Charge-Programmable Alternating Copolymers via Self-Catalyzed Aqueous Polymerization for Tunable Bacterial Translocation and Macrophage Polarization

Abstract

Precise control over sequence and charge distribution is essential for emulating biological macromolecules yet remains synthetically challenging. Here, we introduce a self-catalyzed aqueous amine–epoxy polymerization using amino acid salts, transforming a classical cross-linking chemistry into a versatile route for sequence-defined, charge-programmable alternating copolymers. The salts generate an intrinsic alkaline microenvironment that prevents amine protonation and enables direct ionic incorporation with alternating sequence fidelity. Mechanistic studies reveal the kinetic dominance of deprotonated primary amines in chain propagation among competing aqueous nucleophiles. By orthogonally paring charge-defining amino acid salts and flexibility-tuning diepoxides, we constructed a 20-copolymer library covering cationic, zwitterionic, and anionic regimes. These polymers exhibit programmable biointerfacial behaviors: cationic sequences promote bacterial membrane anchoring, backbone flexibility governs translocation dynamics (seconds to minutes), and zwitterionic or anionic sequences induce M2 macrophage polarization without membrane disruption. This work establishes a unified platform connecting polymer sequence design to predictable bacterial translocation and immunomodulatory functions.

1. Introduction

In natural macromolecules such as DNA and proteins, precise sequence control and charge patterning govern complex biological functions, motivating efforts to replicate these principles in synthetic polymers. − Alternating copolymers are particularly promising in this regard. Unlike random copolymers with heterogeneous arrangements or block copolymers with segregated domains, alternating architectures enforce strict monomer alternation to generate periodic architectures with uniform microenvironments and tunable functional group spacing. − Incorporating cationic, anionic, or zwitterionic units into such well-defined sequences enables direct control over charge placement and spacing, thereby governing interactions with complex biological interfacessuch as bacterial and mammalian cell membranesopening new opportunities for functional biointerface regulation. −

However, the synthesis of ionic alternating copolymers remains a significant challenge. Conventional strategies rely on carefully matched monomer reactivity to construct periodic sequences, greatly restricting the monomers and functional diversity. − Ionic functionalities are typically introduced by postpolymerization, but dense charge incorporation often induces interchain electrostatic repulsion, leading to inefficient modification and microphase separation. − These obstacles restrict access to ionic alternating copolymers with tunable charge programmability and biointerfacial adaptability, underscoring the need for new design strategies.

The polymerization of primary amines with diepoxides proceeds via stepwise ring-opening addition, inherently yielding alternating sequences with click-like efficiencyquantitative conversion, high selectivity, and mild reaction conditions. , This chemistry offers broad monomer diversity and excellent compatibility with ionic functionalities such as guanidinium, carboxylate, and sulfonate. Moreover, the resulting polymer backbones feature tertiary amines as intrinsic ionizable sites, enhancing charge tunability. − However, leveraging this reaction for sequence-defined ionic copolymers remains difficult. (Figure A). In organic media, the basic conditions required to maintain amine nucleophilicity also catalyze epoxide homopolymerization and ether branching, disrupting alternating fidelity. , For ionic monomers, polar aprotic solvents ensure solubility but suppress amine nucleophilicity through protonation, impeding polymerization. , These constraints have long hindered the use of amine–epoxy chemistry for constructing charge- and sequence-defined alternating copolymersarchitectures essential for advanced biointerface design.

1.

(A) Conventional amine–epoxy polymerizations in organic and aqueous media. (B) Plausible competing pathways for the nucleophilic ring-opening of GME in its reaction with GlyS in an aqueous solution. (C) DFT–calculated Gibbs free energy (ΔG) for each pathway in aqueous.

Here, we address this challenge through a self-catalyzed aqueous amin–epoxy polymerization strategy using amino acid salts. These monomers act as built-in activators: their conjugate-base nature spontaneously generates an alkaline microenvironment in water, maintaining the α-amine in a deprotonated, highly nucleophilic state while suppressing hydrolytic side reactions (Figure A). This design simultaneously enables amine activation and reaction selectivity, allowing self-catalyzed polymerization with high sequence fidelity. By decoupling charge identity (via five amino acid salts) from backbone flexibility (via four diepoxides), this approach affords orthogonal control over ionic charactercationic, zwitterionic, and anionicand diverse chain conformations. The resulting sequence-defined copolymers provide a versatile platform to elucidate how alternating sequences encode biointerfacial behaviors, from bacterial membrane translocation kinetics to macrophage polarization, thereby bridging polymer sequence design with predictable biological function.

