Introduction

The design and synthesis of supramolecular architectures with desired structures, properties, and functions on a polymer backbone have been the focus of intense research in recent years [1–8]. Polyrotaxanes, which consist of macrocycles threaded by a linear polymer molecule, are members of this group of molecules due to their special structure and properties [9–12]. Two methods have been described in the literatures for synthesizing side-chain polyrotaxanes: side-chain reactions of the polymer with macrocycles, or polymerization of rotaxane monomers [13–19].

Cucurbit[6]uril (CB[6]), a barrel-shaped host, consists of 6 glycoluril groups and 12 methylene bridges at both ends. The two rims are formed by the glycoluril carbonyl oxygen, so they are negatively charged and participate in ion–dipole interactions with cationic guests [20, 21]. Recently, taking advantage of the structure of cucurbit[n]uril, various mechanically interlocked molecules including rotaxanes and poly(pseudo)rotaxanes were synthesized by Kim [21–38], Buschman [39–42], and others [43–54]. We previously synthesized some side-chain polypseudorotaxanes with cucurbit[n]uril (molecular beads) [55–57].

In the work described herein, we synthesized water-soluble side-chain polypseudorotaxanes with CB[6] threaded onto butyl units. The pH-responsive behavior of P3VBCB was studied by DLS and with a pH meter, and the response of P3VBCB to salt addition was investigated by 1H NMR and DLS. The interaction between P3VBCB and λ-DNA was studied by gel electrophoresis and UV–vis.

Experimental section

Materials and methods

CB[6] was prepared using a method described in the literatures [20, 21]. The monomer N′-3-vinylbenzyl-1,4-diaminobutane dihydrochloride with cucurbit[6]uril (3VBCB) was prepared as reported in our previous paper [58]. 3-Vinylbenzaldehyde (95 %) and triethylamine (90 %) were obtained from Aldrich (St. Louis, MO, USA) without further purification. Potassium persulfate (KPS, Acros, Geel, Belgium) was recrystallized from deionized water before use. NaBH4 (AP), Na2SO4 (AP), NaHCO3 (AP), PPh3 (AP), THF (AP), HCl (CP), methanol (AP), and CH2Cl2 (AP) were used without further purification.

Synthesis of pseudorotaxane monomer (3VBCB)

3-Vinylbenzaldehyde (1.32 g, 0.01 mol) was slowly added into a solution of 1-azido-4-aminobutane (1.36 g, 0.012 mol), triethylamine (1.2 g,), CH2Cl2 (25 mL), and sodium sulfate (10 g) at room temperature and stirred for 20 h at the same temperature. The resulting solution was filtrated and the filtrate was concentrated to give the crude Schiff base. The crude product was dissolved in 20 mL of methanol and NaBH4 (0.45 g) was slowly added. The solution was then stirred at room temperature for 24 h. Next, the reaction solution was concentrated and an aqueous solution of NaHCO3 and CH2Cl2 was added. The organic phase was separated, dried with Na2SO4, concentrated, and then purified by column chromatography using CH2Cl2:MeOH (1:2) as an eluent to give 1.45 g of 1-azido-4-3-vinylbenzylaminibutane (yield: 62 %).

PPh3 (1.58 g) was slowly added into a solution of 1-azido-4-(N-3-vinylbenzyl)aminobutane (1.15 g, 5 mmol) dissolved in 15 mL of THF and 0.2 mL of H2O, which was then stirred for 24 h at room temperature. The reaction solution was concentrated down to half the volume and 5 mmol equivalent of concentrated HCl was added slowly in an ice bath to precipitate the HCl salt, which was isolated by filtration. The product was dried under vacuum to give 1.26 g of N′-3-vinylbenzyl-1.4-diaminobutane dihydrochloride (yield: 82 %).

