Controllable Polymerization of N -Substituted β-Alanine N -Thiocarboxyanhydrides for Convenient Synthesis of Functional Poly(β-peptoid)s

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE27 May 2022Controllable Polymerization of N-Substituted β-Alanine N-Thiocarboxyanhydrides for Convenient Synthesis of Functional Poly(β-peptoid)s Ximian Xiao†, Min Zhou†, Zihao Cong, Longqiang Liu, Jingcheng Zou, Zhemin Ji, Ruxin Cui, Yueming Wu, Haodong Zhang, Sheng Chen, Maoquan Li and Runhui Liu Ximian Xiao† State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 †X. Xiao and M. Zhou contributed equally to this work.Google Scholar More articles by this author , Min Zhou† State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 †X. Xiao and M. Zhou contributed equally to this work.Google Scholar More articles by this author , Zihao Cong Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Longqiang Liu Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Jingcheng Zou Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Zhemin Ji Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Ruxin Cui Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Yueming Wu Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Haodong Zhang Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Sheng Chen Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Maoquan Li Department of Interventional and Vascular Surgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072 Google Scholar More articles by this author and Runhui Liu *Corresponding author: E-mail Address: [email protected] State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.022.202201840 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Poly(β-peptoid) is a class of polypeptide mimics that possesses excellent biocompatibility and resistance to proteolysis. However, the synthesis of poly(β-peptoid)s with functionalities is a long-standing challenge that greatly hinders the functional study and application of poly(β-peptoid)s. We report a controllable and easy synthesis of poly(β-peptoid)s bearing diverse functionalities via the ring-opening polymerization on N-substituted β-alanine N-thiocarboxyanhydrides (β-NNTAs). The polymerization can be carried out in open vessels under mild conditions using amines as the initiators to provide poly(β-peptoid)s with targeted molecular weights, narrow dispersities, and diverse functionalities in the side chains and termini. The β-NNTAs polymerization is even compatible with initiators bearing unprotected hydroxyl groups. The amphiphilic/cationic poly(β-peptoid)s exhibit a broad spectrum and potent antibacterial activities against multidrug-resistant bacteria. In addition, the highly favored stability of β-NNTA monomers for purification and storage highlights the advantages of this β-NNTA polymerization strategy for poly(β-peptoid)s synthesis, functional study, and application as a synthetic mimic of polypeptides. Download figure Download PowerPoint Introduction Poly(β-peptoid)s, as a class of polypeptide mimics, have a polyamide backbone similar to polypeptides but with an extra methylene group and with the proton of nitrogen–hydrogen bond in the backbone substituted by an alkyl group.1–4 Poly(β-peptoid)s have superior biocompatibility and excellent stability against proteolysis and therefore demonstrate promising biological applications in biomimetic scaffolds,3 antifouling properties,5 and antibacterials.6 However, the lack of an effective synthetic strategy is the long-standing challenge that greatly hinders the advance of this field and the application of poly(β-peptoid)s. Conventional solid-phase synthesis is applied to prepare poly(β-peptoid)s using either direct amide bond formation or via aza-Michael addition.7,8 However, no poly(β-peptoid)s with over 10 residues can be obtained using the solid-phase synthesis due to the low efficiency of this chemistry. Compared with the preparation of polypeptides and poly(α-peptoid)s, it is hard to obtain structurally diverse and functional poly(β-peptoid)s. Current solid-phase synthesis of poly(β-peptoid)s is also time-consuming, expensive, and difficult in