Oligo(β-peptoid)s with Backbone Chirality from Aspartic Acid Derivatives: Synthesis and Property Investigation.

Poly(β-peptoid)s (N-substituted poly-β-alanines) are an intriguing class of pseudopeptidic materials for biomedical applications, but the polymers prepared by solution polymerization have restricted diversity and functionality due to synthetic difficulty. Synthesis of structurally diverse poly(β-peptoid)s is highly desirable yet challenging. Herein, we report a new approach to synthesize skeletal chiral β-peptoid polymers from readily available aspartic acid derivatives. Two types of N-substituted β3-homoalanine monomers, i.e., N-(methyl propionate)-Asp-OMe ( N MeP-Asp-OMe) and N-(tert-butyl propionate)-Asp-OMe ( N tBuP-Asp-OMe), were synthesized in high yield via an aza-Michael addition reaction between l-aspartic acid-1-methyl ester (l-Asp-OMe) and acrylate species. Both N-substituted β3-homoalanines can be readily converted into polymerizable N-substituted β3-homoalanine N-carboxyanhydrides (β-NNCAs). Subsequent ring-opening polymerization (ROP) of these β-NNCA monomers provides access to oligo(β-peptoid)s and mPEG-poly(β-peptoid) diblocks with backbone chirality. Their conformations were preliminarily studied by circular dichroism (CD) spectra and Fourier transform infrared spectroscopy (FT-IR). The synthetic strategy would significantly facilitate the development of novel poly(β-peptoid)s with well-defined and diverse structures.

Introduction

Synthetic peptide analogues have been long sought after for their promising applications in biotechnology.[1,2] Poly(β-peptoid)s (N-substipan class="Chemical">tuted poly-β-alanines) are a particularly intriguing class of pseudopeptidic polymers, which can formally be gained by the addition of a methylene unit in the backbone of polypeptoids or the N-substitution of poly(β-peptide)s.[3−5] Tertiary amides in a β-peptoid backbone offer higher proteolytic resistance and less polar as compared to typical peptide amide bonds.[6−11] Benefitting from a combination of biocompatibility, degradability, metabolic stability, and processability, β-peptoids have vast potential for therapeutic applications.[12,13] Furthermore, the extra C–C bond in the β-peptoid backbone would greatly expand the potential diversity of the peptoid shape as compared to α-peptoids.[5,14−16] In this regard, a great deal of effort has been made to develop poly(β-peptoid) materials with interesting structures for property investigation and biological applications in recent years.[14−19]

Generally, β-peptoids are conformationally flexible and do not form stable folding structures due to missing amide protons and chiral centers on their backbones. Therefore, approaches to restricting the structural flexibility of β-peptoids are attractive and significant. It has been demonstrated that β-peptoids with stable helical structures can be achieved by introducing chiral N-substituents.[20] However, the requirement of submonomers with bulky and chiral substituents in this strategy impedes the utility for the synthesis of chemically diverse β-peptoids with well-defined folded structures. In addition to β-peptoids with chiral N-substituents, β-peptoids with backbone chirality have been shown to form defined secondary structures as well.[15−17,19] Specifically, Lim and co-workers reported a series of N-alkylated β2-homoalanine oligomers (α-ABpeptoids) via solid-phase synthesis.[17,19] These α-ABpeptoid oligomers with different side chains showed distinctive circular dichroism (CD) characteristics, indicative of ordered folding conformations. Besides, β-ABpeptoids with chiral groups at β-positions rather than α-positions were prepared via a submonomer solid-phase strategy.[14,16,21] Very recently, Morimoto and co-workers reported that the introduction of bulky substituents at β-carbon- would strongly promote the folding of β-peptoids.[14] All of the abovementioned β-peptoids are synthesized by the monomer or unique “submonomer” method,[4] where the β-peptoid chain is extended in an iterative and stepwise manner. These strategies provide facile access to unique structural control and diversity in a synthetic polymer.[22] However, their multistep synthesis and tedious purification greatly restrict the large-scale synthesis and wide application of β-peptoids.

