer from Varian. FT-IR spectra were recorded using a FT-IR spectrometer Equinox55 from Bruker. Optical micrographs were made using an Olympus microscope BH-2 with a 50X objective.
N 9-(4-vinylbenzyl)adenine VBA
dodecyl methacrylate DMA
The preparation of these new polymers was investigated via both free radical polymerization (RP) and atom transfer radical polymerization (ATRP). Moreover, their solution self-assembly was studied by UV-Vis spectroscopy, FT-IR spectroscopy and light microscopy. Experimental Materials. VBT,4 VBA4 and BPED5 were prepared according to procedures reported in the literature. Ethyl 2-chloropropionate (97%) and DMA (96%) were both purchased from Aldrich and used as received. 2,2’Azobisisobutyronitrile (AIBN) was recrystallized from ethanol, filtered and dried. Copper(I) chloride (Aldrich, 98+%) was washed with glacial acetic acid in order to remove any soluble oxidized species, filtered, washed with ethanol and dried. General procedure for conventional radical polymerization (example). In a Schlenk tube were added VBT (242 mg, 1 mmol), DMA (254 mg, 1 mmol), AIBN (3.3 mg, 0.02 mmol) and 1.2 mL of DMF. The tube was closed with a septum and the solution was purged with dry argon for 20 minutes. Subsequently, the mixture was heated at 60°C in an oil bath. After 20 hours, the experiment was stopped and the resulting polymer was purified by precipitation in methanol, filtrated and dried under vacuum General procedure for atom transfer radical polymerization (example). Copper chloride (4 mg, 0.04 mmol), VBT (242 mg, 1 mmol), DMA (254 mg, 1 mmol) and 1 mL of DMF were added to a flask sealed with a septum. The solution was purged with dry argon for 20 minutes. Then, BPED (21.8 mg, 0.04 mmol) was added through the septum with a syringe and the mixture turned homogeneous. Last ethyl 2-chloropropionate (2.74 mg,
M2b
Temp time conv. (°C) (h) M1 RP VBT DMA 60°C 20 0.85 RP VBA DMA 60°C 20 0.76 ATRP VBT DMA 90°C 20 0.96 ATRP VBA DMA 90°C 66 0.66 a Reaction solvent was DMF in all cases ; RP: ATRP: M1/M2/ECP/CuCl/BPED = 50/50/1/2/2. b monomer 2.
conv. Mw/Mn Mn M2 0.81 41000 2.1 0.64 25000 1.8 0.94 17400 1.35 0.55 7400 1.35 M1/M2/AIBN = 50/50/1 ; M1 = monomer 1, M2 =
Table 1 displays the results obtained for the free radical copolymerization of equimolar mixtures of DMA and either VBT or VBA. In all cases the polymerization could be run homogeneously in DMF since all the monomers and the resulting copolymers P(VBT-co-DMA) and P(VBA-coDMA) are soluble in this solvent. However, for the comonomer pair VBA/DMA, a higher initial dilution was needed as compared to VBT/DMA since VBA is much less soluble in DMF than VBT. Nevertheless, in all cases the nucleic acids functional polymers could be prepared in high yield. Moreover, as targeted, the resulting polymers P(VBT-co-DMA) and P(VBAco-DMA) could be dissolved in a large range of organic solvents. Since the RP of DMA with either VBT or VBA was found to be a straightforward pathway for producing nucleic acids functional polymers, the controlled radical copolymerization (CRP) of VBT/DMA and VBA/DMA was subsequently studied. Among the possible methods of CRP, ATRP was selected since this technique was already reported to be efficient for polymerizing nucleic acids functional monomers.6, 7 Nevertheless, a catalyst system based on copper chloride and the ligand N,N’-bis(pyridin-2-ylmethyl 3-hexoxo-3-oxopropyl)ethane-1,2-diamine (BPED) was selected for the present work since it was reported to be a very efficient system for controlling the ATRP of problematic functional monomers.5 As illustrated by Table 1, this catalytic system also allowed the successful preparation of well-defined P(VBT-co-DMA) and P(VBA-co-DMA). SEC results confirmed that the
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All the formed supramolecular self-assemblies were found to be sensitive to temperature. The intermolecular H-bonds associations could be disrupted by heating the solutions at 50°C-60°C. In that case clear solutions of soluble polymers were observed. This process is reversible and the initial superstructures were recovered when the solutions were cooled down to room temperature.