scale up.9 To overcome the shortcomings of solid-phase synthesis in preparing poly(β-peptoid)s, polymerization strategies have been explored. Jia et al. reported a successful synthesis of poly(β-peptoid)s from the copolymerization of N-alkylaziridines and carbon monoxide using cobalt catalysts (Figure 1a).1,10 Nevertheless, this polymerization relies on high-pressure reaction conditions and a metal catalyst. Since 1954, to realize a mild synthesis of poly(β-peptoid)s, amine-initiated polymerization on N-substituted β-alanine N-carboxyanhydrides (β-NNCAs) has been explored (Figure 1b).2,11,12 However, this strategy suffers from the instability of β-NNCA monomers.12 In addition, the above two polymerization strategies for poly(β-peptoid)s synthesis are both highly sensitive to moisture and have structural diversity limited to N-alkyl and/or N-aryl substitution, which has been a serious obstacle to the synthesis, functional study, and application of poly(β-peptoid)s. Therefore, we need to develop a convenient, mild, and open vessel synthesis of poly(β-peptoid)s. Meanwhile, it is also vital to explore structurally diverse poly(β-peptoid)s for their functions and applications. Figure 1 | Polymerization strategy for the synthesis of poly(β-peptoid)s. Download figure Download PowerPoint Herein, we report the synthesis of poly(β-peptoid)s from a controllable ring-opening polymerization on N-substituted β-alanine N-thiocarboxyanhydrides (β-NNTAs) (Figure 1c). This controllable and living polymerization on β-NNTAs can be achieved using variable amines as the initiator to obtain poly(β-peptoid)s with predictable molecular weight (Mn) controllable degree of polymerization (DP) and low dispersity (Đ) without using any catalyst. This polymerization can be operated conveniently in an open vessel. Moreover, β-NNTA monomers can be easily obtained with a variety of N-substitutions, which enables a breakthrough in the structural diversity of resulting poly(β-peptoid)s. In addition, a variety of terminal functional groups can be introduced into poly(β-peptoid)s via the amine initiators. This strategy for poly(β-peptoid)s synthesis from controllable and living polymerization on β-NNTAs addresses the long-lasting challenges in this field and provides a convenient alternative to facilitate the synthesis, functional study, and application of poly(β-peptoid)s. Experimental Methods All reagents and solvents were purchased from Adamas Chemical Reagent Co., Ltd. (Shanghai, China) and used as received unless otherwise specified. Petroleum ether (PE), ethyl acetate (EtOAc), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), toluene (Tol), acetonitrile (MeCN), dioxane, dimethyl sulfoxide (DMSO), and 1-methyl-2-pyrrolidinone (NMP) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. and used as received without purification. Synthesized intermediates were purified using a SepaBean machine equipped with Sepaflash columns produced by Santai Technologies (Changzhou, China) Co. Ltd. Nuclear magnetic resonance (NMR) spectra were collected on a Bruker spectrometer at 400 MHz or 600 MHz, using CDCl3 or CF3COOD as solvents. Chemical shifts are referenced to residual protons in the deuterated NMR solvents. High-resolution electrospray ionization time-of-flight mass spectrometry (HRESI-TOF-MS) was collected on a Waters Xevo G2 TOF mass spectrometer (Waters Technologies Shanghai Ltd) and high-resolution electron ionization time-of-flight mass spectrometry (HREI-TOF-MS) was collected on a Waters GCT. High-performance liquid chromatography (HPLC) analysis was carried out on a Shimadzu LC-20AR HPLC System (Shimadzu (Shanghai) Global Laboratory Consumables Co., Ltd.) equipped with a Gemini 5 μm NX-C18 column. Gel permeation chromatography (GPC) was performed on a Waters GPC instrument equipped with a Brookhaven BI-MwA multiangle light-scattering detector (BI-MwA, Brookhaven Instruments Shanghai Representative Office) and a refractive index detector (Waters 2414) using DMF, supplemented with 0.01 M LiBr, as the mobile phase at a flow rate of 1 mL/min at 50 °C. The GPC were equipped by a Tosoh TSKgel Alpha-2500 column (particle size 7 μm), a Tosoh TSKgel Alpha-3000 column (particle size 7 μm), and a Tosoh TSKgel Alpha-4000 column (particle size 10 μm, 300 × 7.8 mm) linked in series. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) experiments were conducted on an AB Sciex 4800 Plus MALDI TOF/TOF analyzer (Shanghai AB Sciex Analytical Instrument Trading Co., Ltd.) using a 355 nm laser, a Shimadzu MALDI-7090TM TOF-TOF mass spectrometer (Shimadzu (Shanghai) Global Laboratory Consumables Co., Ltd.) with 2,5-dihydroxybenzoic acid (DHB) as the matrix. Dynamic light scattering (DLS) measurements were used to measure hydrodynamic diameters of assembled polymers using a Zetasizer Nano-ZS instrument, Model ZEN3600 (Malvern Instruments Ltd., Malvern, United Kingdom). Transmission electron microscopy (TEM) measurements were conducted using a JEOL JEM-1400 TEM (JEOL (Beijing) Co., Ltd. Shanghai Branch) at an acceleration voltage of 100 kV. Time-lapse fluorescent confocal imaging was performed using a Leica TCS SP8 system (Leica Microsystems) equipped with LAS X v3.5.2. software (Leica Microsystems) for acquisition control. Fluorescence spectra of the samples were performed on a Varian Cary Eclipse fluorescence spectrophotometer (Varian China Ltd., Shanghai Office). Results and Discussion Using pFBn β-NNTA ( 1a) as a model, we conducted the ring-opening polymerization on β-NNTA using 4-tert-butylbenzylamine (tBuBnNH2) as the initiator in variable solvents including DMF, THF, Tol, dioxane, MeCN, DMSO, and NMP ( Supporting Information Scheme S13, Figure S1, Table S1). Poly(N-pFBn-β-ala) was successfully obtained in polymerization using most common solvents as a single peak of GPC trace with narrow dispersity (Đ = 1.04–1.11), except that a bimodal peak of GPC trace was observed for polymerization using DMSO and NMP as the solvent. Among all solvents that are compatible with β-NNTA polymerization, DMF afforded the resulting poly(β-peptoid) with a polymer length (obtained DP = 21) almost identical to the expectation (calculated DP = 20). Considering that DMF normally provides excellent solubility and is a widely used solvent for polypeptide and polypeptoid synthesis, we chose DMF as the solvent for continuous β-NNTA polymerization studies. To examine if the polymer length of amine-initiated β-NNTA polymerization is controllable, we tried the polymerization on pFBn β-NNTA ( 1a) in DMF using variable [M]0/[I]0 ratios. The results showed that poly(β-peptoid)s were obtained with an incrementally increased chain length from 22 mer (Mn = 4.1 kDa) to 150 mer (Mn = 27.1 kDa) with narrow dispersities (Đ = 1.18–1.20) and DP almost identical to the prediction (Table 1, entries 1–5, Figure 2a). These results demonstrate the excellent controllability of the β-NNTA polymerization. Kinetics study showed that the polymerization had an evolution of Mn that showed in linear correlation with monomer conversion and kept narrow dispersity (Đ = 1.09–1.18) throughout the whole process (Figure 2b). It is worth mentioning that the polymerization showed first-order kinetic characteristics (kp[I] = 0.018 h−1) and over 90% conversion of β-NNTA monomer within 14 h (Figure 2c). The polymerization on pFBn β-NNTA in DMF was completed within 20 h. Table 1 | Poly(β-peptoid)s Synthesized from β-NNTAs Polymerizationa Entry M M0/I Mn, th (kDa) Mn (kDa)b DPb Ðb 1 1a 20 3.7 4.1 22 1.19 2 1a 40 7.3 7.3 40 1.20 3 1a 80 14.6 14.6 80 1.19 4 1a 100 18.1 17.9 99 1.18 5 1a 150 27.0 27.1 150 1.20 6 1b 20 3.4 3.2c 19c 1.12d 7 1c 20 4.3 4.6 21 1.13 8 1d 40 7.4 7.2 39 1.18 9 1e 20 2.4 2.1c 17c 1.15d 10 1f 20 2.7 2.4 18 1.17 11 1g 20 2.7 3.6c 27c 1.08d 12 1h 20 4.8 3.3 14 1.09 13 1i 20 5.1 4.0 16 1.13 14 1a-r- 1d 40/40 14.6 13.9 76 1.17 15 1a1st 20 3.7 3.7 20 1.16 1d2nd 20 7.4 7.3 40 1.15 aAll polymerizations were conducted using tBuBnNH2 as initiator in a sealed vessel at 60 °C. [M]0 at 2 M for entry 5, [M]0 at 1 M for other entries. bMn, DP, and Đ were determined by GPC using DMF as the mobile phase at a flow rate of 1 mL/min. cMn and DP were determined by NMR. dĐ was determined by MALDI-TOF-MS. Mn,th is the theoretical number average molecular