2. Results and Discussion

2.1. Self-Catalyzed Aqueous Amine–Epoxy Polymerization toward Ionic Alternating Copolymers

To elucidate the intrinsic reactivity of amine–epoxy coupling in waterwhere multiple nucleophiles competewe employed amino acid salts as self-activating monomers. Acting as conjugate bases of weak acids, these salts create an intrinsic alkaline microenvironment (pH ≈ 11 for GlyS) that ensures the α-amine remains deprotonated and highly nucleophilic while maintaining aqueous solubility. Glycine sodium salt (GlyS), devoid of side-chain interference, was chosen as the model system. Its reaction with glycidyl methyl ether (GME) in water (amine hydrogen: epoxide = 1:1) proceeded to near-complete conversion at room temperature (Figures A and S1–S3). Notably, real-time pH monitoring during the reaction revealed that the solution pH was stably maintained at approximately 11 throughout the reaction course (Figure S4), indicating the formation of a persistent alkaline microenvironment. Liquid chromatography–mass spectrometry (LC–MS) analysis revealed the bis-adduct 1 as the predominant product (>98%), with only trace monoadduct 1′ (1.4%; Figures B, S1A and S5). This remarkable preference for the bis-adduct under stoichiometric conditions demonstrates that the deprotonated primary amine acts as a highly efficient, persistent nucleophile capable of two successive ring-opening in water without any catalyst.

2.

Mechanistic elucidation and kinetic analysis of the aqueous amine–epoxy ring-opening reaction. A small-molecule model of the GlyS–GME reaction was used to elucidate the aqueous amine–epoxy ring-opening mechanism. (A) ¹H NMR,¹³C NMR, and (B) LC–MS spectra of the lyophilized crude product. (C) DFT–calculated Gibbs free-energy profile for the reaction in aqueous solution. The kinetic behavior of the polymerization between GlyS and DEP3 was further investigated. (D) Schematic representation of the step-growth process in water at room temperature. (E) Time-dependent ¹H NMR spectra in D₂O. (F) ¹³C NMR spectrum of the crude mixture after 24 h. (G) Conversion of DEP3 (blue) as a function of time, based on ¹H NMR integration, and number-average molecular weight (M _n, green) evolution obtained from GPC.

To compare the nucleophilicity of the primary amine in GlyS and the secondary amine intermediate, a model reaction was conducted under GlyS-rich conditions (amine hydrogen: epoxide ≈2.5:1). This excess created a competitive environment where both amine species reacted with a limited amount of epoxide. Proton nuclear magnetic resonance (¹H NMR) spectroscopy revealed the coexistence of the bis-adduct 1 and monoadduct 1 ′ (Figure S2 and Table S1). Quantitative integration of characteristic signalsthe d/d′ methylene (δ = 2.4–2.8 ppm), e/e′ methylene (δ = 3.10–3.25 ppm), and a/a′ methyl (δ = 3.35–3.40 ppm)showed that 1 accounted for only ∼40 mol %, confirming the predominance of the monoaddition pathway. These data confirm that the free primary amine of GlyS is more nucleophilic than the secondary amine in intermediate 1 ′, leading to preferential GME consumption by excess GlyS. Consequently, the second ring-opening step from 1 ′ to 1 is kinetically suppressed, leaving the monoadduct as the major product.

