Brønsted base mediated one-pot synthesis of catechol-ended amphiphilic polysarcosine-b-poly(N-butyl glycine) diblock copolypeptoids

Materials

Tetrahydrofuran (THF) was purchased from Sigma-Aldrich and distilled from Na/K. DMF was purchased from Sigma-Aldrich, dried over CaH₂, and subsequently distilled in vacuo onto molecular sieves (3 Å). Dichloromethane (99.5%, DCM) was distilled over CaH₂ under an argon atmosphere. Sar-NCA [24] and Bu-Gly-NCA [30] monomers were prepared following the modified literature procedure. Dopamine hydrochloride and other substances were used as received. Manganese chloride, MnCl₂·4H₂O (TCI, 98%), sodium oleate (TCI, 98%) and 1-octadecene (TCI, 95%) were used to synthesize oleate-capped MnO-NPs.

Characterizations

¹H NMR spectra were recorded on a Bruker ARX-250 spectrometer with 400 MHz at room temperature and the data were analyzed by MestreNova software. Melting points of Sar-NCA monomer was determined using a melting point apparatus. Size exclusion chromatography (SEC) was performed in N,N-Dimethylformamide (DMF) using an SSI 1500 pump equipped with Waters column (5 mm, 300×7.8 mm) at a flow rate of 0.07 mL min⁻¹ at 25°C, and a Wyatt Optilab rEX differential refractive index (DRI) detector with a 658 nm light source. DMF containing 0.05 mol L⁻¹ LiBr was used as the eluent. Both the column and the detector temperature were kept at 25°C. All data were analyzed by Wyatt Astra V 6.1.1 software. The number average molecular weight (M_n) and molecular weight distributions (M_w/M_n) were determined at dn/dc values of 0.0987. Matrix assisted laser desorption/ionization time-of-flight mass spectra (MALDI-ToF MS) were performed on a mass spectrometer (ultraflextreme; Bruker Co.) with Smartbeam/Smartbeam II modified Nd: YAG laser. Mass spectra of 500 shots were collected for the spectra at a 25 kV acceleration voltage. The polymer sample was dissolved in CHCl₃ at a concentration of 5.0 mg mL⁻¹, the matrix 2,5-DHB (2,5-dihydroxybenzoic acid) was dissolved in solution of trifluoroacetic acid and acetonitrile (volume ratio=70/30) in water (1.0%, 10 μL). The self-assembly of block copolymers was analyzed by Dynamic Light Scattering (DLS) Malvern Instrument Zetasizer Nano S90 with a He-Ne laser (633 nm, 4 mW) at room temperature. The sample solutions were prepared in distilled water with a concentration of 5.0 mg mL⁻¹. To observe the size and morphologies of the aggregates, transmission electron microscopy (TEM) measurement was performed using JEM-1200 TEM instrument operating at 120 kV. A drop of MnONPs@cat-PSar-b-PGly^Bu solution in ethanol was placed on a carbon-coated copper grid followed by solvent evaporation at room temperature.

General procedure for the synthesis of polysarcosine (PSar₅₀)

Sar-NCA (0.2647 g, 2.3 mmol) was dissolved in 2.3 mL dry DMF. Followed by the addition of dopamine hydrochloride (8.72 mg, 0.046 mmol) as initiator and 25 μL of TEA (0.18 mmol) as catalyst. The reaction mixture was stirred at room temperature under inert atmosphere (N₂ flow) and continuous stirring. The polymerization was quenched in Et₂O and the product was dried under vacuum oven. ¹H NMR (500 MHz; D₂O): δ=2.89–3.3 (150H, CH₃-), 4.20 (104H, -CH₂-CO-), 6.3–6.5 (3H, C₆H₃O₂H₂).