weight; Mn,obtained is the obtained number average molecular weight; DP is the obtained degree of polymerization; Đ means the dispersity. Figure 2 | (a) Plots of Mn and Đ as a function of the [M]0/[I]0 ratio for pFBn β-NNTA. Inset: Overlay of GPC profiles at different [M]0/[I]0 ratios corresponding to entries 1–5 in Table 1. (b) Profiles of Mn and Đ as a function of pFBn β-NNTA conversions at the [M]0/[I]0 ratio of 20/1. Inset: Overlay of GPC profiles at different monomer conversions. (c) Kinetic profiles of conversion and ln([M]0/[M]) vs time at the [M]0/[I]0 ratio for pFBn β-NNTA of 20/1 using tBuBnNH2 as the initiator. (d) GPC traces of the block copolymer corresponding to entry 15 in Table 1. (e) The ring-closure synthesis for β-NNTAs. Reaction condition: 1.0 equiv. PBr3, anhydrous CH2Cl2, 0 °C to r.t., 4 h. (f) The purification of Bu β-NNTA via silica gel chromatography (9:1 PE:EtOAc). (g) Stability analysis of β-NNTA and β-NNCA at r.t. (h) Open-vessel polymerization of ThEt β-NNTA at 2 g scale at 60 °C in DMF, [M]0 = 1 M. Mn, DP and Đ were determined by GPC. Download figure Download PowerPoint The side-chain functionality or structural diversity of peptide mimics plays an essential role in defining their activity, function, and application.13–17 However, for poly(β-peptoid)s, current synthetic strategies using either the copolymerization of N-alkylaziridines and carbon monoxide or the ring opening polymerization of β-NNCAs can only afford poly(β-peptoid)s with N-alkyl or N-aryl functional groups.1,2 This challenge encouraged us to explore the synthesis of functional poly(β-peptoid)s from the polymerization on β-NNTAs. Structurally diversified β-NNTAs were easily synthesized from primary amines (Figure 1c, β-NNTA 1a– 1i, Supporting Information Schemes S1–S10 and Figures S32–S57). In addition, tBuBnNH2 successfully initiated the polymerization of all those β-NNTAs in our study to afford corresponding poly(β-peptoid)s in narrow dispersities (Đ = 1.09–1.18) and with diversified structures or side-chain functional groups including aromatic groups (4-fluorobenzyl in pFBn β-NNTA, benzyl in Bn β-NNTA, (4-chlorophenyl)ethyl in pCPE β-NNTA), heterocycles in ThEt β-NNTA, alkyl groups (cyclopropyl in cPr β-NNTA, isobutyl in iBu β-NNTA, and n-butyl in Bu β-NNTA), carboxylates in Cbz-Et β-NNTA, and amines in Cbz-AE β-NNTA (Table 1, entries 1–13, Figure 2a, and Supporting Information Figures S2–S9). The β-NNTA polymerization was also feasible for copolymerization. A random copolymerization on the mixture of pFBn β-NNTA and ThEt β-NNTA afforded poly[(N-pFBn-β-ala)-r-(N-ThEt-β-ala)] with the expected polymer length (DP = 76) and a narrow dispersity (Đ = 1.17) (Table 1, entry 14, Supporting Information Scheme S14 and Figure S10). Sequential addition of pFBn β-NNTA and ThEt β-NNTA in one-pot synthesis gave the first block of poly(N-pFBn-β-ala) at 20 mer with a narrow dispersity (Đ = 1.16) and the expected final block copolymer poly(N-pFBn-β-ala)-b-poly(N-ThEt-β-ala) at 40 mer with a low dispersity (Đ = 1.15) (Table 1, entry 15, Figure 2d, and Supporting Information Scheme S15). All these results demonstrate that the amine-initiated β-NNTA polymerization has excellent controllability to prepare poly(β-peptoid)s with diversified structures and side-chain functionalities. The stability of β-NNTA is vital to its purification and storage, ultimately contributing to structural and functional diversity of poly(β-peptoid)s. We found that β-NNTA displayed stability superior to β-NNCA with the same side chain. It is worth mentioning that we can obtain all β-NNTAs with high yield (81−93%) during the ring-closing process via silica gel chromatography (Figure 2e). Purification of β-NNTA via silica gel chromatography makes it possible to easily obtain pure oily β-NNTA monomers (representative Bu β-NNTA in Figure 2f), whereas β-NNCA is less stable and will decompose on silica gel, making the purification of oily β-NNCA monomers a headache ( Supporting Information Scheme S11 and Figures S58–S60). In addition, during storage, the purity of β-NNTA such as pFBn β-NNTA was maintained above 99% over 27 