CB[6] (1.68 mmol) was added into a solution (30 mL) of N′-3-vinylbenzyl-1.4-diaminobutane dihydrochloride (0.40 g) in a small portions, and the resulting solution was stirred for 12 h at room temperature. Excess CB[6] was filtered out to provide a clear solution that was evaporated to leave about 2 mL of solution. Ethanol (50 mL) was then added to the resulting solution to generate the precipitate, which was isolated by filtration to obtain 1.72 g of the desired pseudorotaxane monomer (3VBCB; yield: 96 %). 1H NMR (D2O): δ0.62 (m, 4H), δ2.35 (t, J = 7.5 Hz, 2H), δ2.46 (t, J = 7.5 Hz, 2H), δ4.38 (s, 6H), δ4.43 (s, 6H), δ4.46 (s, 2H), δ5.38 (d, J = 12Hz, 1H), δ5.67 (s, 12H), δ5.74 (s, 6H), δ5.79 (s, 6H), δ5.97 (d, J = 18 Hz, 1H), δ6.88 (dd, J = 12 Hz, 6 Hz, 1H), δ7.54 (t, J = 9 Hz, 1H), δ7.60 (d, J = 9 Hz, 1H), δ7.70 (d, J = 9 Hz, 1H), δ8.01 (s, 1H).

Synthesis of polypseudorotaxanes (P3VBCB)

Under an inert N2 atmosphere, 5 mL of H2O and a small magnetic stir bar were added to a 10 mL round-bottomed reaction flask. The monomer 3VBCB (0.5 g) was added to the flask to generate a solution, and the flask was then placed in an oil bath at 65 °C. After thermal equilibrium had been reached and the solution had been bubbled for 0.5 h, KPS (dissolved in 0.5 mL of H2O) was added to the solution. After 24 h, the flask was removed from the oil bath and cooled to room temperature. The resulting solution was precipitated and washed with ethanol and then dried under reduced pressure to give 0.32 g of side-chain polypseudorotaxanes (P3VBCB; yield: 83 %).

Characterization techniques

All 1H-NMR experiments were performed on a Bruker (Rheinstetten, Germany) Avance 400 NMR spectrometer.

Multiangle laser light scattering (MALLS) measurements were performed with a Wyatt Technology (Santa Barbara, CA, USA) DAWN HELEOS 18-angle (from 15° to 165°) light scattering detector using a Ga-As laser (658 nm, 40 mW). The refractive index increment (dn/dc) of P3VBCB in 0.1 mol/L NaCl was determined at 25 °C using an Optilab Rex interferometeric refractometer (Wyatt Technology) at a wavelength of 658 nm, and the concentrations of P3VBCB determined in 0.1 mol/L NaCl for use in a Zimm plot were 1.32 × 10−4, 2.52 × 10−4, 4.92 × 10−4, 4.92 × 10−4, and 7.48 × 10−4 g/mL.

DLS measurements were performed on the DAWN HELEOS, Wyatt QELS, and Optilab DSP instruments. The water used for light scattering measurements was all filtered through 0.22 μm hydrophilic membranes (Millipore, Billerica, MA, USA) before use. Solutions were prepared by the weighing method.

The RLS measurements were performed at 25 °C on a Hitachi (Tokyo, Japan) F-4500 fluorescence spectrophotometer.

The pH experiments were performed on a pHS-3 pH meter (Leica Instrument Co., Shanghai), with an E-201-C glass electrode.

Conductivity experiments were performed on the E-201-C glass electrode together with a model 232 Hg electrode used as a reference electrode.

Electrophoresis experiments were performed in TAE buffer (pH 7.4) on a DYY-6c electrophoresis system (Pioway Medical Lab Equipment Co., Beijing, China) using ethidium bromide as a chromogenic reagent (U = 70 v, T = 37 °C, t = 40 min).

The thermal denaturation of λ-DNA was studied at λ = 260 nm by a UV3100 spectrophotometer (Shimadzu, Tokyo, Japan), increasing the temperature from 25 °C to 90 °C using a constant-temperature water bath.

Results and discussion

Preparation of polypseudorotaxanes (P3VBCB)

The pseudorotaxane monomer 3VBCB, consisting of cucurbit[6]uril (CB[6]) and N′-3-vinylbenzyl-1,4-diaminobutane dihydrochloride (compound 2), was prepared according to Fig. 1. First, acetalization of 3-vinylbenzyl aldehyde with 1-azido-4-aminobutane followed by reduction with NaBH4 produced compound 1. The azide reduction of compound 1 was carried out using the triphenylphosphine method, and the resulting solution was then neutralized using concentrated hydrochloride at 0 °C to give compound 2. 3VBCB was synthesized in high yield by threading CB[6] onto compound 2 in aqueous solution.