Synthesis of polysarcosine₅₀-block-poly(N-butyl glycine)₂₅ diblock copolypeptoids

Initially, Sar-NCA (0.2647 g, 2.3 mmol) was dissolved in 2.3 mL DMF. Dopamine hydrochloride (8.72 mg, 0.046 mmol) and 25.08 μL TEA (0.18 mmol) were added to the flask. The reaction was carried out under nitrogen flow. After 24 h, some portion of the reaction mixture was precipitated in diethyl ether and kept in vacuum oven for further characterization. To the flask containing PSar, Bu-Gly-NCA [30] (0.133 g, 1.15 mmol) was dissolved in 0.87 mL DMF and added to the flask containing the first block. The reaction mixture was stirred for additional 24 h under N₂ flow. Then, the polymerization reaction was terminated by diethyl ether. ¹H NMR (400 MHz; DMSO-d₆): δ=6.57 (3H, C₆H₅O₂), 4.19–3.88 (108H, C₄H₉-N-CH₂-CO- and CH₃-NH-CH₂-C₆H₃(OH)₂), 3.27 (36H,CH₃-CH₂-CH₂-), 3.03–2.79 (159H, CH₃), 1.44 (25H, CH₃-CH₂-CH₂-CH₂-), 1.24 (29H,CH₃-CH₂-C₂H₄), 0.88 (37H, CH₃-C₃H₆-).

Functionalization of MnO nanoparticles

The synthesis and functionalization of oleate capped MnO-NPs was carried out following literature report [35]. cat-PSar-b-PGly^Bu (30.0 mg) was dissolved in chloroform (20.0 mL). The oleate-capped MnO-NPs (10.0 mg) were dissolved in chloroform (5.0 mL) and methanol (5.0 mL) followed by dropwise addition to the polymer solution. The solution was stirred overnight at room temperature and subsequently concentrated to 10.0 mL under reduced pressure. The product was obtained by the addition of hexane (10.0 mL) followed centrifugation (6 min, 6000 min⁻¹) and redissolved in 5.0 mL acetone. The precipitate was washed three times and dried in a vacuum oven. The aqueous solubility of the cat-PSar–b-PGly^Bu functionalized MnO-NPs were elucidated in hexane/water solvent systems.

Catechol initiator

Dopamine hydrochloride initiator has two acidic sites comprising the catechol moiety and the ammonium salt (-CH₃CH₂NH₃Cl) (Scheme 3, 1). Notably, the pK_a value of the ammonium salt in H₂O was estimated to be 8.59 and catechol moiety contains one intramolecularly bonded hydrogen with pK_a value about 14.08 in H₂O and one free hydrogen with pK_a value about 11.16 in H₂O [37]. The free hydrogen of catechol moiety is much more acidic than that of intramolecularly bonded hydrogen. In this contribution, a Brønsted base mediated ROP of Sar-NCA using catechol initiator was suggested. The Brønsted base coordinates with the free acidic hydrogen which result in the formation of strong intramolecular H-bonding (Scheme 3, 2). Most importantly, the Brønsted base plays a dual role that is (1) prevent the intervention of catechol moiety (2) control the activity of the primary amine in the polymerization process. The ROP of Sar-NCA was carried out using dopamine hydrochloride initiator and triethylamine as Brønsted base. Initially, 1.0/2.0 ratios of dopamine hydrochloride initiator and TEA was used for ROP Sar-NCA such that 1.0 equiv. of TEA is supposed to react with the ammonium salt to generate the active amine end latter on the other 1.0 equiv. TEA react with free hydrogen of catechol moiety. The primary amine in dopamine was used to initiate the ROP of Sar-NCA.

Scheme 3:

A representative reaction for one-pot synthesis of Brønsted base mediated ROP of Sar-NCA using catechol initiator and copolymerization with Bu-Gly-NCA.