days at room temperature and showed no significant decomposition even after half a year at −20 °C (Figure 2g, Supporting Information Figure S11), whereas the purity of the corresponding β-NNCA sharply decreased to 23% after 27 days (Figure 2g). The open-vessel polymerizations have been reported in the synthesis of polypeptides and polypeptoids.18–27 The excellent stability of β-NNTA encouraged us to explore its polymerization in an open flask outside of a glove box. A gram-scale β-NNTA polymerization was achieved successfully in an open flask to give the resulting poly(N-ThEt-β-ala)20 in 90.1% yield (Figure 2h). The living and controllable polymerization of β-NNTAs encouraged us to explore the reaction mechanism. We proposed that the polymerization is initiated by nucleophilic addition of primary amine at the C1 carbonyl in a β-NNTA ring, followed by the ring opening of β-NNTA and release of the small molecule carbonyl sulfide (COS) to give an intermediate 1 that has an N-terminal secondary amine as the reactive center to attack another β-NNTA for chain propagation (Figure 3a).24,26,28,29 To provide mechanistic insights into chain initiation and propagation, Fourier transform infrared (FT-IR) characterization on a 1∶1 molar ratio mixture of tBuBnNH2 and pFBn β-NNTA was conducted (Figure 3b). Compared with pFBn β-NNTA, an above 1∶1 mixture had a shifted signal of the C1 carbonyl group from 1715.9 to 1642.1 cm−1 (C1′ in the mixture). The disappearance of C2 carbonyl signal at 1648.8 cm−1 indicated the ring opening of β-NNTA and subsequent release of COS. In addition, proton NMR characterization of an immediately prepared mixture of pFBn β-NNTA and tBuBnNH2 solution in CDCl3 at variable ratios showed a reduction of Ha and Hb peaks in the β-NNTA ring and the emergence of upfield-shifted Ha′ and Hb′ peaks in the β-NNTA ring opening product simultaneously, which indicated a quick nucleophilic addition of tBuBnNH2 to the C1 carbonyl in the β-NNTA ring at the initiation step (Figure 3c). Figure 3 | (a) Proposed mechanism of primary amine-initiated β-NNTA polymerization. (b) FT-IR spectra of β-NNTA, tBuBnNH2 and a 1∶1 β-NNTA/tBuBnNH2 mixture (DMF, room temperature, [M]0=0.5 M). (c) Instantaneous 1H NMR titration spectra on a mixture of β-NNTA/tBuBnNH2 in variable ratio (CDCl3, 25°C. [M]0=0.2 M). (d) HRESI-TOF-MS analysis on the 1∶1 β-NNTA/tBuBnNH2 mixture (DMF, room temperature, [M]0=0.5 M). (e) MALDI-TOF-MS of poly(N-pFBn-β-ala)20 (entry 1 in Table 1) with the corresponding chemical structures. Download figure Download PowerPoint HRESI-TOF-MS characterization on the above 1∶1 mixture showed a clear spectrum and high intensity fragment at m/z 343.2187 for intermediate 1 (Figure 3d), consistent with the result in the above NMR analysis and confirming nucleophilic addition of tBuBnNH2 to the β-NNTA ring during initiation. This was further confirmed by examining the final poly(β-peptoid)s (poly(N-pFBn-β-ala)20) using MALDI-TOF-MS characterization. The 179 Da difference between peaks reflected successful incorporation of the β-peptoid residues. Specific mass corresponding to each peak revealed the incorporation of a C-terminal tBuBnNH group via nucleophilic addition during initiation (Figures 3a and 3e). These results echoed the controllable β-NNTA polymerization and living polymerization with a normal amine mechanism. The terminal functionalization is important for the structural diversity and function of peptide mimics, for instance, in bioactive molecule conjugation and fluorescence labelling.30–39 The aforementioned open-vessel polymerization encouraged us to further explore C-terminal functionalization in an open vessel ( Supporting Information Scheme S16). Using primary amines as the initiator for the ring-opening polymerization on β-NNTAs allows the easy incorporation of diverse functionalities into the C-terminal of poly(β-peptoid) chains including alkenyl, alkynyl, adamantane, bromophenyl, azide, protected thiol, protected amine, methoxy, polyethylene glycol (Figure 4a, 2a− 2j), unprotected base, unprotected hydroxyl groups, aggregation-induced emission luminogen (AIEgen), and