Ring-opening polymerization (ROP) of α-N-carboxyanhydrides (α-NCAs) represents an efficient and expedient pathway to obtain poly(α-peptide)s[23] and poly(α-peptoid)s[24,25] with controlled structures and compositions.[26] In contrast to poly(α-peptide)s and poly(α-peptoid)s, studies on the ROP of β-NNCAs were rarely reported.[12,27−29] In 1954, Birkofer et al. described the successful synthesis and polymerization of N-p-tolyl-β-alanine-NCA by both thermal and p-toluidine initiation.[28] Later on, they reported the polymerization of N-phenyl-β-alanine-NCA, initiated with water or by heating above the melting point.[29] Zilkha and co-workers demonstrated the synthesis of poly(N-benzyl-β-alanine)s from ROP of corresponding β-NNCAs.[27] A long time later, Luxenhofer et al. demonstrated the living character of β-NNCA polymerization from kinetic studies.[12] Homo and block copolymers with Poisson distribution were successfully obtained via aza-Michael addition. However, the poor solubility limits further investigation. Recently, an alternative route for the synthesis of β-peptoids was reported through copolymerization of N-alkylaziridines[13,30−32] with carbon monoxide using a metal-mediated ROP approach. Although a few β-peptoid materials have been prepared via different synthetic routes based on ROP, synthetic versatility of functional monomers and polymers is still challenging. Especially, β-peptoids with backbone chirality have never been prepared by the ROP strategy in the literature to the best of our knowledge. There is still a significant lack in our understanding of the synthesis and physicochemical properties of β-peptoids. Hence, it is particularly intriguing and highly desirable to develop a novel and effective strategy to obtain structurally diverse β-peptoid materials.

Herein, we report an efficient way to make N-substituted β3-homoalanines using an pan class="Chemical">aza-Michael addition reaction between commercially available l-aspartic acid 1-methyl ester (l-Asp-OMe) and acrylate species (Scheme ). Two types of optically active N-substituted β3-homoalanine monomers, i.e., N-(methyl propionate)-Asp-OMe (MeP-Asp-OMe) and N-(tert-butyl propionate)-Asp-OMe (tBuP-Asp-OMe), were synthesized in high yield. Both of the N-substituted β3-homoalanines can be readily converted into polymerizable N-substituted β3-homoalanine N-carboxyanhydrides (β-NNCAs). Subsequent ROP of these β-NNCA monomers provides access to oligo(β-peptoid)s and mPEG-poly(β-peptoid)s diblocks. The key point of our design is the introduction of the chiral backbone from aspartic acid derivatives using the efficient aza-Michael addition reaction. Aspartic acid, the only naturally occurring proteinogenic β-amino acid, is readily available and shares many excellent properties of natural amino acids. Aza-Michael addition can proceed efficiently under benign conditions and has high tolerance of functional groups. The synthetic strategy represents an efficient methodology to prepare poly(β-peptoid)s with well-defined and diverse structures.

Scheme 1

Synthetic Routes to Oligo(β-peptoid)s from l-Asp-OMe

Results and Discussion

Synthesis of N-Substituted l-Asp-OMe

Scheme shows the synthetic procedures of N-substituted l-aspartic acid derivatives. Generally, pan class="Chemical">l-aspartic acid 1-methyl ester (l-Asp-OMe) reacted with acrylate species (methyl acrylate and tert-butyl acrylate) via the aza-Michael addition reaction to give β-peptoid monomers. The aza-Michael conjugate addition was conducted in methanol under weak basic conditions to avoid hydrolysis of the ester bond. The reactions proceeded efficiently at ambient temperature to give clean products of N-(methyl propionate)-Asp-OMe (MeP-Asp-OMe) and N-(tert-butyl propionate)-Asp-OMe (tBuP-Asp-OMe) as white powders in excellent yields, which can be easily purified by washing. The structures of MeP-Asp-OMe and tBuP-Asp-OMe were characterized by 1H and 13C NMR spectra (Figure S1a–d). All of the peaks were well assigned, indicative of the successful N-substitution of l-Asp-OMe. Furthermore, electrospray ionization tandem mass spectrometry (ESI-MS) results confirmed the successful preparation of MeP-Asp-OMe and tBuP-Asp-OMe (Figure S2). Note that there were no disubstituted byproducts, possibly due to the large steric hindrance, relatively low activity of the α-amino group, and mild reaction conditions.[12] Aspartic acid, the only naturally occurring proteinogenic β-amino acid, is readily available and shares many excellent properties of natural amino acids.[33] The synthetic protocols are facile and readily scalable in high yield, significantly facilitating the synthesis of optically active β-peptoid monomers and relevant conformational study of β-peptoid polymers.

