Novel Synthesis and Characterization of Bioconjugate Block

Biomacromolecules 2005, 6, 2328-2333

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Novel Synthesis and Characterization of Bioconjugate Block Copolymers Having Oligonucleotides Atsushi Noro,† Yutaka Nagata,† Masaki Tsukamoto,‡ Yoshihiro Hayakawa,‡ Atsushi Takano,† and Yushu Matsushita*,† Department of Applied Chemistry, Graduate School of Engineering, and Laboratory of Bioorganic Chemistry, Graduate School of Information Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan Received April 5, 2005; Revised Manuscript Received April 27, 2005

Novel and efficient synthesis of polymers terminated with nucleotides via the phosphoramidite method has been developed. A hydroxyl-terminated polymer was converted into a polymer capped with a nucleotide in three steps, where the conversion of the reactions was very high, almost 100%. By repetition of this synthetic method, a block copolymer composed of a synthetic polymer, polystyrene, and biological oligonucleotides with thymidine units has been successfully synthesized. A microphase-separated structure of this block copolymer was observed by both transmission electron microscopy and small-angle X-ray scattering, and a cylindrical structure was confirmed. I. Introduction Many syntheses of end-functionalized polymers have been performed1-6 because these polymers have practically and potentially numerous applications. End functionalizations such as a vinyl group,1 a carboxyl group,2 a sulfonyl group,4 an amino group,5 and a hydroxyl group6 were done precisely in combination with living anionic polymerizations. The procedure of synthesizing end functional polymers by way of living anionic polymerization has two advantages: one consists of very simple end-functionalizing reaction in the same flask just after polymerization, and the other is very high conversion because living anionic ends are very reactive. However, this method cannot be applied to many chemical species because reactivity of living ends against terminal group is limited severely. Recently, several end functional polymers that are more complicated than the examples cited in a preceding paragraph were synthesized from basic end-functionalized polymers. In many cases, hydroxyl-terminated polymers are preferred because a hydroxyl group can be converted into many other functional groups with milder reactions than living anionic ones. Meijer et al.7,8 reported on the synthetic method of endfunctionalized polymers with 2-ureido-4-pyrimidone groups, which are introduced onto hydroxyl end-functionalized polymers in two-step reactions. Their unique polymers have self-assembled to form reversible hydrogen-bonded supramolecular polymer networks. Yamauchi et al.9 also introduced 2-ureido-4-pyrimidone groups into hydroxyl endcapped polymers so as to produce multiple hydrogen bondings and investigated their glass transition temperatures and viscosity. Moreover, they introduced heterocyclic and * Corresponding author. Tel.: +81-52-789-4604. Fax: +81-52-7893210. E-mail: [email protected]. † Graduate School of Engineering. ‡ Graduate School of Information Science.

complemental base units, such as adenine, 2,6-purine, and thymine via Michael addition using acryloyl-teminated polystyrene.10 Binder et al.11-14 have also used heterocyclic bases such as thymine, cytosine, and 2,6-diaminotriazine, 2,4diamino-1,3,5-triazine. These polymers are very interesting because they may show the novel features of biological parts as well as those of synthetic polymers by introducing biological units onto synthetic polymers. Actually, they observed peculiar structures caused by hydrogen bonding from biological components and the related interesting properties. Thus, bioconjugate polymers, especially nucleobase-terminated polymers, can be designed as building blocks of high-functional supramolecular materials. However, both Yamauchi’s and Binder’s methods can add only one unit of heterocyclic bases on each polymer end so that the applicability of their methods is limited considerably. Moreover, their synthetic conditions are relatively severe; for example, one method of Yamauchi et al. for thymine-PS10 requires 60 °C for 24 h, while another one of Binder et al. for diaminotriazine-terminated PEK requires a higher temperature, 165 °C for 48 h,12 and these conditions limit severely both polymer species and biological parts. To proceed the studies effectively on interesting bioconjugate polymers such as peptide/protein hybrid materials,15-16 a simple and widely applicable synthetic method with milder conditions is required. Thus, in this paper, we report on a simple, efficient, and widely available synthetic method of introducing a nucleotide on a polymer chain end via the phosphoramidite method, whose conditions are mild and whose reactions can be quickly performed. Furthermore, this method can be extended to the synthesis of a block copolymer consisting of a polystyrene block, which is a typical synthetic polymer, and oligonucleotides by repeating quick and mild reactions.

