Optically Active Nucleobase-Functionalized Polynorbornenes Mimicking Double-Helix DNA

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Jun 2021Optically Active Nucleobase-Functionalized Polynorbornenes Mimicking Double-Helix DNA Li Wang, Nan Lu, Shuai Huang, Meng Wang, Xu-Man Chen and Hong Yang Li Wang School of Chemistry and Chemical Engineering, Jiangsu Province Hi-Tech Key Laboratory for Bio-Medical Research, Institute of Advanced Materials, Southeast University, Nanjing 211189 , Nan Lu College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018 , Shuai Huang School of Chemistry and Chemical Engineering, Jiangsu Province Hi-Tech Key Laboratory for Bio-Medical Research, Institute of Advanced Materials, Southeast University, Nanjing 211189 , Meng Wang School of Chemistry and Chemical Engineering, Jiangsu Province Hi-Tech Key Laboratory for Bio-Medical Research, Institute of Advanced Materials, Southeast University, Nanjing 211189 , Xu-Man Chen School of Chemistry and Chemical Engineering, Jiangsu Province Hi-Tech Key Laboratory for Bio-Medical Research, Institute of Advanced Materials, Southeast University, Nanjing 211189 and Hong Yang *Corresponding author: E-mail Address: [email protected] School of Chemistry and Chemical Engineering, Jiangsu Province Hi-Tech Key Laboratory for Bio-Medical Research, Institute of Advanced Materials, Southeast University, Nanjing 211189 https://doi.org/10.31635/ccschem.020.202000358 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail The development of synthetic polymers that mimic the double-stranded helical structure of DNA is a fascinating topic in polymer science. In this study, we designed and synthesized two chiral norbornene monomers containing adenine and thymine, which were mixed to form a hydrogen-bonding complementary complex, by which a ring-opening metathesis polymerization (ROMP) was performed. High-resolution transmission electron microscopy (HRTEM) undoubtedly showed the self-assembly of the optically active complementary nucleobase-functionalized polynorbornenes into a double helix. Computer simulations and a two-dimensional nuclear Overhauser effect spectroscopy (2D-NOESY) experiment showed that the double-stranded helical polynorbornene was derived from a copolymer of alternating adenine and thymine units. Download figure Download PowerPoint Introduction Many biological properties and functions are attributed to the superhelical structures of self-assembled biomolecules. The carrier of genetic information, DNA, is a fascinating biological macromolecule in which the Watson–Crick double-helix structure is formed by two complementary polynucleotide chains held together by hydrogen-bonding interactions between paired nucleobases [adenine–thymine (A–T) and guanine–cytosine (G–C)].1–3 The unique molecular replication, self-assembly properties and biological functions of DNA have motivated intense scientific activity on the development of synthetic polymers that mimic the double-stranded helical structure of DNA.4–17 Thus far, self-assembly into double-stranded helical structures has only been found in a few exquisite polymeric structural motifs based on peptide nucleic acids,4–6 metal-ion-coordinated helicates,7,8 aromatic oligoamides,9,10 crescent-shaped m-terphenyl oligomers,11 poly(m-phenylene)s,12 acyclic ethynyl aromatic oligomers,13,14 oligopeptide-functionalized poly(diacetylene),15 and ladder polymers.16,17 DNA comprises an alternating deoxyribose ring (i.e., an optically active five-membered, three-stereocentered heterocyclic ring) and a rigid, chiral phosphate backbone (Figure 1a). Inspired by its macromolecular structure, we designed a new polymer with a polynorbornene backbone that forms a double-stranded helical superstructure through the self-complementary hydrogen-bonding interactions of two polymer chains. Polynorbornenes are a representative class of cyclic olefin polymers prepared by ring-opening metathesis polymerization (ROMP),18,19 a living chain-reaction polymerization. However, most known polynorbornenes are optically inactive, because