Preparation and Characterization of β-NNCAs

Monomers of MePpan class="Chemical">-Asp-OMe and tBuP-Asp-OMe were then converted into the corresponding β-NNCAs by adopting a modified procedures, as outlined in Scheme . Note that anhydrous tetrahydrofuran (THF) was applied as a solvent for the preparation of Boc-protected precursors due to the hydrolysis of the ester bond in an alkaline aqueous solution. Subsequently, Boc-protected precursors underwent ring closure into β-NNCAs using PCl3 in anhydrous dichloromethane (DCM) under N2 purge. The obtained two β-NNCAs were generally viscous oils at ambient temperature, which were then fractionally precipitated in hexane from a THF solution (THF/hexane = 1:3) to yield samples as colorless oils in 64 and 59% yields, respectively. Both β-NNCA monomers were readily soluble in common solvents such as ethyl ether, toluene, THF, ethyl acetate, DCM, and dimethylformamide (DMF), except hexane. Their structures were unambiguously characterized by 1H and 13C NMR (Figure S3a–d). All of the chemical shifts in MeP-Asp-OMe and tBuP-Asp-OMe NCAs agreed well with the designed structures, indicating the successful cyclization of MeP-Asp-OMe and tBuP-Asp-OMe monomers. Furthermore, Fourier transform infrared spectroscopy (FT-IR) spectra confirmed the successful preparation of two types of β-NNCA monomers with the appearance of a new peak at about 1805 cm–1 (Figures S4 and S5).

Ring-Opening Polymerization of β-NNCAs

It was reported that the N-substituted β-alanine NCAs can undergo living nucleophilic polymerization.[12] We then focus on the ROP of the N-substituted l-Asp-OMe NCA. We have previously prepared several N-substituted α-amino acids and corresponding NNCAs via Schiff base and reductive amination reactions.[34,35] Unfortunately, previous studies showed that the ROP of NNCAs was not successful due to the steric hindrance from the double substitution at the nitrogen atom as well as the C3 atom. For the case of β-NNCAs, which have slightly large ring size and reduced steric hindrance, we hence expected that double substituted β-NNCAs might undergo feasible ROP under appropriate conditions to prepare poly(β-peptoid)s as well as copoly(β-peptoid)s with backbone chirality.

We first studied the effects of the solvent on the ROpan class="Chemical">P of N-substituted l-aspartic acid NCAs with benzylamine as an initiator. All of the reactions were conducted at 50 °C under reduced pressure to remove the generated carbon dioxide. FT-IR was used to monitor the progress of the reaction. 1H NMR and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra were used to confirm the chemical structures and the degree of polymerization (DP) of the polymers. Considering the solubility of the polymers, polymerization in polar solvents such as DMF, N-methyl-2-pyrrolidone (NMP), and THF was first studied. At predetermined time intervals, a small portion of the reaction solution was taken out for FT-IR measurement. FT-IR spectra suggest the (nearly) complete conversion of β-NNCA monomers within 3 days in DMF and NMP. Also, the monomers in THF underwent complete consumption within 2 days. Surprisingly, only oligopolymers with DPs around 3 were obtained regardless of solution concentrations (20–100 mg mL–1) and the initial monomer-to-benzylamine ratio ([M]0/[BnNH2]0 = 50:1 or 100:1) (Figure a and Table S1). Note that extension of the reaction time did not result in polymers with higher DPs, indicating the absence or limited extent of the interchain coupling reaction. Also, polymerization in toluene (TOL), which is a typical nonpolar solvent for the ROP of NNCAs, has been investigated.[36,37] During the polymerization process, precipitates in the form of paste appeared and accumulated on the wall of the Schlenk tube while maintaining the reaction solution in a clear state. The monomer was consumed within 1.5 days with the disappearance of an absorbance peak at about 1805 cm–1 and the appearance of a new peak at 1645 cm–1 (Figure S4) and the products were precipitated using THF/ether to afford oligo(β-peptoid)s with a DP of around 7. In addition, the mixture solvents of THF and TOL (v/v = 1:1) were also attempted, giving oligo(β-peptoid)s with DP around 5 within 2 days. Apparently, TOL appeared to be the best solvent to produce oligomers in a shorter time among these solvents. The possible reason was that β-peptoid precipitated during polymerization due to its poor solubility in TOL, which confined active chain ends to react with monomers.[38] The solvent dependence of the polymerization behavior has been reported previously by other groups.[39,40] Furthermore, polymerization of MeP-Asp-OMe NCA in the absence of any solvent did not work well to form P(MeP-Asp-OMe) (Table S1). We also attempted the polymerization in TOL using other initiator systems such as BnNH2/DBU, BnOH/TU/TBD, n-hexylamine, and hexamethyldisiloxane (HMDS). Unfortunately, only oligomers with DP < 10 were obtained (Table S1).