Density functional theory (DFT) calculations further corroborated the experimentally observed reactivity trend (Figure C). The deprotonated α-amine preferentially attacks the less hindered epoxide carbon with a low activation barrier (ΔG ^‡ = 19.0 kcal mol^–1), whereas attack at the sterically hindered site is less favorable (23.4 kcal mol^–1). The built-in alkalinity of GlyS thus secures both selectivity and kinetic dominance, as competing nucleophiles exhibit higher barriers (−COO^–, 23.1 kcal mol^–1; H₂O, 32.2 kcal mol^–1; and OH^–, 72.0 kcal mol^–1) (Figures B and C). The resulting oxyanion intermediate (INT1) undergoes rapid intramolecular proton transfer to form a secondary amine (1′), which serves as the exclusive nucleophile for the subsequent ring-opening. Consistently, the barrier for the initial attack by GlyS (ΔG ^‡ = 19.0 kcal mol^–1) is slightly lower than that for the secondary amine (ΔG ^‡ = 19.2 kcal mol^–1), confirming the higher nucleophilicity of the primary amine. Together, these results delineate a self-catalyzed pathway enabling selective, linear amine–epoxy coupling in water while intrinsically suppressing ether-type branching.

To evaluate structural generality and identify kinetic determinants for polymer growth, model reactions were extended to four representative amino acid saltsArgS, PheS, GluS, and CyaSencoding cationic, hydrophobic, carboxylate, and sulfonate side groups (Figures S6–S17). Quantitative ¹H NMR and LC–MS analyses confirmed near-quantitative conversion (>99.5%) across all systems, confirming the universality of the first ring-opening event. However, LC–MS revealed pronounced structure–reactivity differences in the second ring-opening step, which mimics chain propagation (Figure S1). Monomers with low steric hindrance (ArgS and GluS) afforded bis-adducts in high yields (95% and 98%), whereas those bearing bulky substituents (PheS and CyaS) gave lower yields (85% and 88%), with the monoadduct as the major byproduct. The bis-adduct serves as the difunctional growth unit for high-molecular-weight formation, while the monoadduct functions as a chain terminator. Thus, the bis-to-mono ratio predicts attainable molecular weight, establishing the amino acid side chain as a programmable determinant of both chemical functionality and kinetic reactivity.

Building on these insights, GlySexhibiting the highest bis-adduct yield and minimal terminationwas paired with DEP3, a diepoxide analogous of GME, for step-growth polymerization in D₂O (Figure D). Real-time ¹H NMR spectroscopy showed the decay of epoxide signals (2.73 and 2.93 ppm), concurrent growth of amine ring–opened methylene (2.53–2.70 ppm), and hydroxyl-associated methine (3.85 ppm) resonances, tracing continuous chain extension (Figure E). Conversion reached ∼90% within 6 h, indicating fast coupling. Kinetic analysis revealed two district stages: initial monoadduct formation (≤1 h, ∼60% M-DEP) by primary amines, followed by secondary-amine participation (∼3 h) generating bis-adducts (B-DEP) and triggering chain growth (Figure S18 and Table S2). This mono- to bis-adduct transition marks the onset of polymer propagation. Gel permeation chromatography (GPC) confirmed step-growth behavior, with molecular weight increasing sharply only at high conversion to M _n = 16,400 and Đ = 2.1 after 24 h (Figure G and Table S3). The dispersity aligns with the theoretical limit for step-growth systems near completion. Furthermore, ¹³C NMR verified the alternating architecture, showing DEP3-derived carbons adjacent to Gly tertiary amines (Figure F). The coexistence of α-carboxylate and tertiary amine functionalities imparts a zwitterionic backbone, which prevented us from obtaining reliable matrix–assisted laser desorption/ionization time–of–flight mass spectrometry (MALDI–TOF MS) signals. These results confirm that self-catalyzed aqueous synthesis of high-molecular-weight, sequence-defined ionic alternating copolymers.

A library of 20 copolymers was synthesized by systematically pairing five amino acid salts (Unit A) with four diepoxides (Unit B), spanning backbones from rigid alicyclic to highly flexible polyether structures (Figure A). Despite the limited aqueous solubility of the diepoxides, all polymerizations proceeded efficiently at room temperature. The initially biphasic mixtures homogenized within 1–2 h via reaction-induced self-solubilization, likely driven by in situ-formed amphiphilic oligomers acting as transient surfactants. This process yielded pseudohomogeneous systems with high conversion (>93%) and well-defined alternating sequences, as confirmed by ¹H NMR (Figures S19–S23). Unit A governs the ionic nature through side-chain identity, while Unit B independently modulates backbone flexibility. Within the DEP series, backbone flexibility increases monotonically from DEP1 to DEP4 as flexible linkages are progressively introduced, establishing a clear structural gradient for correlating chain dynamics with material function. This decoupled design enables orthogonal control over backbone dynamics and side-chain–mediated functionality.