A Brønsted base (B) is used to mask the activity of catechol via intramolecular H-bonding and controlled the ROP of Sar-NCA (Scheme 3). Briefly, about 1.0 equiv. of B first react with the ammonium salt to provide active amine (2) and conjugate acid (BH⁺). Latter, another 1.0 equiv. of B coordinates with the free acidic hydrogen from catechol moiety (1) which result in the formation H-bonding between catechol moiety (3) and the Brønsted-base (B). The ROP of Sar-NCA takes place following normal amine mechanism [38], [39]. The primary amine in dopamine (3) as a nucleophile attacks the electron deficient carbonyl carbon of Sar-NCA monomer followed by decarboxylation of the carbamic acid to yield PSar (4). The secondary amine in PSar terminus as macroinitiator triggers the ROP of Bu-Gly-NCA monomer. Followed by the formation of catechol end-functionalized amphiphilic cat-PSar-b-PGly^Bu diblock copolymers (5). To obtain cat-PSar-b-PGly^Bu with free catechol moiety (6), (5) treated with 1.0 mol L⁻¹ HCl (aq.).

Catechol functionalized polysarcosine

Ring-opening polymerization of α-amino acid N-carboxyanhydrides (NCAs) is well established method to prepare polypeptides [40], [41], [42]. The strategy showed success in polypeptoid synthesis from NCAs of N-substituted glycine [30], [43]. The incorporation of catechol moiety to PSar-based amphiphilic block copolypeptoids demands a precise control over the molecular weights of the designed polymers. Previously, primary amine hydrochlorides were used to control the ROP of NCAs through protonation of NCA anions [44], [45]. Thornton et al. reported the polymerization of NCAs using dopamine hydrochloride initiator with slow rate [46]. Later, Schlaad et al. disclosed a controlled ROP of NCAs with relatively enhanced rate using a primary amine/tertiary amine catalytic system [47]. Herein, Brønsted base mediated ring-opening polymerization strategy was suggested to overcome catechol interference and modulate the polymerization. We disclosed TEA mediated ROP of Sar-NCA by dopamine hydrochloride initiator to obtain catechol end-functionalized polysarcosine with fast polymerization rate [46], [47], [48]. In the initiation step, the ROP of Sar-NCA triggered by nucleophilic attack of primary amine on carbonyl carbon at C5 (Scheme 4) result in the formation of carbamic acid followed by decarboxylation to afford PSar with secondary amine terminus [41], [49].

Scheme 4:

ROP of Sar-NCA using dopamine in DMF at 25°C to obtain catechol functionalized Psar.

The ROP of Sar-NCA monomer was performed in DMF at 25°C using dopamine hydrochloride initiator and TEA catalytic system. The rate of polymerization of R-NCA monomers by dopamine hydrochloride without the addition of a catalyst is quite slow and no polymer peak appeared in ¹H NMR spectrum after 24 h (Fig. S3). After 96 h reaction time, monomer conversion was less than 10.0% and product was not observed in diethyl ether (Et₂O). Afterwards, dopamine hydrochloride/TEA catalytic system were used for ROP of Sar-NCA to enhance rate of polymerization of the monomer. The reaction was terminated by Et₂O after 24 h. The product was clearly observed and kept in vacuum oven for further investigation. The presence of TEA significantly enhanced the polymerization rate of Sar-NCA. The degree of polymerizations (DPs) and the molecular weights were calculated from ¹H NMR spectra by comparing the integral values of aromatic protons (δ=6.5–6.8 ppm) with methylene protons (δ=3.7–4.5 ppm) of PSar (Fig. 1). The monomer conversion reached 99.9% after 24 h as confirmed by ¹H NMR and the monomer peak was no longer available. The ¹H NMR of the sample polymers were in accordance with the proposed structure of the repeating unit and DPs.

Fig. 1:

¹H NMR spectrum of catechol moiety end-functionalized PSar₅₀ in D₂O.

To prepare well-defined polypeptoids, the polymerization reaction was carried out at various monomer to initiator feed ratios. Low molecular weight PSar (M_n,SEC=1.1 kg mol⁻¹) with narrow dispersity (Đ<1.1) and monomodal SEC traces obtained at M/I/TEA=20/1/1.1 (Fig. S4). However, as the M/I ratios increased to 30, 50, 75 and 100, keeping I/TEA same, the SEC traces are found to be unreliable and a shoulder appeared. Even though, the conversion reached 99.9% and M_n,NMR nicely agreed with M_n,theo but M_n,SEC values obviated (Table S1). In a quest to obtain high M_n,SEC, the ROP of Sar-NCA was carried out at different ratios of TEA.