Figure 1

Influence of (a) solvents (at 50 °C) and (b) reaction temperatures (in TOL) on the degree of polymerization (DP) and polymerization time for MeP-Asp-OMe NCA ([M]0/[BnNH2]0 = 50:1). All samples were prepared at 100 mg mL–1.

Subsequently, polymerization of MeP-Asp-OMe NCA in toluene was investigated with benzylamine at various reaction temperatures. Figure b shows the DP and polymerization time (the time approaching ∼100% monomer consumption) versus temperature plots for MeP-Asp-OMe NCA. Apparently, the DP slightly increased from 4 to 10 as the temperature increased from 25 to 80 °C. Meanwhile, the polymerization time gradually decreased, indicating accelerated monomer consumption at elevated temperatures. Further increasing the temperature from 80 to 120 °C, however, leads to oligomers with almost no further increased DPs. Representative MALDI-TOF analysis of P(MeP-Asp-OMe)7 is shown in Figure S6, confirming the chemical structure of oligo(β-peptoid) (mass of repeat unit, Δm = 215.1 Da, [C9H13NO5]+ = 215.1 g mol–1; DP = ∼ 7; residual mass, r.m. = 106.1 (BnNH, C7H8N)). As for tBuP-Asp-OMe NCA, only oligomers with 3–4 repeat units were obtained under all conditions studied. The characteristics of the prepared oligomers are summarized in Table S1. We assumed that three possible reasons led to low DPs. In contrast to α-NCAs and α-NNCAs, the less ring strain in β-NNCAs makes them less active to undergo ROP.[12] Moreover, the steric hindrance of the propagating species limits the further chain propagation of the disubstituted β-peptoids. Besides, spontaneous polymerizations induced by solvents, water, or other nucleophilic impurities could also result in uncontrollable polymerizations with low DPs and yields (Figure S7).[41,42]

Diblock Copoly(β-peptoid)s Synthesis

To obtain polymers with high molecular weights, we synthesized diblock copolymers of mPEG-b-P(MeP-Asp-OMe) and mPEG-b-P(tBuP-Asp-OMe) via ROP of β-NNCAs using mPEG-NH2 as a macromolecular initiator. Note that the copolymerization in THF and TOL was also investigated, giving similar results to the homopolymerization reactions. Thus, the copolymerizations were all conducted in TOL. The corresponding diblocks were characterized using gel permeation chromatography (GPC) and 1H NMR (Figures , S8, and S9). The molecular characteristics of obtained polymers are listed in Table S2. Figure gives the typical GPC traces of mPEG45-b-P(MeP-Asp-OMe)13 and mPEG113-b-P(MeP-Asp-OMe)22. Obviously, both diblock copolymers showed monomodal distributions with dispersities (Đs) of 1.17 and 1.08, respectively. The DPs were calculated to be 13 and 22 according to GPC, which is similar to the 1H NMR results (Figure S9c,d). Besides, mPEG45-b-P(tBuP-Asp-OMe)9 and mPEG113-b-P(tBuP-Asp-OMe)18 have been successfully obtained and their structures have been confirmed by GPC and 1H NMR, giving acceptable Đs of 1.18 and 1.25, respectively (Figures S8 and S9e,f). These results indicate the successful copolymerization of β-NNCAs and give a complementary approach to access poly(β-peptoid)s with well-defined and complex structures.