10.1021/bm0502462 CCC: $30.25 © 2005 American Chemical Society Published on Web 06/04/2005

Block Copolymers Having Oligonucleotides Scheme 1. General Phosphoramidite Method, Scheme for Synthetic DNA

II. Strategy for Synthesis of Polymer Terminated with Nucleotides The phosphoramidite method is one of the most useful DNA syntheses. Reaction conditions of this method have been investigated by several researchers;17-21 it is summarized in Scheme 1. At first, a 5′ OH group of a deoxynucleoside couples with a phosphoramidite, R2NP(OR)2, and a phosphite of the type P(OR)3 can be obtained. Second, oxidation to a phosphate of the type OdP(OR)3 is executed. This phosphate is a stable product for acid; therefore, acid treatment is performed to deprotect dimethoxytrityl (DMTr), and we can obtain a phosphate with a 5′ free hydroxyl group (Scheme 1). By repeating these steps, we can add nucleotides one by one, and polynucleotides can be synthesized by cutting off from the support. We apply this phosphoramidite method to obtain the specified nucleotide-terminated polymer easily and quantitatively by focusing on not a polymer as a support of DNA synthesis but the terminated polymer itself. The most important process of phosphoramidite method adopted in this work consists of the coupling reaction between a phosphoramidite and a hydroxyl-terminated polymer, which can be synthesized precisely by anionic polymerization (Scheme 2). Although we may obtain a specified polymer terminated with a nucleotide by carrying out the above method, it is very difficult to determine chain-end structures of the obtained polymers and also the introduction yield of an end group by 1H NMR if the molecular weights of polymers are high. Therefore, we use deuterated polymer chain so as to analyze the end structure or introduction yield accurately. III. Experimental Section Materials. Thymidine has the simplest structure of four typical nucleosides in DNA but has suitable properties as a

Biomacromolecules, Vol. 6, No. 4, 2005 2329 Scheme 2. Synthetic Scheme of Polystyrene-d8 Terminated with One Nucleotide, Thymidine Phosphate (PSd8-T1-OH)

nucleoside, so in this work a phosphoramidite with thymidine was used. Styrene-d8 monomer was purchased from Aldrich. 5′-O-(p,p′-Dimethoxytrityl)-2′-thymidine 3′-(2-cyanoethyl N,N-diisopropylphosphoramidite) was purchased from Transgenomic. BIT (benzimidazolium triflate) was synthesized as in the procedure reported22 and used as a promoter of amidite-coupling reaction. tert-Butyl hydroperoxide as an oxidation reagent23 was supplied from Aldrich. Trichloroacetic acid (TCA) was obtained from Kishida Chemical Co. Ltd. and diluted with dichloromethane into approximately 3 vol % and used for detritylation, i.e., deprotection of DMTr groups. Anhydrous tetrahydrofran (THF) as a solvent for coupling reaction was purchased from Aldrich. Dichloromethane as a solvent for detritylation and methanol as a solvent for reprecipitation were purchased from Kishida Chemical Co. Ltd. Preparation of Hydroxyl-Terminated Polymer. Polystyrene-d8 terminated with hydroxyl group was prepared by sequential two-step living anionic polymerization in THF at -78 °C with sec-butyllithium as an initiator.24 A small amount of polystyrene-d8 solution was separated as a precursor to check the molecular weight and molecular weight distribution.25 A living polystyryl-d8 anion was terminated with ethylene oxide, and the reaction solution was directly precipitated into methanol to purify polystyrene-d8 terminated with a hydroxyl group (PSd8-OH).6,10 These polymers were dissolved in benzene and freeze-dried to remove remaining methanol or THF completely. Preparation of Polymer Terminated with Thymidine Phosphates. PSd8-OH (1.00 g) and a thymidine phosphoramidite (8 equiv) were maintained under reduced pressure for 10 min. Successibly, THF (6.5 mL) was added under nitrogen gas atmosphere. When the solution became homogeneous, an effective promoter (BIT, 8 equiv) was added as shown in Scheme 2. After 15 min, an oxidizer (tert-butyl hydroperoxide, 16 equiv) was added; after another 10 min, the reaction solution was directly poured into methanol (100 mL) and a white solid was precipitated. This material was collected and dried in vacuo. To purify this crude product adequately, the powder was dissolved in THF and reprecipitated into methanol at least two more times. The ratio of end-capping was confirmed to be nearly 100% by NMR