commercially available norbornene derivatives and monomers, though bear chiral carbons, they tend to be racemic compounds.20–23 Thus, previously reported nucleobase-functionalized polynorbornenes usually self-assemble into cylindrical20 or spherical21,23 micelles. Although polybisnorbornene-based ladder polymers with chiral ferrocene linkers could form a one-handed double helix,16,17 the covalently bound dimeric structure, unfortunately, lacked the reversible binding-dissociation function, fundamental for DNA replication. Therefore, we synthesized novel nucleobase-functionalized polynorbornenes with an optically active, "all-carbon" polynorbornene backbone. The mimicry strategy, as illustrated in the schematic in Figure 1b, consisted of using a chiral (1S,2S,4S)-cyclopentane ring to replace the five-membered three-stereocentered deoxyribose ring of DNA and replace the phosphate linkers with internal alkene groups efficiently formed by ROMP.18,19 As in DNA, nucleobases were laterally attached to the chiral polynorbornene backbone, which consequently possessed hydrogen-bonding recognition capability. Figure 1 | Molecular structures: (a) DNA with an optically active polydeoxyribose–phosphate backbone constructed by polycondensation, and (b) a novel polymer with an optically active polynorbornene backbone constructed by living polymerization. Download figure Download PowerPoint Experimental Methods The instrumentation, computer simulations, starting materials, detailed synthetic procedures, and NMR spectra ( Supporting Information Figures S1–S29) of compounds 3, 4, 5, 8, 9, 10, 11, monomer 12, monomer 13, complex 13.12, poly(norbornene–adenine) ( PN-A), poly(norbornene–thymine) ( PN-T), and polynorbornene nucleic acid ( PNNA) are provided in the Supporting Information. Typical PNNA synthesis procedure Monomer 12 (48.0 mg, 0.20 mmol), monomer 13 (46.0 mg, 0.20 mmol), and anhydrous dichloroethane (30 mL) were added to a Schlenk flask under a nitrogen atmosphere and stirred at room temperature for 3 h to form a hydrogen-bonding-induced complex 13.12. A Grubbs second-generation catalyst (17.0 mg, 0.02 mmol) was added to the reaction mixture and stirred at 40 °C for 24 h. One drop of ethyl vinyl ether was added to quench the reaction. Then methanol was added to the reaction mixture and centrifuged at 10000 r/min for 10 minutes. The precipitate was washed three times with anhydrous methanol and dried to obtain PNNA (89.0 mg, yield: 95.10%) as a light yellow powder. The product was characterized by proton nuclear magnetic resonance (1H NMR), using the following conditions: 1H NMR (300 MHz, DMSO-d6, δ): 11.171 (s, 1H), 8.107 and 8.075 (d, 2H), 7.437 (s, 1H), 7.196, 5.343 (s, 4H), 4.075 (d, 2H), 3.898 (s, C2H4Cl2), 3.461 (s, 2H), 3.334 (s, H2O), 2.943 (s, 1H), 2.649 (s, 3H), 2.355 and 2.287 (d, 2H), 1.880 (s, 2H), 1.715–1.633 (m, 5H), 1.133 (s, 4H). Results and Discussion The syntheses of optically active polynorbornenes started from the enantioselective syntheses of chiral norbornene monomers. The binding of nucleobases to chiral norbornenes would result in chiral amplification and transmission at the molecular level under the chiral torsional forces of three chiral centers of the (1S,2S,4S)-cyclopentane ring after ROMP, which would eventually induce the formation of helical nanostructures.24 The synthetic process of these chiral monomers is presented in Scheme 1a. An esterification reaction between acryloyl chloride ( 1) and chiral auxiliary D-pantolactone ( 2) produced the chiral compound 3, which was further treated with freshly redistilled cyclopentadiene and a TiCl4 catalyst to carry out a highly endoselective asymmetric Diels–Alder reaction to prepare the key intermediate compound 4, whose NMR spectra and optical rotation values were well matched with data from the literature.25 Compound 4 was reduced by lithium aluminum hydride (LiAlH4) in dry tetrahydrofuran (THF) to remove the chiral auxiliary to yield (−)-(1S,2S)-5-norbornene-2-methanol ( 5). The measured optical rotation [ α ] D 20 of –92o of compound 5 (c = 1.0 g·L−1, 98% ethanol) was