Fig. 1
figure 1

Synthesis of the polypseudorotaxanes P3VBCB

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The polypseudorotaxanes P3VBCB were prepared using K2S2O8 as the initiator and 3VBCB as the monomer in water under an N2 atmosphere by free-radical polymerization, as shown in Fig. 1. Following purification using ethanol, the polymeric product P3VBCB was obtained with an 83 % yield.

In static light scattering, we can obtain the weight-average molar mass M w of polymer chains from the angular dependence of the excess absolute scattering intensity, known as the Rayleigh ratio R(θ), on the basis of

$$ \frac{K*C }{{R\left( \theta \right)}}=\frac{1}{{{M_{\text{w}}}}}\left( {1+\frac{1}{3}\left\langle {r_{\text{g}}^2} \right\rangle {q^2}} \right)+2{A_2}C, $$
(1)

where \( K*={{{4\pi {{{\left( {{{\text{d}}n \left/ {{\text{d}}c } \right.}} \right)}}^2}{n_0}}} \left/ {{\left( {{N_{\text{A}}}\lambda_0^4} \right)}} \right.} \) and \( q=\left( {{{{4\pi {n_0}}} \left/ {{{\lambda_0}}} \right.}} \right)\sin \left( {{\theta \left/ {2} \right.}} \right), \) where n 0, dn/dc, λ 0, and θ are the solvent refractive index, the specific refractive index increment, the wavelength of the incident light in vacuum, and the scattering angle, respectively. Figure 2 shows a typical static Zimm plot of P3VBCB in 0.1 mol/L NaCl, where C ranges from 1.32 × 10−4 to 7.48 × 10−4 g/mL. Employing the projection θ = 0 and C = 0, the weight-average molar mass M w was calculated using Eq. 1. The static light scattering measurements indicated that the value of M w for P3VBCB is 1.87 × 103 g/mol.

Fig. 2
figure 2

Typical static Zimm plot for P3VBCB in 0.1 mol/L NaCl

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1H NMR spectra

The 1H NMR spectrum of P3VBCB clearly indicated the formation of polypseudorotaxanes consisting of not only rotaxane but also diaminobutane ions, as shown in Fig. 3a. Following the preparation of P3VBCB, the olefinic proton signals from the vinylbenzyl group disappeared, which suggested that free-radical polymerization of the monomer 3VBCB had taken place. The effects of adding different salts (BaCl2 and Na2SO4) on P3VBCB were also studied by 1H NMR. Figure 3 shows the 1H NMR spectra of (a) P3VBCB, (b) P3VBCB with adding BaCl2, and (c) P3VBCB with adding Na2SO4. When P3VBCB (5 mg) was added to a solution of BaCl2 (0.1 mol L−1), Fig. 3b shows that the methylene signals of the butyl units in the side chains of P3VBCB shifted downfield to 1.7 and 3.0 ppm. This indicates that CB[6] was jettisoned from the side chains of P3VBCB upon the addition of BaCl2, because the binding constant of CB[6] with Ba2+ (1.7 × 105 L/mol) is higher than that of CB[6] with butanediamine units [36]. This in turn provides a theoretical basis for the application of P3VBCB in drug release, because the quaternary ammonium salt shielded by CB[6] is nontoxic comparing with the physiological toxicity of the free quaternary ammonium salt. Upon the removal of Ba2+ as precipitate, CB[6] would be able to thread back onto the side chains. Indeed, when Na2SO4 was added into a BaCl2 solution of P3VBCB, CB[6] threaded back onto the side chains, as shown in Fig. 3c. This experiment was repeated three times to prove the reversibility of the threading and dethreading of CB[6] in the side chains of P3VBCB with the addition of Ba2+. The experimental results were consistent with the changes in the 1H NMR spectrum seen in Fig. 3, so it is clear that the threading and dethreading of CB[6] in the side chains can be controlled by the addition of salt.

Fig. 3
figure 3

1H NMR spectra of P3VBCB (a), P3VBCB with the addition of BaCl2 (b), and a BaCl2 solution of P3VBCB with NaSO4 added (c)

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Effect of the concentration of P3VBCB on the aggregate size

Figure 4 shows the hydrodynamic radius distributions and RLS spectra of different concentrations of P3VBCB in 0.1 mol/L NaCl. The distribution of the hydrodynamic radius and the RLS intensity of P3VBCB at the maximum scattering wavelength of 394 nm were found to increase as the concentration of P3VBCB increases, as shown in Fig. 4. This indicates that the aggregate sizes increases as the concentration of P3VBCB increases, because the intermolecular interaction strength for P3VBCB increases with the concentration of P3VBCB increasing.