Figure 2

GPC traces of mPEG45-b-P(MeP-Asp-OMe)13 (red line) and mPEG113-b-P(MeP-Asp-OMe)22 (blue line) polymerized in TOL.

Solubility

We first studied the solubility of the samples and the corresponding data are summarized in Table S3. It was found that both oligo(β-peptoid)s and diblocks have good solubility in common organic solvents such as THF, DCM, DMF, and NMP. We assumed that the pendant ester groups at the nitrogen atom as well as at the α-C atom provide these β-peptoids good solubility in organic solvents. In turn, the enhanced solubility facilitates further property investigation as well as processibility. Although the samples displayed similar solubility in organic solvents, they had different water solubilities. In particular, both P(MeP-Asp-OMe) and mPEG-b-P(MeP-Asp-OMe) could be readily dissolved in water, while P(tBuP-Asp-OMe) and mPEG-b-P(tBuP-Asp-OMe) showed poor water solubility. The reason is that tert-butyl groups in α-C side chains can offer strong hydrophobicity.

Conformational Studies

It is known that the introduction of backbone chirality can offer ordered folding structures for β-peptoids.[14,15,19,43] Thus, circular dichroism (CD) spectroscopy was utilized to investigate the potential folding structures of the samples. The CD spectra of oligo(β-peptoid)s were obtained in H2O at 25 °C in the varying length (190–280 nm). As shown in Figure a, P(MeP-Asp-OMe)10 displayed characteristic CD signals with intense minima around 208 nm and shallow minima around 230 nm at pH ∼ 7, which were identical in shape to those of oligo(β-peptoid)s prepared by solid-phase synthesis.[15,16] The diblock copolypeptoids of mPEG45-b-P(MeP-Asp-OMe)13 and mPEG113-b-P(MeP-Asp-OMe)22 showed nearly identical CD signals to P(MeP-Asp-OMe)10, indicating the similarly ordered structure (Figure S11b,c). Also, mPEG45-b-P(tBuP-Asp-OMe)9 and mPEG113-b-P(tBuP-Asp-OMe)18 presented ordered structures with intense minima around 195 nm and shallow minima around 228 nm (Figure S11d,e).

Figure 3

(a) CD spectra of P(MeP-Asp-OMe)10 in H2O (1 mg mL–1) (pH = 2.0, 7.0, and 10.1), (b) CD spectra of P(P-Asp-OH)10 in H2O (1 mg mL–1) (pH from 3.0 to 11.0), (c) FT-IR spectra of P(P-Asp-OH)10 (pH from 3.0 to 10.0), and (d) CD spectra of mPEG45-b-P(P-Asp-OH)13 in H2O (1 mg mL–1) (pH = 2.0, 7.0, and 10.0).

We then studied the concentration dependence of the CD signals of β-peptoipan class="Chemical">ds. The spectral shapes and intensities of P(MeP-Asp-OMe)10, mPEG45-b-P(MeP-Asp-OMe)13, mPEG113-b-P(MeP-Asp-OMe)22, mPEG45-b-P(tBuP-Asp-OMe)9, and mPEG113-b-P(tBuP-Asp-OMe)18 remained almost unchanged in water under various concentrations, i.e., 0.1, 0.2, 0.5, and 1 mg mL–1 (Figure S11a–e). Moreover, P(MeP-Asp-OMe)10 and mPEG45-b-P(P-Asp-OH)13 showed identical spectral signals in methanol under various concentrations (Figure S12). These results indicate that the spectral shape is not due to the aggregation of the peptoids.[14] Note that the CD spectra of P(tBuP-Asp-OMe) was not given due to the poor solubility in water. Next, we investigated the stability of the peptoids under different pH values. Typically, the CD spectrum of P(MeP-Asp-OMe)10 was almost unchanged in shape as the pH increased from 2 to 7 (Figure a). However, distinct features were observed for P(MeP-Asp-OMe)10 as the pH value was further increased from 7 to 10, where the minima moved from 208 to 220 nm and the shallow minima around 230 nm almost disappeared. The changes possibly arise from the increased deprotonation degree of the amino groups at the chain end of the polymer, which influence intramolecular hydrogen bonds.[14] Also, similar pH dependence was observed for the diblock copolymer of mPEG45-b-P(MeP-Asp-OMe)13 (Figure S13b). CD spectra of N-terminal capped P(MeP-Asp-OMe)10 were almost unchanged as the pH increased from 3.7 to 6.9 and to 10.4, confirming the effect of terminal amino groups on folding propensities (Figure S14).