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Figure 1. 1H NMR charts: (a) PSd8-OH, (b) polymer-d8 terminated with one thymidine phosphate (PSd8-T1-OH), (c) PSd8-T5-OH. Two boxes drawn with dotted lines at (a) are showing the peaks of dimethyl in sec-butyl group (-CH(CH3)CH2CH3) as the initiator section and of methylene in ethylene alcohol (-CH2CH2OH) as another end section, respectively.

Figure 2.

31P

NMR charts: (a) PSd8-T1-OH, (b) PSd8-T5-OH.

measurements (yield 0.98 g). This polystyrene-d8 terminated with one thymidine phosphate (PSd8-T1-DMTr) was further treated for deprotection. To detach the DMTr group from PSd8-T1-DMTr, a dichloromethane solution of TCA (10 equiv) was added into a dichloromethane solution of PSd8T1-DMTr (0.95 g). After reprecipitation and freeze-drying, the product was confirmed to be PSd8-T1-OH (0.93 g) by NMR (Figure 1b, Figure 2a). Using a thymidine phosphoramidite, amidite-coupling reactions were performed to add thymidine units on PSd8T1-OH. PSd8-T1-OH (0.90 g) was coupled with a thymidine phosphoramidite (8 equiv) by the aid of BIT (8 equiv) and oxidized with an oxidizer (16 equiv). After treating with TCA (10 equiv), PSd8-T2-OH (0.85 g) was produced. Repetition of this procedure mentioned here led to PSd8T3-OH, PSd8-T4-OH, and finally PSd8-T5-OH (0.50 g) was obtained. NMR Measurements and Characterizations. 1H NMR measurements were performed on each step of synthesis using Varian UNITY INOVA 700 MHz to confirm the molecular structures. Polystyrene-d8 terminated with nucleotides dissolves well in chloroform; therefore, chloroformd1 was used as a solvent of NMR measurements. Relatively concentrated solution was used to improve the S/N ratio (solute 30 mg/solvent 0.85 mL). 31P NMR measurements