Fig. 4
figure 4

a Hydrodynamic radius distributions and b RLS spectra for different concentrations of P3VBCB in 0.1 mol/L NaCl

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Effect of the salt concentration on P3VBCB

NaCl (C < 0.15 mol/L) has no effect on the threading and dethreading of CB[6] [59], so the effect of NaCl on the aggregation behavior of P3VBCB was studied by DLS. Figure 5 shows the hydrodynamic radius distributions of P3VBCB (0.2 mg/mL) in different concentrations of NaCl. The hydrodynamic radius distribution of P3VBCB was found to increase as the concentration of NaCl was increased from 0.05 to 0.13 mol L−1, which indicates that the aggregate size increases with increasing NaCl concentration. This is mainly because the Na+ shields the CB[6] beads threaded on side chains from each other, decreasing the electrostatic repulsive interactions among the side chains of P3VBCB [59]. Therefore, increasing the concentration of NaCl effectively increases the attraction among P3VBCB molecules, thus leading to an increase in aggregate size.

Fig. 5
figure 5

Hydrodynamic radius distributions of P3VBCB (0.2 mg/mL) at different concentrations of NaCl

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The pH-responsive behavior of P3VBCB

Figure 6a shows the effect of pH on the hydrodynamic radius of P3VBCB. The experimental data show that the hydrodynamic radius of P3VBCB initially increases but later decreases with increasing pH. This is because interchain association occurs in acidic solution, whereas the small CB[6] beads dethread from the polypseudorotaxanes and enter solution at alkaline pH.

Fig. 6
figure 6

a Effect of pH on the hydrodynamic radius of P3VBCB (0.2 mg/ml), and b pH and electrical conductivity curves for an aqueous solution of P3VBCB (13 mL, 0.160 mg/mL) upon adding 0.1 mol/L NaOH

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To further study the pH-responsive behavior of P3VBCB, HCl (1 mol/L) was initially added to a 0.2 mg/ml solution of P3VBCB, and then 0.1 mol/L NaOH was added dropwise to this acidic solution of P3VBCB, while measuring the pH and electrical conductivity of the solution of P3VBCB. Figure 6b shows the effect of the concentration of NaOH on the pH and electrical conductivity of the solution of P3VBCB. Initially, as the NaOH (0.1 mol/L) solution is added, the pH of the solution of P3VBCB does not change. This indicates that the H+ concentration in the solution does not change, so the OH− ions from the added NaOH must first neutralize the H+ from the protonated 1,4-diaminobutane moiety of P3VBCB, neutralizing P3VBCB. When the volume of NaOH added reaches 0.85 mL, the pH of the P3VBCB solution increases rapidly because the OH− ions from the added NaOH begin to neutralize the free H+. Finally, when the volume of NaOH increases beyond 1.05 mL, the pH remains relatively constant. At the same time, the electrical conductivity of the solution of P3VBCB jumps when the volume of NaOH added is between 0.85 mL and 1.05 mL because the added Na+ can bind with CB[6], which leads to the dethreading of CB[6] from the side chains. The jumps in pH and electrical conductivity seen upon NaOH addition clearly indicate that P3VBCB presents pH-responsive behavior.

The interaction between λ-DNA and P3VBCB

Polymers with quaternary ammonium salts can bind with DNA. Therefore, in order to determine whether the quaternary ammonium shielded could bind with DNA, the interaction between λ-DNA and P3VBCB was studied by electrophoresis. Figure 7 shows the gel electrophoretic patterns of λ-DNA (lanes 1 and 11) and λ-DNA after treatment with different concentrations of P3VBCB. When the concentration of P3VBCB decreases from 1 mg/mL to 0.125 mg/mL, there is no electrophoresis of λ-DNA, but the fluorescence intensity of the electrophoresis of λ-DNA increases as the concentration of P3VBCB decreases further, down to 0.0039 mg/mL, as shown in Fig. 7. These results indicate that P3VBCB could form a stable complex with λ-DNA, because CB[6] threaded onto the side chains of P3VBCB can bind with the amidogen groups of λ-DNA via hydrogen bonding between the amidogen groups of λ-DNA and the carbonyl groups of CB[6], while the quaternary ammonium units of P3VBCB can neutralize the basic groups of λ-DNA.