consistent with the literature value.26 Two norbornene-functionalized nucleobase monomers, norbornene–adenine and norbornene–thymine, were synthesized, as shown in Scheme 1a. N,N-di-Boc-protected adenine ( 8), and N-Boc-protected thymine ( 9) were first synthesized according to literature protocols.27,28 The weak acidity of the unprotected imino groups of compounds 8 and 9 resulted in the formation of new C–N bonds with the optically active alcohol 5 via the Mitsunobu reaction. Deprotection of the Boc groups of the corresponding compounds 10 and 11 under alkaline conditions produced the desired two monomers, norbornene–adenine ( 12) and norbornene–thymine ( 13). The optical rotations [ α ] D 20 of norbornene–adenine and norbornene–thymine were measured as –43° (c = 1.0 g·L−1, 98% CHCl3) and –64° (c = 1.0 g·L−1, 98% CHCl3). Scheme 1 | (a and b) Synthetic routes for two nucleobase-functionalized norbornene monomers and the corresponding polynorbornene PN-A, PN-T, and PNNA. Download figure Download PowerPoint We applied ROMP to the chiral norbornene monomers ( 12 and 13) to synthesize two PNNA homopolymers, that is, PN-A and PN-T, respectively, as shown in Scheme 1b. The ROMP was carried out utilizing a Grubbs second-generation catalyst in 1,2-dichloroethane at 40 °C for 24 h. The initial molar ratio of the norbornene–nucleobase monomers and the Grubbs second-generation catalyst was set to 20:1. The experimental results showed a quantitative synthesis of PN-T and a low reaction yield of PN-A (∼23.9%). This outcome might have derived from the poor solubility of the monomer 12 in 1,2-dichloroethane or the presence of the free amino group of monomer 12, which might have interfered with the catalytic activity of the Grubbs second-generation catalyst. The PN-A and PN-T obtained were insoluble in chloroform and 1,4-dioxane, slightly soluble in methanol and acetonitrile, and soluble in dimethylformamide (DMF) and dimethyl sulfoxide (DMSO). Gel permeation chromatography (GPC) of these PNNAs was carried out using DMF as the eluent. As illustrated in Supporting Information Table S1, PN-A had low molecular weights, whereas the much higher Mn values of PN-T implied the potential for PN-T to form aggregates in DMF through intermolecular hydrogen bonding. To prepare the desired double-stranded PNNA copolymers, we first mixed norbornene–adenine and norbornene–thymine monomers in a one-to-one molar ratio to form a stable hydrogen-bonded complex, the bisnorbornene monomer 13.12 ( Supporting Information Figures S22 and S29), as confirmed by the electrospray ionization mass spectrometry (ESI-MS) spectra [m/z: 512.23752 (monomer 12 + monomer 13 + K)+, Supporting Information Figure S23] and the observation of a strong nuclear Overhauser effect (NOE) (Ha–Hg, adenine–thymine) between equal molar amounts of monomers 12 and 13 in the two-dimensional (2D) 1H,1H nuclear Overhauser effect spectroscopy (NOESY) spectrum ( Supporting Information Figure S24). The measured optical rotation [ α ] D 20 of the complex 13.12 was –55° [c = 1.0 g·L−1, 98% CHCl3).Subsequently, we performed ROMP of the complex 13.12 by using a Grubbs second-generation catalyst in 1,2-dichloroethane at 40 °C for 24 h. (The H-bonds of complex 13.12 were stable at 40 °C, Supporting Information Figure S30.) The initial molar ratio of the two norbornene–nucleobase monomers and the Grubbs second-generation catalyst was set to 20:1. The resulting PNNA exhibited a single peak in the GPC profile ( Supporting Information Figure S40c). The molecular weight peak at 3994.287 in the matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) data ( Supporting Information Figure S43) implied that PNNA was formed by two complementary polymer chains, where the degree of polymerization of complex 13.12 was ∼8–9. We also raised the initial molar ratio of the monomers and the Grubbs catalyst to 40:1 and 60:1; however, the dramatic decrease in the solubility of the corresponding polymers in DMF and DMSO precluded accurate characterization by GPC. All subsequent experiments were performed using the