were also performed for each prepared solution with multiplication for one night to confirm the presence of phosphate qualitatively (Varian Mercury 300 MHz). PSd8-OH. 1H NMR (700 MHz, CDCl3, δ): 0.6-0.8 ppm (-CH(CH3)CH2CH3: 6H), 0.8-1.0 ppm (-CH(CH3)CH2CH3: 3H), 0.8-2.1 ppm (poly(styrene)-d8 backbone and -CH2CH2OH: 3H × repeating units × (1 - deutrated ratio) and 2H), 3.2-3.4 ppm (-CH2CH2OH: 2H), 6.3-6.9 ppm (poly(styrene)-d8 backbone: 2H × repeating units × (1 deutrated ratio)), 6.9-7.2 ppm (poly(styrene)-d8 backbone: 3H × repeating units × (1 - deutrated ratio)); cf., Figure 1a. PSd8-T1-OH. 1H NMR (700 MHz, CDCl3, δ): 0.6-0.8 ppm (-CH(CH3)CH2CH3: 6H), 0.8-1.0 ppm (-CH(CH3)CH2CH3: 3H), 0.8-2.1 ppm (poly(styrene)-d8 backbone and -CH2CH2OH: 3H × repeating units × (1 - deutrated ratio) and 2H), 1.9 ppm (-CH3 of thymine: 3H), 2.3-2.7 ppm (H-2′,2′′ and -OCH2CH2CN: 4H), 3.6-4.1 ppm (-OCH2CH2CN and -CH2CH2OP and H-5′,5′′ and H-4′: 7H), 5.0 ppm (H-3′: 1H), 6.1 ppm (H-1′: 1H), 6.3-6.9 ppm (poly(styrene)-d8 backbone: 2H × repeating units × (1 deutrated ratio)), 6.9-7.2 ppm (poly(styrene)-d8 backbone and H-6 of thymine: 3H × repeating units × (1 - deutrated ratio) and 1H), 8.0 ppm (H-3 of thymine: 1H). 31P NMR (300 MHz, CDCl3, δ): -2.5 ppm (-OdP(OR)3); cf., Figure 1b and Figure 2a. PSd8-T5-OH. 1H NMR (700 MHz, CDCl3, δ): 0.6-0.8 ppm (-CH(CH3)CH2CH3: 6H), 0.8-1.0 ppm (-CH(CH3)CH2CH3: 3H), 0.8-2.1 ppm (poly(styrene)-d8 backbone and -CH2CH2OP: 3H × repeating units × (1 - deutrated ratio) and 2H), 1.6-2.0 ppm (-CH3 of thymine: 3H × repeating units), 2.1-3.0 ppm ((H-2′,2′′ and -OCH2CH2CN: 4H × repeating units), 3.4-4.8 ppm (-OCH2CH2CN and -CH2CH2OP and H-5′,5′′ and H-4′: 5H × repeating units

Block Copolymers Having Oligonucleotides

and 2H), 4.8-5.4 ppm (H-3′: 1H × repeating units), 5.96.3 ppm (H-1′: 1H × repeating units), 6.3-6.9 ppm (poly(styrene)-d8 backbone: 2H × repeating units × (1 deutrated ratio)), 6.9-7.2 ppm (poly(styrene)-d8 backbone and H-6 of thymine: 3H × repeating units × (1 - deutrated ratio) and 1H × repeating units), 9.8-10.6 ppm (H-3 of thymine: 1H × repeating units). 31P NMR (300 MHz, CDCl3, δ): -2.2 to -3.2 ppm (-OdP(OR)3); cf., Figure 1c and Figure 2b. GPC Measurements. The molecular weight and molecular weight distribution of a precursor polystyrene-d8 as a main chain of PSd8-OH were estimated by gel permeation chromatography (GPC) using HLC-8020 of TOSOH Co. with three columns of G4000HXL, G3000HHR, and G3000HXL.25 They were calibrated with polystyrene standards. GPC was also used to confirm the occurrence of side-reactions such as dimerization of polymers, and decomposition of polymers in the course of reactions and workup. Morphological Observation. PSd8-T5-OH has a fairly large volume of T sequence so that it may behave as a diblock copolymer. The sample film for morphological observation was prepared by solvent-casting from benzene solution for 1 day without further annealing. Small-angle X-ray scattering (SAXS) was performed for this sample film using the Rigaku SAXS system, Nano Viewer, operated at 45kV and 60 mA. Two camera lengths were adopted, that is, 202 and 982 mm,26 and two profiles are connected. For reference of PSd8-T5-OH, the film of PSd8-T1-OH was prepared and SAXS measurement for this film was also performed. Bulk structure of PSd8-T5-OH was also observed by transmission electron microscopy (TEM) using H-800 of Hitachi for an ultrathin section stained with I2 vapor, which selectively stains thymidine units.25,26 IV. Results and Discussion The molecular weight and the molecular weight distribution index of the polystyrene-d8 main chain section of PSd8OH were determined to be 1.1 × 104 and 1.05, respectively, by GPC. The introduction ratio of a hydroxyl end group on PSd8 was confirmed by end-group method with 1H NMR (Figure 1a). PSd8-OH has an end sec-butyl group (-CH(CH3)CH2CH3) as the initiator section and a hydroxyethyl group (-CH2CH2OH) as another end section. Peaks for 6H of -CH(CH3)CH2CH3 and 2H of -CH2CH2OH stand out at 0.6-0.8 and 3.2-3.5 ppm, respectively. Because every polymer molecule has the initiator section, the introduction fraction of the hydroxyl end was estimated as almost 100% from 3 times the integral values at 3.2-3.5 ppm divided by integral values at 0.6-0.8 ppm. The same concept was applied to polystyrene-d8 terminated with nucleotides to evaluate the introduction ratios. The synthetic scheme of polystyrene-d8 having a thymidine phosphate is shown in Scheme 2. “Thy” in the nucleoside denotes thymine. 1H NMR charts for polystyrene-d8 terminated with one thymidine phosphate (PSd8-T1-OH) are shown in Figure 1b. The principal peaks are assigned as follows: 6H at 0.6-0.8 ppm stands for dimethyl of -CH(CH3)CH2CH3, 1H at 6.1 ppm for 1′ proton, 1H at 5.0 ppm