aggregation-caused quenching (ACQ) fluorophore (Figures 4b and 4c, 2k− 2q). In addition to terminal functionalization, all poly(β-peptoid)s were successfully synthesized with excellent control of chain length and narrow dispersities (Đ = 1.06−1.08) ( Supporting Information Figures S12−S29). Figure 4 | Primary amine-initiated β-NNTA polymerization in an open vessel. (a) Different functional primary amines as C-terminal groups. (b) Representative primary amines with nucleophilic groups. (c) Functional demonstration of C-terminal modified poly(β-peptoid)s. Download figure Download PowerPoint What is more, the polymerization on β-NNTAs can be initiated by primary amines that have an unprotected base (pyridine, tertiary amine) and hydroxyl group (phenol, alcohol) (Figure 4b, 2k– 2n). The tolerance of unprotected reactive groups of β-NNTA polymerization facilitated the synthesis of poly(β-peptoid)s with function groups without tedious protection and deprotection steps. This encouraged us to explore a more challenging β-NNTA polymerization using unprotected 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin (NH2-β-CD) as the initiator. Surprisingly, the polymerization also tolerated multiple hydroxyl groups to give the expected poly(β-peptoid)s smoothly with a narrow dispersity (Đ = 1.15, GPC analysis) and a terminal β-CD moiety in the polymer chain as characterized by MALDI-TOF MS (Figure 4c, 2o, Supporting Information Figure S27). The β-CD grafted poly(β-peptoid)s spontaneously self-assembled in the mixing solvent of water/DMSO, indicating the potential application of this polymerization in drug delivery (Figure 4c, 2o).25,40–46 This mild β-NNTA polymerization also enabled us to synthesize poly(β-peptoid)s with terminal fluorescence groups such as the AIEgen tetraphenylethylene ( 2p-poly(N-pFBn-β-ala)20) ( Supporting Information Scheme S12 and Figures S61 and S62) and the ACQ fluorophore morpholino-naphthalimide ( 2q-poly(N-pFBn-β-ala)20), with the expected polymer length and low dispersity (Figure 4c, 2p and 2q). 2p-poly(N-pFBn-β-ala)20 and 2q-poly(N-pFBn-β-ala)20 displayed AIE and ACQ characteristics, respectively. They showed the strongest photoluminescence (PL) intensity at 90% and 0% water content, respectively, which indicates a potential application of this β-NNTA polymerization and resulting poly(β-peptoid)s for imaging.30,47–51 Poly(β-peptoid)s have backbone structures similar to polypeptides, favorable biocompatibility, and excellent stability against proteolysis, which strongly suggests the possible use of poly(β-peptoid)s as a synthetic mimic of polypeptides for a large variety of applications. However, current functional study and application of poly(β-peptoid)s are very limited due to the obstacle of poly(β-peptoid) synthesis.28,52 To the best of our knowledge, no antibacterial application of poly(β-peptoid)s has been reported, likely due to the inaccessibility to poly(β-peptoid)s via polymerization.6,9,52 The mild and controllable synthesis of poly(β-peptoid)s from β-NNTA polymerization enables us to prepare structurally diverse poly(β-peptoid)s for functional studies including antibacterials. Using Cbz-AE β-NNTA and pFBn β-NNTA as models for proof-of-concept demonstrations, we synthesized amphiphilic poly(β-peptoid)s (poly[(N-Cbz-AE-β-ala)-r-(N-pFBn-β-ala)]20) that were composed of both the cationic subunit (aminoethyl functionality) and the hydrophobic subunit (4-fluorobenzyl functionality) with an incrementally increased ratio of the cationic subunit as synthetic mimics of host defense peptides (Figures 5a and 5b).13,23,53–62 These amphiphilic poly(β-peptoid)s were obtained at about 20 mer at low dispersities (Đ = 1.12−1.13) (Figures 5a and 5b, GPC traces in Supporting Information Figure S30). Figure 5 | Open-vessel polymerization for synthesis of poly(β-peptoid)s for antibacterial activity studies. (a) A library of amphiphilic poly(β-peptoid)s. (b) Characterizations of poly(β-peptoid)s. (c) The MIC values of the amphiphilic poly(β-peptoid)s against 5 strains of drug-resistant bacteria. (d) GPC characterization of side-chain protected poly[(N-Cbz-AE-β-al