To investigate the charge state on folding structures, pan class="Chemical">P(MeP-Asp-OMe)10 was hydrolyzed using LiOH to obtain the completely deprotected oligomer of P(P-Asp-OH)10 (Scheme ). The chemical structures of P(P-Asp-OH)10, mPEG45-b-P(P-Asp-OH)13, and mPEG113-b-P(P-Asp-OH)22 were characterized by 1H NMR (Figure S10a–c). After hydrolysis, P(P-Asp-OH)10 showed decreased solubility in organic solvents, while increased solubility in water. The folding propensity of P(P-Asp-OH)10 was explored in aqueous solutions under diverse pH values. As revealed by CD (Figure b), the deprotected P(P-Asp-OH)10 displayed a maximum at near 195 nm and a minimum at about 215 nm at pH ∼ 7, which were distinct from unhydrolyzed P(MeP-Asp-OMe)10. As the pH was raised from 3 to 11, the intensities of both maximum and minimum bands gradually increased with the maxima and minima shifting from 193 to 197 nm and from 207 to 216 nm, respectively. It should be noted that there was a sharp increase as the pH value increased from 6 to 7, possibly due to the increased deprotonation degree of −COOH moieties.[33] The structure changes were also investigated by FT-IR spectra (Figure c), in which the carbonyl at 1721 cm–1 gradually reduced as the pH increased from 3 to 7 and almost disappeared for samples at pH values of 8, 9, and 10. Further, the carbonyl at 1633 cm–1 exhibited a red shift as the pH increased from 3 to 10. The deprotected diblock copolypeptoid of mPEG45-b-P(P-Asp-OH)13 showed identical pH-dependent transitions in aqueous solutions of P(P-Asp-OH)10 (Figure d). Moreover, all of the deprotected samples show almost unchanged spectral shapes with concentration (Figure S15a–c).

Scheme 2

Deprotection of Oligo(β-peptoid)s

To investigate the −COOH content on the folding properties, pan class="Chemical">mPEG45-b-P(tBuP-Asp-OMe)9 was partially deprotected by CF3COOH to remove t-butyl groups on the side chains but reserve the methyl ester bonds (Scheme ). The partially deprotected product mPEG45-b-P(P-Asp-OMe)9 was characterized by 1H NMR (Figure S10d). The CD spectrum of mPEG45-b-P(P-Asp-OMe)9 presented intense minima around 195 nm and shallow minima around 228 nm at pH ∼ 7 (Figure S15c), which are identical to undeprotected mPEG45-b-P(tBuP-Asp-OMe)9. We assumed that partial deprotection cannot change the folding structure. Further, CD measurements were conducted at different pH values. Obviously, the CD spectrum of mPEG45-b-P(P-Asp-OMe)9 was almost unchanged in shape as the pH increased from 2 to 7 (Figure S13c). As the pH increased from 7 to 10, however, the minima moved from 195 to 217 nm and the shallow minima around 228 nm almost disappeared, mostly due to the increased deprotonation degree of −COOH groups at the side chains.