3.

Library of ionic alternating copolymers derived from amino acid salts and diepoxides. (A) Representative structures of amino acid salts and diepoxide monomers used in the alternating polyaddition and Chemical structures of the resulting ionic alternating copolymers. (B) Experimentally determined pKa1 values of ionic alternating copolymers, measured by potentiometric titration. (C) Net charge per repeating unit at pH 7.4, calculated from Henderson–Hasselbalch equation using experimentally determined pKa values.

Monomer structure exerted a decisive influence on polymer yield and molecular weight (Table S3 and Figure S24). Linear diepoxides (DEP2–DEP4) afforded high yields (60–95%), with ArgS, GlyS, and GluS producing polymers of M _n = 10,000–20,000 and yields >80%. In contrast, PheS and CyaS gave lower M _n (7,000–12,000) and yields (60–76%), reflecting steric impediments of chain propagation. This amino acid dependence disappeared with the rigid DEP1, which uniformly limited yields (40–60%) and M _n (5800–10,000) across all monomers, likely due to restricted conformational freedom during chain extension. The method also exhibited structural selectivity: amino acids with nucleophilic side chains (lysine, histidine, and cysteine) induced cross-linking, whereas proline failed to initiate polymerization. These results delineate the structural scope of the aqueous polyaddition. By leveraging the built-in alkalinity of amino acid salts to sustain α-amine nucleophilicity, this self-catalyzed process achieves sequence-controlled polymerization under mild conditions. Despite steric limitations and moderate molecular-weight control, the clear correlation between monomer structure and polymerization outcome provides a predictive framework for designing ionic alternating copolymers and interpreting their unit-specific biointerfacial behavior.

2.2. Alternating Sequence–Programmable Charge Encoding toward Tunable Ionic Identities

The programmable charge of these alternating copolymers arises from three ionizable sites per repeat unit: (i) the backbone tertiary amine (pKa1) adjacent to Unit B, (ii) the α-carboxyl group (pKa2), and (iii) side-chain functionalities (pKa3) such as guanidinium (PArg), carboxylate (PGlu), and sulfonate (PCya). Potentiometric titration revealed a dichotomy in pKa1 tunability: PPhe, PGlu, and PCya displayed narrow distributions (6.5–7.2), whereas PArg and PGly exhibited broader ranges (6.5–9.0) (Figure B and S25–S29, and Table S3). We attribute this dichotomy to how the side chain of Unit A modulates the backbone amine’s sensitivity to the electronic character of Unit B. PPhe, PGlu, and PCya create stable electronic microenvironments that shield the amine from backbone variations. In contrast, the guanidinium group in PArg withdraws electron density, reducing backbone nitrogen basicity and enhancing its sensitivity to Unit B’s electron-donating effects, while PGly, lacking side-chain shielding, exposes the amine to both electronic and steric influences. In both cases, Unit B dominates pKa1 tuning. Meanwhile, pKa2 of the α-carboxyl group remained consistently acidic (1.8–2.9), while the pKa3 of permanent charged groups (guanidinium, sulfonate) fell outside the measurable titration window. ,

The synergy between these ionizable groups enables precise charge programming. Mapping the pKa values via the Henderson–Hasselbalch equation yielded a quantitative charge-profile heatmap at pH 7.4 ranging from strongly cationic (e.g., PArg3, +0.9) through zwitterionic (e.g., PGly4, approximately 0) to strongly anionic (e.g., PCya4, – 1.9) (Figure C and Table S3). This visualization reveals that the Unit A defines the charge regime (cationic, anionic, or zwitterionic), whereas the combination of Units A and B modulates its magnitude: the PArg series spans the cationic regime, PGly and PPhe are tunable from anionic to zwitterionic, and PGlu and PCya remain strongly anionic.