2.5 2.0
Absorbance
prepared copolymers both possess a controlled molecular structure, although measured apparent molecular weights and molecular weight distributions should be cautiously interpreted due to the high structural discrepancies between the analyzed copolymers and the SEC standards. Nevertheless, the present study indicates that ATRP can be successfully used for preparing P(VBT-co-DMA) and P(VBA-co-DMA). Thus, the macromolecular engineering capabilities of ATRP can be applied to the polymers presented here e.g. blocks copolymers, star copolymers, telechelics or brushes can be prepared. Self-assembly in CHCl3 of P(VBT-co-DMA) and P(VBA-co-DMA). The self-assembly in chloroform solutions of either P(VBT-co-DMA), P(VBA-co-DMA) or a P(VBT-co-DMA)/P(VBA-co-DMA) mixture was studied by light microscopy. In every cases, micron size spherical superstructures (most probably polymersomes) were observed. As an example, Figure 1 shows the morphology observed for a solution of P(VBT-co-DMA). Such observations correlate with the results of Rotello and coworkers concerning the self-assembly of diamidopyridine and thymine functionalized polymers.8, 9 The observed size of the spherical objects was further confirmed by dynamic light scattering measurements, which indicated an average hydrodynamic radius of around 1 micron for the homopolymers spheres and of 0.5 micron for the mixture. All the observed superstructures were found to be very stable with time. The latter indicates an efficient stabilization of these structures in chloroform due to the presence the long alkyl chains of DMA.
1.5 1.0 0.5 0.0 240
260
280
300
320
Wavelength (nm) Figure 2. UV absorption spectra of equimolar mixtures of PVBT-co-DMA and PVBA-co-DMA in chloroform (full lines) compared to the additional absorption spectra of separate chloroform solutions of PVBT-co-DMA and PVBA-co-DMA (dashed lines). Conclusions A new family of nucleic acids substituted polymers was studied. These new polymers P(VBT-co-DMA) and P(VBA-co-DMA) could be easily prepared in high yield via either RP or ATRP. In chloroform, the resulting copolymers P(VBT-co-DMA) and P(VBA-co-DMA) as well as P(VBT-coDMA)/P(VBA-co-DMA) mixtures were found to spontaneously self-assemble in micron size superstructures (most likely polymersomes). Such assembly process was proven to be driven by H-bond formation (i.e. T-T, A-A or A-T interactions). Acknowledgements. Fraunhofer society is gratefully acknowledged for financial support. In addition, the authors thank Marlies Gräwert, Olaf Niemeyer, Silke Koch, Dr. Knut Rurack, Monika Spieles, Dr. Klaus-Werner Brzezinka and Anka Kohl for analytical measurements
Figure 1. Optical micrographs recorded at 25°C for a solution of PVBT-coDMA in chloroform (3 mg.mL-1). The cohesion inside the superstructures is insured by intermolecular Hbonds occurring between the polymer segments, (i.e. T-T interactions in the case of P(VBT-co-DMA) solutions, A-A interactions in the case of P(VBAco-DMA) solutions and A-T in the case of polymer mixtures). The latter was evidenced by either FT-IR or UV-vis spectroscopy studies. In the case of a P(VBT-co-DMA) solution, the FT-IR spectrum clearly indicated the existence of intermolecular T-T interactions (a typical band at 3184 cm-1 due to the Hbonded NH stretching vibration in thymine was observed).10 Similar results were obtained for the self-assembly of P(VBA-co-DMA) in chloroform. The FT-IR spectra of P(VBA-co-DMA) in chloroform at 25°C clearly exhibits intense bands at 3485 cm-1, 3315 cm-1 which are both due to the stretching vibrations of the bonded NH2 group of adenine.10 Nevertheless, UV-vis spectroscopy confirmed that the superstructures formed in chloroform by a P(VBT-co-DMA)/P(VBA-co-DMA) mixture are resulting from intermolecular A-T interactions. In Figure 2, the UV absorption spectrum of a mixture P(VBT-co-DMA)/P(VBA-co-DMA) was compared to the additional spectra of separate solutions of both P(VBT-co-DMA) and P(VBA-co-DMA). A lower absorbance (hypochromicity) was observed for the mixture in comparison to the additional spectra of the single copolymers solutions.11 Indeed, stronger supramolecular interactions are formed in the copolymer mixture as compared to the individual P(VBT-co-DMA) and P(VBA-co-DMA) solutions. The latter is easily understandable since it is known that A-T supramolecular interactions are much stronger than A-A or TT interactions.
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