Fig. 7
figure 7

Gel electrophoretic patterns of λ-DNA (lanes 1 and 11) and λ-DNA after treatment with different concentrations of P3VBCB at pH 7.4 for 12 h: (2) 1.0 mg/mL, (3) 0.5 mg/mL, (4) 0.25 mg/mL, (5) 0.125 mg/mL, (6) 0.0625 mg/mL, (7) 0.031 mg/mL, (8) 0.0156 mg/mL, (9) 0.0078 mg/mL, (10) 0.0039 mg/mL

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The unraveling of the double helix structure of DNA and the degeneration of DNA with heating are due to the damages of the hydrogen-bonding network, base stacking forces, and electrostatic forces within DNA. The UV absorption of DNA at 260 nm increases with the degeneration of DNA. Figure 8a shows the thermal denaturation curves of λ-DNA and λ-DNA after treatment with P3VBCB. The UV absorption intensity of λ-DNA increases after treatment with P3VBCB at 260 nm (OD260 nm), and the temperature at which the curve for λ-DNA jumps decreases after treatment with P3VBCB, as shown in Fig. 8a.

Fig. 8 a–b
figure 8

Thermal denaturation (a) and f ss (b) curves with increasing temperature for λ-DNA and for λ-DNA after treatment with P3VBCB

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The thermal melting temperature of DNA is a measure of the stability of the DNA double helix upon heating. An increase in the thermal melting temperature (T m) indicates an interaction between DNA and P3VBCB. The parameter f ss can be calculated for the λ-DNA system as follows:

$$ {{f}_{\mathrm{ss}}}={{{\left( {A-{{A}_0}} \right)}} \left/ {{\left( {{{A}_{\mathrm{f}}}-{{A}_0}} \right)}} \right.}. $$
(2)

Here, A f is the max absorbance of λ-DNA at 260 nm (indicating completely melted—or unraveled—λ-DNA), and A 0 is the minimum absorbance of λ-DNA at 260 nm (indicating completely unmelted λ-DNA; i.e., double-helix DNA). When f ss is equal to 0.5, the corresponding temperature is the melting temperature of DNA (T m). In the present case, thermal melting studies were carried out and T m values were determined by monitoring the absorbance of DNA at 260 nm as a function of temperature. The melting temperature of DNA (T m) in the presence of a binding molecule can also be used to distinguish between intercalative and external binding modes. Usually, classical intercalation gives rise to higher ΔT m values than either groove binding or outside stacking [60]. The T m value for pure λ-DNA was found to be 59.5 °C under our experimental conditions. Under the same set of conditions, the double-helix λ-DNA structure degraded at 38 °C in the presence of P3VBCB, as shown in Fig. 8b. The experimental data show that P3VBCB can destroy the double-helix structure of λ-DNA through the insertion of the rotaxane structure of P3VBCB into base pairs of λ-DNA, as shown in Fig. 9, which leads to a decrease in the T m value of λ-DNA with the addition of P3VBCB. Thus, the water-soluble polypseudorotaxanes P3VBCB can perform DNA scission.

Fig. 9
figure 9

Schematic illustration of the damage caused by P3VBCB to the double-helix structure of λ-DNA

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Conclusions

We synthesized the rotaxane monomer 3VBCB, consisting of cucurbit[6]uril (CB[6]) and N′-3-vinylbenzyldiaminobutane dihydrochloride. Side-chain polypseudorotaxanes P3VBCB were obtained by polymerizing the monomer 3VBCB. The 1H NMR data showed that the threading and dethreading of CB[6] onto the side chains of P3VBCB can be controlled by adding BaCl2 and Na2SO4. DLS results showed that the aggregate size increases as the concentration of P3VBCB or NaCl is increased. pH and electrical conductivity experimental data showed that P3VBCB presents pH-responsive behavior, and gel electrophoresis and DNA thermal melting data showed that P3VBCB can destroy the double-helix structure of λ-DNA, leading to a decrease in the value of T m of λ-DNA upon the addition of P3VBCB. Further research on the toxicity of P3VBCB to cell cultures would be worthwhile.