The polymerization was done at M/I/TEA=50/1 by varying the ratios of TEA to obtain relatively close M_n,SEC to the predicted one. As the ratio of TEA increased from 1.1 to 2.1, the M_n,SEC changed dramatically from 1.6 kg mol⁻¹ to 2.0 kg mol⁻¹. Further increase in TEA ratios to 2.5, 2.7, 2.9 and 3.1 reveals high M_n,SEC. Table 1 entry 3 displays close agreement in M_n,SEC 3.4 kg mol⁻¹ as the TEA ratio increased to 2.3. Besides, high M_n,SEC=4.3 kg mol⁻¹ and M_n,NMR=4.1 kg mol⁻¹ obtained at ratios I/TEA of 1/2.9 which is higher than the predicted M_n,theo (Table 1 entry 6). Hence, the results indicate that TEA worked a dual role including to mask catechol and control the activity of active amine-end. The traces of 50/1/2.3 are better than that of 50/1/1.1 which confirms the catechol masking. M_n,SEC is in close agreement with that of M_n,NMR for the polymerization which takes place at the initiator to catalyst ratio of 1/2.3 (Fig. 2d). The polymerization is not well controlled at higher TEA ratios i.e. 2.9 and 3.1 which result in broad SEC traces with shoulder.

Fig. 2:

SEC traces of cat-PSar₅₀ at different initiator to TEA ratios (a) 1/2.5 (b) 1/2.9 (c) 1/3.1 (d) 1/2.3.

Catechol initiator was successfully attached to PSar end and the mass differences (∆m/z) between individual signal peaks attributed to the molecular weight of the Sar repeating unit which is 71.04 g mol⁻¹ [50] (Fig. 3). The results suggested that dopamine hydrochloride initiated the ROP of Sar-NCA to afford catechol end PSar. TEA coordinates with the acidic hydrogen of catechol initiator via H-bonding interaction. We reasoned that the Brønsted base coordinated with the free acidic hydrogen via H-bonding. As a result the other intramolecularly bonded hydrogen gets stronger in such a way that catechol masked throughout the polymerization. Water and unknown end groups also initiated the monomer. The presence of other peaks of the PSar is due to impurities and side reactions.

Fig. 3:

MALDI-ToF mass spectrum of catechol end-functionalized PSar₅₀ ([I]/[TEA]=1:1.1).

Catechol functionalized polysarcosine-block-poly(N-butyl glycine)

The switch of N-substituents from methyl to n-butyl turned hydrophilic PSar into hydrophobic poly(N-butyl-glycine). The synthesis of end-chain modified polypeptoids can be achieved by different ways using functional initiators [51], monomers [52], and postpolymerization reactions [53], [54], [55]. In this work, a functional initiator dopamine hydrochloride was employed to prepare catechol end-functionalized amphiphilic diblock copolypeptoids. The Brønsted base, triethylamine, controls the activity of the active amine end. Thereafter, PSar with secondary amine terminus as macroinitiator triggers the ROP of Bu-Gly-NCA (Scheme 5, 1) to offer catechol end-functionalized amphiphilic diblock copolypeptoids (2).

Scheme 5:

ROP of Bu-Gly-NCA in DMF at 50°C to obtain catechol end-functionalized amphiphilic PSar-b-PGly^Bu diblock copolypeptoids.

The polymerization of Bu-Gly-NCA was carried out at 50°C under N₂ flow. After 48 h, the final product was precipitated in diethyl ether. The molecular structures of the block copolymers were confirmed by ¹H NMR in DMSO-d₆. In the ¹H NMR spectrum (Fig. 4), the proton resonances at 4.04–4.54 ppm and 2.88–3.14 ppm are assigned to the methylene proton and methyl proton of the PSar. The molecular structures of the amphiphilic polypeptoids and their corresponding monomers are in accordance with the literature report [24]. M_n,NMR of the obtained block copolypeptoids are in agreement with that of the theoretical values.