PNNA ([M]/[C] = 20:1) sample. We verified the existence of adenine–thymine hydrogen-bonding pairs in the PNNA sample by performing a variable-temperature 1H NMR experiment in DMSO-d6 ( Supporting Information Figure S31). Supporting Information Figure S31a shows a chemical shift of the −NH– group of thymine from 11.152 to 10.698 ppm during the heating process from 30 to 60 °C, and was recovered to 11.146 ppm by dropping the temperature from 60 to 30 °C (cooling process). Moreover, Supporting Information Figure S31b shows chemical shifts of the −NH2 group of adenine from 7.114 (30 °C) to 6.887 ppm (60 °C) during the heating process and back to 7.113 ppm (30 °C) during the cooling process, which were consistent with those of the −NH– group. During the heating process, the upfield chemical shifts of the –NH– and −NH2 groups indicated the gradual opening of the H-bonds of the adenine–thymine units. During the cooling process, the chemical shifts of the –NH– and −NH2 groups recovered fully to the original values, which indicated that the H-bonds could be recognized and reconnected. Thus, the formation of the double-stranded PNNA–PNNA was a reversible process. We quantitatively studied the hydrogen-bonding stability using an NMR titration experiment ( Supporting Information Figures S34 and S35) based on the Benesi–Hildebrand model,29–31 whereby, the association constant Ka of the complex 13.12 was measured. This value was calculated at 24.64 M−1, which was in good agreement with the regular association constant data (∼10–100 M−1, CDCl3) for the adenine–thymine base pair recognition reported in the literature.24,32–34 We used 2D 1H,1H NOESY, to investigate the PNNA structure. The NOESY spectra of PNNA in Figure 2 and Supporting Information Figure S28 show clear NOE signals (Ha–Hd) between the protons of amino groups of adenine units and imino groups of thymine units, proving the existence of hydrogen bonds between the adenine and thymine units. Additionally, the significant NOEs signals for PNNA appearing between the protons c–g, b1–f1, and b1–f2 indicated an alternating adenine and thymine structure for PNNA. Also, the 2D 1H,1H NOESY data in Figure 2 shows that the internal olefin protons had intense NOEs with almost all the protons in the high field. However, no obvious NOE signals (Ha–Hb2) of the Hoogsteen base pairs35 were found (Figure 2 and Supporting Information Figures S37–S39). According to the literature, the Hoogsteen base pairs were low-populated, short-lived,36 and only existed transiently.37 Therefore, we used Watson–Crick mode in this manuscript. Figure 2 | 2D 1H,1H NOESY spectrum of PNNA obtained in DMSO-d6 at 25 °C. Download figure Download PowerPoint This alternating adenine and thymine structure (Figure 3a) of PNNA could derive from the steric hindrance effect. Although a repeating structure for a portion of PNNA could not be ruled out (Figure 3b), an alternating adenine and thymine structure were hypothesized to be the predominant PNNA structure. We verified this hypothesis by performing quantum chemical calculations using the density functional theory (DFT) M06-2X38 function of the Gaussian 03 program.39 The stereostructures of the two isomers are shown in Figure 3. A comparative analysis (see Supporting Information) showed that the alternating adenine and thymine structure of PNNA were more stable than the repeating structure. All the data indicated above proved that the adenine units and thymine units were polymerized alternately and that hydrogen bonds stabilized the two paired polymer chains. Figure 3 | Stereostructures of (a) alternating adenine–thymine and (b) repeating isomers of PNNA optimized at M06-2X/6-31G* level of theory in the gas phase; red balls represent oxygen atoms, blue balls represent nitrogen atoms, gray balls represent carbon atoms, and the white balls represent hydrogen atoms. Download figure Download PowerPoint The UV–vis absorption spectra of all the monomers and their complex in acetonitrile (25 °C) were illustrated in Supporting Information Figure S44, showing the appearance of the