Conclusions

In summary, we have demonstrated a facile approach to synthesize backbone chiral β-peptoid polymers from readily available pan class="Chemical">aspartic acid derivatives via an aza-Michael addition reaction. Two types of N-substituted β3-homoalanine monomers, i.e., N-(methyl propionate)-Asp-OMe (MeP-Asp-OMe) and N-(tert-butyl propionate)-Asp-OMe (tBuP-Asp-OMe), were synthesized in high yield. Successful cyclization and polymerization of these N-substituted β3-homoalanines provide access to oligo(β-peptoid) and mPEG-poly(β-peptoid) diblocks with backbone chirality. It was found that both oligo(β-peptoid)s and diblocks have good solubility in common organic solvents but behave differently in water due to their structural differences. Their conformations were preliminarily studied by CD and FT-IR. Definitely, there are still many questions regarding ROP and material properties but the synthetic strategy represents a facile and efficient methodology to prepare poly(β-peptoid)s with well-defined and diverse structures.

Experimental Section

Materials and Instruments

Hexane, tetrahydrofuran (THF), dichloromethane (DCM), and toluene (TOL) were first dried with calcium hydride and purified by passing through activated alumina columns prior to use. l-Aspartic acid 1-methyl ester was purchased from GL Biochem (Shanghai) Ltd. Methyl acrylate, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) were purchased from Energy Chemical. Methoxypolyethylene glycol amine (mPEG45-NH2, Mw = 2000 Da; mPEG113-NH2, Mw = 5000 Da) was purchased from Jenkem Technology Co, Ltd. (Beijing, China). Bis(3,5-bis[trifluoromethyl]phenyl) thiourea (TU) was prepared as previously reported.[44] The rest of the chemicals were purchased from commercial suppliers and used without further purification unless otherwise stated. NMR spectra were recorded on a Bruker DMX-500 FT-NMR spectrometer for 1H NMR at 400 MHz and for 13C NMR at 101 MHz. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was carried out on a Bruker BIFLEX III MS spectrometer equipped with a 337 nm nitrogen laser. The polymer was dissolved in THF (10 mg mL–1); dithranol in THF (20 mg mL–1) was used as the matrix and CF3COONa (10 mg mL–1) was used as the cationizing agent. The polymer was tested after mixing in a volume ratio of 5:10:1. Infrared spectroscopy tests were carried out on a JASCO FI-IR-4700 spectrometer recorded from 4000 to 400 cm–1. The solution was dropped on a KBr plate, and the solvent evaporated before performing the measurements. Gel permeation chromatography (GPC) was performed at 50 °C using an SSI pump connected to a Wyatt Optilab DSP with 0.02 M LiBr in DMF as the eluent at a flow rate of 1.0 mL min–1. All GPC samples were prepared at concentrations of ∼7 mg mL–1. Circular dichroism (CD) spectra were recorded on an Applied Photophysics Chirascan CD spectrometer (Applied Photophysics Ltd., United Kingdom). The solution was placed into a quartz cell with a path length of 0.1 cm. Electrospray ionization tandem mass spectrometry (ESI-MS) was recorded on a Bruker Impact II spectrometer operating in the positive ion mode.

General Procedure for the Synthesis of N-Substituted l-Aspartic Acid 1-Methyl Ester (MeP-Asp-OMe or tBuP-Asp-OMe)

A typical example is described below. Methyl acrylate (10.9 mL, 122.0 mmol) was added dropwise into a mixpan class="Chemical">ture of l-aspartic acid-1-methyl ester (15 g, 102.0 mmol), methanol (200 mL), and triethylamine (15.6 mL). After stirring at room temperature for 12–18 h, the solution changed from turbid to clear, suggesting completion of the reaction. After solvent evaporation, the crude product was washed twice with 100 mL of diethyl ether. A white powder was obtained via filtration and dried under vacuum to constant weight (23.2 g, 97.6% yield). 1H NMR (400 MHz, D2O) δ 4.20 (t, 1H), 3.74 (s, 3H), 3.65 (s, 3H), 3.35 (t, 2H), 2.81 (m, 4H). 13C NMR (101 MHz, CD3OD) δ 48.24, 48.03, 47.82, 47.60, 47.39, 47.18, 46.97.