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Figure 3. GPC chromatographs: (a) PSd8-OH, (b) PSd8-T1-OH, (c) PSd8-T5-OH.

for 3′ proton, and 1H at 8.0 ppm for 3 position of thymine. The ratio of the integral values of these peaks is almost 6:1: 1:1; therefore, a thymidine was attached to the polymer end quantitatively. 31P NMR detects a peak at -2.5 ppm (H3PO4 aqueous solution as an external standard) as shown in Figure 2a, supporting the presence of a phosphate, and no other peaks are shown. GPC chromatograms of PSd8-OH and PSd8-T1-OH are compared in Figure 3. Both chromatograms are monodisperse and similar, and hence side reactions such as dimerization or decomposition of polystyrene did not occur in the reactions or workup. From all of these facts, we are convinced that PSd8-T1-OH was successfully synthesized. Thus, obtained PSd8-T1-OH was used for further reactions. Scheme 3 shows the synthesis of polystyrene-d8 terminated with nucleotides more than two. All of the products, that is, PSd8-T2-OH, PSd8-T3-OH, PSd8-T4OH, PS-T5-OH, were detected by 1H NMR and 31P NMR. To determine the introduction ratios of thymidine unit for each reaction step, 6H for the initiator unit and XnH for 3′ proton were used where Xn denotes the observed number of thymidine units for the molecule PSd8-Tn-OH (n ) 1, 2, 3, 4, 5). The evaluated Xn values are 1.00 (n ) 1), 1.98 (n ) 2), 2.95 (n ) 3), 3.77 (n ) 4), and 4.33 (n ) 5), respectively. We notice that Xn deviates from the ideal value with increasing n; however, it is still sufficiently high at n ) 5. The reason for this deviation may due to the incompleteness in the amidite-coupling or detritylation reactions. A 31P NMR chart for PSd8-T5-OH of Figure 2b gives a broad peak at -2.7 ppm (H3PO4 standard), which may suggest the overlapping of some phosphates under similar environment. A GPC chromatogram of PSd8-T5-OH is also