Fig. 4:

¹H NMR spectrum of catechol end-functionalized PSar₅₀-b-PGly^Bu₁₂ in DMSO-d₆.

However, the molecular weights of diblock copolypeptoids, M_{n,SEC ar} are not in agreement with the M_n,theo and M_n,NMR (Table 2 and Fig. S6). This may be due to the impurities and side reaction, the initiation of Bu-Gly-NCA monomer by the secondary amine macroinitiator is slower and resulted in relatively lower M_n,SEC of the second block [39]. Further optimization of Bu-Gly-NCA and addition of the Brønsted base are suggested to control the polymerization. Overall, the substituents of N-alkyl glycines can be derivatized by changing the R group in the amide backbone for click and other postpolymerization reactions.

Amphiphilic N-substituted polypeptoids have shown the ability to disperse a hydrophobic model compound in aqueous media [24]. Catechol initiator was successfully incorporated to amphiphilic PSar-b-PGly^Bu diblock copolymers. The prepared amphiphilic PSar-b-PGly^Bu block copolypeptoids bearing catechol moiety were soluble in water due to their aqueous self-assembly thereby further used for surface functionalization of MnO-NPs [2], [56], [57]. The final product cat-PSar-b-PGly^Bu with free catechol end was released by its acidification with 1.0 mol L⁻¹ HCl (aq.) (Scheme 5, 3). Furthermore, the prepared amphiphilic diblock copolypeptoids with free catechol moiety were used for aqueous solubility and MnO-NPs surface coatings.

Aqueous dispersion of manganese oxide nanoparticles

Monodisperse MnO-NPs are prepared by thermal decomposition of manganese-oleate complex. Herein, we employed grafting technique to functionalize the pre-made MnO-NPs by catechol end-functionalized amphiphilic PSar-b-PGly^Bu diblock copolypeptoids. Catechol was used as anchoring group to the surface of the MnO-NPs. The diblock copolypeptoids provide further stability and aqueous dispersion. Since the density of the hydrophobic segment is much lesser than that of the hydrophilic, the NPs coated by block copolymers can self-assemble in aqueous solution. Aqueous dispersability of olaete capped MnO-NPs and catechol end-functionalied PSar-b-PGly^Bu are shown in Figs. 5 and S8. Figure 5a-left and 5C display the solubility of oleate-coated MnO-NPs in hexane and its corresponding TEM image, respectively. Figure 5a-right shows the aqueous phase transition of MnO-NPs coated by catechol-ended functionalized amphiphilic PSar-b-PGly^Bu diblock copolypeptoids.

Fig. 5:

(a) Solution behavior of oleate-capped MnO-NPs (left); after functionalization by catechol end-functionalized PSar₅₀-b-PGly^Bu₁₂ (right) (b) DLS spectrum of MnO-NPs coated by cat-PSar-b-PGly^Bu in aqueous solution (c) TEM image of oleate-capped MnO-NPs (d) TEM image of cat-PSar-b-PGly^Bu coated MnO NPs.

The self-assembly nature of cat-PSar-b-PGly^Bu played a key role for the dispersion of MnO-NPs in water with no precipitation (Fig. 5a right). DLS results demonstrated the aggregate behavior of the cat-PSar-b-PGly^Bu into nanostructures in aqueous solution with average hydrodynamic diameter of 64 nm (Fig. 5b). In addition, TEM image displays the formation of monodisperse cat-PSar-b-PGly^Bu capped MnO-NPs with average size of about 12 nm (Fig. 5d). The size difference might be attributed to the solvation shell [58] along with the MnO-NPs surface. Overall, the resulting MnO@cat-PSar-b-PGly^Bu nanocomposites have showed self-assembly driven excellent aqueous dispersability [56], [59] and stabilization.