General Procedure for the Synthesis of NNCAs (MeP-Asp-OMe and tBuP-Asp-OMe NCAs)

A typical example is described below. Triethylamine (60 mL) and pan class="Chemical">di-tert-butyl dicarbonate (Boc2O, 46.8 g, 214.5 mmol) was added into the solution of MeP-Asp-OMe (14.1 g, 60.5 mmol) in dry THF (350 mL) under a nitrogen atmosphere. The reaction was stirred overnight at room temperature. After the solvent was removed by a rotavapor, the mixture was dissolved in 200 mL of deionized water, followed by washing with 100 mL of hexane twice to remove unreacted di-tert-butyl decarbonate. Then, the pH was adjusted to 2 using 4 M aqueous HCl. The aqueous phase was extracted with 100 mL of ethyl acetate three times. The organic phase was subsequently washed with brine and dried with anhydrous MgSO4. The colorless oil (Boc-MeP-Asp-OMe) was collected after the solvent was removed under reduced pressure (15.5 g, 75.2% yield).

A total of 15 g of Boc-MeP-Asp-OMe was dissolved in anhydrous CH2Cl2 (250 mL) under nitrogen. PCl3 (5.4 mL) was added dropwise to the reaction solution at 0 °C, and then the system was stirred for 0.5 h in an ice bath and for another 3 h at RT. After the solvent was removed under vacuum, the oil was extracted twice with CH2Cl2 (2 × 20 mL) and filtered. The filtrate was evaporated to give a colorless oil. After three times dissolving/precipitating with THF/hexane in a glovebox, the oil of MeP-Asp-OMe NCA was obtained (7.5 g, 64.4% yield). 1H NMR (400 MHz, CDCl3) δ 4.55 (s, 1H), 3.98–3.86 (m, 1H), 3.81 (s, 3H), 3.70 (s, 3H), 3.56–3.46 (m, 1H), 3.20–2.98 (m, 2H), 2.98–2.86 (m, 1H), 2.74–2.59 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 172.92, 169.39, 162.75, 148.65, 56.47, 53.51, 52.04, 46.17, 32.46, 32.40.

General Procedure for the Synthesis of Oligo(β-peptoid)s [P(MeP-Asp-OMe) and P(tBuP-Asp-OMe)]

The ROP of NNCAs was performed in toluene (TOL) using benzylamine as an initiator. A benzylamine/TOL (0.1 M) solution was injected into NNCA/TOL (100 mg mL–1) under nitrogen. The solution was then stirred at a designed temperature under vacuum. The progress of the reaction was monitored by FT-IR. The reaction system was then precipitated in cold diethyl ether. The polymer was isolated by centrifugation and drying. The yield was about 20%, and the oligomer was characterized using MALDI-TOF and 1H NMR.

General Procedure for the Synthesis of Diblock Copolymers [mPEG-b-P(MeP-Asp-OMe) and mPEG-b-P(tBuP-Asp-OMe)]

Typically, mPEG45-NH2 (77.5 mg, 0.0386 mmol) was pumped under high vacuum at 80 °C for 12 h and then dissolved in 2 mL of anhydrous pan class="Chemical">TOL in a Schlenk glass tube under a N2 atmosphere. Five milliliters of an NNCA/TOL solution (100 mg mL–1) was added to the glass tube, and the solution was then stirred at 50 °C for 48 h under vacuum. The reaction mixture was precipitated in cold diethyl ether, centrifuged, and dried. A white solid was obtained with a 35% yield, and the copolymers were characterized using GPC and 1H NMR.

General Procedure for Hydrolysis of Methyl Ester Groups

Typically, 0.18 g of P(MeP-Asp-OMe)10, LiOH·H2O (0.18 g, 4.3 mmol), and 10 mL of deionized water were added in a flask, and the mixture was stirred at RT for 4 h. Then, the solution mixture was put into a suitable dialysis bag and dialyzed in distilled water for 2 days with changing water eight times. Dialyzed solutions were lyophilized to obtain P(P-Asp-OH)10 as a white solid.

General Procedure for Hydrolysis of tert-Butyl Ester Groups

Typically, 0.15 g of mPEG45-b-P(tBuP-Asp-OMe)9 was dissolved in 15 mL of CF3COOH (TFA) at 0 °C, and the mixture was stirred at RT for 2 h. The solution was added to 15–20 mL of water and loaded into a dialysis bag, and then the solution was dialyzed in distilled water for 2 days with water changing eight times. Dialyzed solutions were lyophilized to obtain mPEG45-b-P(P-Asp-OMe)9 as a white solid.