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Scheme 3. Repeating Process of Amidite-Coupling Reaction to Obtain PSd8-T5-OH

monodisperse as shown in Figure 3c, and this means that no critical side-reactions occurred in all processes, supporting the results from NMR studies strongly. Figure 4 shows a small-angle X-ray scattering profile of PSd8-T1-OH. One broad peak is observed at a q of 0.85 nm-1, where q ()4π sin θ/λ) is the scattering vector. This broad peak in the profile for PSd8-T1-OH may be thought to be a correlation hole peak because electron density of the chain-end group is different from that of PSd8. Even though we did not observe the structures of PSd8T2-OH, PSd8-T3-OH, and PSd8-T4-OH, it is conceived this polymer series tend to microphase-separate with increasing number of T units.27 A transmission electron micrograph of PSd8-T5-OH is shown in Figure 5. From this figure, we can safely state that this molecule shows microphaseseparated two-phase structure, although it is somewhat difficult to determine the manner of domain packing.27 The size of microdomain is roughly estimated as 12 nm; this value is reasonable considering the total molecular weight of this copolymer, that is, 1.3 × 104 because the experimental result of lamellar domain spacing (D) versus molecular weight, D ) 0.0337Mn0.64, for poly(styrene-block-2-vinylpyridine)28 gives 14 nm. A small-angle X-ray scattering profile of PSd8-T5-OH is displayed in Figure 6. The experimental scattering profile has many broad peaks such as at 0.634, 1.10, and 1.91 nm-1;

Figure 4. A small-angle X-ray scattering profile of PSd8-T1-OH.

Figure 5. A transmission electron micrograph of PSd8-T5-OH. Scale bar is 50 nm. The brighter phase is for polystyre-d8 part, while the darker one is for the thymidine phosphate part (T5-OH).

Figure 6. A small-angle X-ray scattering profile of PSd8-T5-OH. Solid line is for an experimental profile. Bars are for theoretical lattice scattering intensities at each diffracted plane assuming hexagonally packed cylindrical structure, with its domain spacing of 11.4 nm and the volume fractions of 0.21/0.79.

their relative magnitudes of scattering vectors are approximately 1, 31/2, and 91/2. Existence of the peak at 31/2 might indicate that this bioconjugate molecule forms hexagonally packed cylindrical structure. In reality, diffracted intensities were calculated based on hexagonally packed cylinders by adjusting both the locations of peaks and the relative magnitudes of the intensities associated with a certain structure factor. In Figure 6, a set of results are shown as the vertical bars, where two important parameters, that is, 11.4 nm for a distance of cylinders and 0.21/0.79 for volume ratio of two polymers, are used for calculation. It is evident that the calculated results agree well the observed intensities from Figure 6. Therefore, this bioconjugate molecule, PSd8T5-OH, is thought to form cylindrical microphase-separated structure from TEM and SAXS just as a block copolymer does.

Block Copolymers Having Oligonucleotides

In conclusion, we have successfully prepared a polymer terminated with a nucleotide by applying the phosphoramidite method, which is a useful synthetic method for DNA. Furthermore, oligonucleotides up to five units were quantitatively added to the polymer end by repeating the amiditecoupling reaction. This procedure gives a block copolymer composed of a synthetic polymer and biological oligonucleotides. As a matter of fact, microphase-separated cylindrical structure was observed by small-angle X-ray scattering and also by TEM. In the future, we will show more information of syntheses and characterizations of polymer terminated with all of the other typical deoxyribonucleotides and further applications of these bioconjugate polymers. Acknowledgment. We thank Dr. S. Arai at the Ecotopia Science Institute in Nagoya University for his help in taking transmission electron micrographs and Mr. T. Hikage at High Intensity X-ray Diffraction Laboratory, Nagoya University, for his help in measuring small-angle X-ray scattering. We also thank Mr. K. Kondo at the Department of Applied Chemistry in Graduate School of Engineering, Nagoya University, for his help with NMR measurements. This work was partially supported by the Ministry of education, science, sports, and culture, Grant-in-Aid program #17651066, and was also supported by the 21st century COE Program entitled “The Creation of Nature-Guided Materials Processing”. We thank them for their financial assistance. References and Notes (1) (2) (3) (4) (5) (6)

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