corresponding absorption peaks at 259 nm (monomer 12) and 268 nm (monomer 13), respectively. When monomers 12 and 13 were mixed in a 1∶1 molar ratio, the absorption peaks of the corresponding complex 13.12 appeared at 261 nm. A suitable solvent was required to investigate the variable-temperature UV (VT-UV) spectra of PN-A, PN-T, and PNNA. Although these polymers displayed good solubility in DMF and DMSO, the maximum UV absorption wavelengths of pure DMF and DMSO at 270 and 265 nm, respectively, strongly interfered with those of PN-A, PN-T, and PNNA.24 Besides, we attempted to use 1,4-dioxane to dissolve PN-A, PN-T, and PNNA; however, these polymer compounds were nearly insoluble in this solvent. Fortunately, we found that these PNNAs were soluble in acetonitrile. Thus, the VT-UV data of PN-A, PN-T, and PNNA were obtained in acetonitrile over a 10–75 °C temperature range at a heating rate of 0.5 °C·min−1. As shown in Figures 4a–4c, the absorption peaks of PN-A, PN-T, and PNNA in acetonitrile appeared at 266 ( PN-A), 270 ( PN-T), and 272 nm ( PNNA), respectively. The observed trends revealed that the absorbance of PN-A, PN-T, and PNNA decreased gradually along with increasing temperatures, matching the characteristics of thermosensitive polymers.40–42 This temperature-sensitive property originated from the H-bonds between the nucleobases.24,43,44 Figure 4 | VT-UV spectra of (a) PN-A, (b) PN-T, (c) PNNA measured in acetonitrile (∼50 mg·L−1) in a temperature range from 10 to 75 °C, and VT-CD spectra of (d) PN-A, (e) PN-T, (f) PNNA measured in acetonitrile (∼50 mg·L−1) in a temperature range from 10 to 70 °C. Download figure Download PowerPoint It was well known that the circular dichroism (CD) technique was very sensitive to the helical structure of DNA in solution. Acetonitrile was also used to study the CD spectra. The concentrations of all the solutions were set at 50 mg·L−1 (25 °C). As demonstrated in Supporting Information Figure S45, the negative Cotton effect was observed in the CD spectra of all the monomers at the wavelengths, in accordance with their UV–vis absorption regions, proving the molecular chirality of the monomers. The ROMP method was used to confer monomeric chirality to the polymeric backbone. To demonstrate this, acetonitrile was employed for the evaluation of the variable-temperature CD (VT-CD) spectra of PN-A, PN-T, and PNNA. We obtained VT-CD data for PN-A, PN-T, and PNNA at temperatures of 10, 30, 50, and 70 °C, respectively (Figures 4d–4f). As shown in Figure 4d, the multisignate Cotton bands were observed in the CD spectra of adenine-functionalized PN-A over the 240–300 nm range. Moreover, thymine-functionalized PN-T (Figure 4e) was not analogous to PN-A and exhibited broad Cotton bands with a negative peak at ∼248 nm, a positive peak at ∼267 nm, and a zero-crossing point at ∼257 nm, implying a preferential handed helical stacking in this macromolecule.45,46 The CD spectra of PN-A and PN-T were similar to those reported previously in single-stranded DNA systems, that is, poly(deoxyadenylic acid) [poly(dA)] and poly(deoxythymidylic acid) [poly(dT)].47,48 Furthermore, PNNA (Figure 4f) exhibited broad Cotton bands with peak shapes similar to those of PN-A. A positive Cotton effect at ∼268 nm and a negative Cotton effect at ∼252 nm were observed. These results indicated the existence of chiral structures in the polynorbornene backbones of PN-A, PN-T, and PNNA. Similar temperature dependence of the CD signals of PN-A, PN-T, and PNNA suggested that the hydrogen-bonding interactions in nucleobases were more intensive at lower temperatures than at higher temperatures.45,49,50 Transmission electron microscopy (TEM) experiments were further conducted to study the self-assembly behaviors of PN-A, PN-T, and PNNA. All the samples were dispersed at a dilute concentration of 60 mg·L−1 in CHCl3. The resulting solutions were cast on copper grids, which were placed in the TEM instrument for observation after the solvent was evaporated. As shown in Figures 5a–5i, PN-A, PN-T, and PNNA self-assembled into filamentous nanostructures in CHCl3. The diameters of the filaments of these PNNA homopolymers were ∼6–48 nm. In addition, the filaments intertwined to form larger-diameter filaments. For example, two 16-nm diameter filaments of PN-A were wound into a 22-nm diameter filament (Figure 5c), a 32-nm diameter filament and a 20-nm diameter filament of PN-T intertwined to form a 48-nm diameter filament (Figure 5e), and two 18-nm diameter filaments of PNNA intertwined into a 24-nm diameter filament (Figures 5g and 5i). Moreover, a large number of distinct helical fibers were found in the PN-T sample (Figures 5d–5f), whereas the helical nanostructures were blurry in the PN-A and PNNA samples. Figure 5 | TEM images of self-assembled morphologies of (a–c) PN-A, (d–f) PN-T, and (g–i) PNNA in CHCl3. Download figure Download PowerPoint Eventually, high-resolution TEM (HRTEM) experiments were performed to observe the self-assembled morphology of PNNA (Figure 6) in DMF. PNNA was dissolved in pure DMF at a concentration of 60 mg·L−1. After casting the solution on a copper grid and evaporation of the solvents, the sample was placed in the HRTEM instrument to obtain the HRTEM images of PNNA. Fortunately, we clearly observed the helically arranged nanofiber structures that were apparent in PNNA, as shown in Figures 6a–6d. Especially in Figures 6a and 6d, the double-stranded helix was visible. The average helix diameter and pitch lengths were ∼2.0 and 8.3 nm, respectively. Here, one pitch possessed ∼27–28 pairs of nucleobases, which was estimated from quantum chemical calculations (Figure 6e, the distance between two adjacent pairs of nucleobases was estimated as ∼0.304 nm by DFT using M06-2X38 function of the Gaussian 03 program39). In general, the double-helical structure of DNA has a diameter of 2.0 nm and a pitch of 3.4 nm, where one pitch comprises 10 pairs of nucleobases.1–3 Compared with DNA, the helix of PNNA had a similar diameter, with a much longer pitch length (8.3 nm), which implied that the chiral twisting power of the chiral (1S,2S,4S)-cyclopentane ring (unidirectional chiral torsion) of PNNA might be much weaker than that of the chiral deoxyribose ring chiral torsion) of This has to the pitch length of the double-helix by the of the chiral Figure | HRTEM images of self-assembled double-stranded helical structures of PNNA dissolved in DMF and (e) schematic of double-stranded helix of PNNA. Download figure Download PowerPoint We synthesized two pure norbornene monomers adenine and thymine units, which were mixed to form a hydrogen-bonding complementary bisnorbornene complex to which ROMP was applied to prepare three optically active nucleic by an of the self-assembly of these novel The homopolymers and the self-assembly into helical were observed by HRTEM demonstrated the self-assembly of the optically active nucleic acid with an alternating adenine–thymine structure. the corresponding optically active polynorbornene presented a of double-helix These a new on the development of double-helical polymeric to mimic the structures of These polymers with nucleobase recognition might have potential in and Supporting Information Supporting Information is of is no of to The the of and the Jiangsu of for the of this of A for of the of 3. of of DNA by with a to the by and of of of and of of and of a of a with and of the by the of in and of on of of from to in of Yang Yang Huang Chen Chen Yang Chen Yang Grubbs and Grubbs via and into and of by the and of into Wang Wang Yang with Yang with an Active and in and to with and of the of from NMR of Molecular of and Li of of with and of as of the of and and the of Hoogsteen Hoogsteen in the of in Hoogsteen in of for and Two and of and 12 Li Chen with and 5, on the of and of 50, and Molecular of with Huang Chen Nucleobase-Functionalized as for and of UV CD of DNA on and of the in the with and and of the of DNA and a of 4, of and Synthetic Li Huang of 5, and and for the of Molecular 50, of DNA 5, Information Chemical the of and the Jiangsu of for the of this times

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