2.3. Alternating Sequence–Encoded Membrane Engagement Governing Bacterial Translocation Depth and Dynamics

We first assessed how ionic alternating sequences affect bacterial surfaces by measuring the zeta potential of Pseudomonas aeruginosa (P. aeruginosa), a model Gram-negative pathogen, after polymer treatment (256 μg/mL), using polymers standardized to a molecular-weight range (3–10 kDa) for comparability. At pH 7.4, untreated P. aeruginosa showed a zeta potential of approximately – 15 mV. Neutral (PGly, PPhe) and anionic (PGlu, PCya) polymers caused only slight shifts (−11 to −7 mV), indicating weak electrostatic interaction. In contrast, Arg-containing polymers (net repeat–unit charges +0.1 to +0.9) significantly increased surface potential. Especially, PArg2, PArg3, and PArg4-bearing flexible backbones–elevated the potential to −3 to −1 mV, approaching neutrality and reflecting strong electrostatic binding. However, the rigid PArg1 had minimal effect (−11 mV), despite similar charge density (Figure A). Note that within the tested concentration range, PArg1 showed no significant self-aggregation. Thus, its weak membrane-binding effect is mainly due to restricted main-chain conformation, not reduced contact area (Figure S30). These data reveal that cationic charge from Unit A is required for the initial electrostatic anchoring, while sufficient backbone flexibility from Unit B dictates multivalent, conformationally adaptive contact.

4.

Interactions of ionic alternating copolymers with bacteria. (A) Zeta potentials of P. aeruginosa after 24 h incubation with polymers (256 μg/mL, 37 °C). Relative membrane depolarization and permeability of P. aeruginosa, assessed by (B) DiSC₃(5) and (C) PI fluorescence assays. (D) Outer-membrane permeability evaluated by NPN uptake after treatment with PArg polymers. Data in (A–D) are normalized to PBS control and shown as mean ± SD (n = 3). (E) Time–lapse CLSM images FITC–labeled PArg polymers interacting with Cy3–labeled P. aeruginosa at 10, 30, 60, and 600 s. (F) Flow cytometry of Cy3–labeled P. aeruginosa after 24 h incubation with FITC–labeled PArg polymers, with Q2 quadrant indicating dual positive (FITC⁺/Cy3⁺) populations. (G) Schematic illustration of copolymer–bacteria interaction modes modulated by alternating sequence flexibility and charge.

To further elucidate the interaction mechanisms, membrane potential (3,3′-dipropylthiadicarbocyanine iodide, DiSC₃(5)), outer-membrane permeability (N-phenyl-1-naphthylamine, NPN), and membrane integrity (propidium iodide, PI) were monitored under identical conditions (Figure B–D). Neutral (PGly and PPhe) and anionic (PGlu and PCya) polymers induced no detectable changes, confirming that net positive charge is essential for measurable membrane interaction. Within the cationic series, PArg2 and PArg3 exhibited concurrent NPN uptake (>2 × PBS) and DiSC₃(5) depolarization (≈12–14-fold), indicative of coordinated engagement of both outer and inner membranes. In contrast, PArg4 increased NPN fluorescence with minimal DiSC₃(5) variation, suggesting predominant outer-membrane association, whereas rigid PArg1 produced negligible responses. Consistently negative PI signals ruled out membrane lysis, supporting a sublytic, adhesion-based mechanism. Collectively, these data move beyond surface charge neutralization to establish a charge–flexibility dependence on spatial depth of engagement: Unit A mediates electrostatic anchoring, the flexibility of Unit B dictates the depth of engagementdetermining whether interactions remain surface-bound or penetrate the inner membrane.

To resolve engagement kinetics and spatial progression, real-time confocal laser scanning microscopy (CLSM) was performed using FITC-labeled PArg polymers and Cy3-labeled P. aeruginosa (Figures E and S31). Rigid PArg1 exhibited negligible binding, whereas moderately flexible PArg2 gradually penetratedaccumulating at the periphery by 10 s and fully translocating by 60 sand PArg3 generated intracellular fluorescence within 10 s. In contrast, highly flexible PArg4 remained surface-confined throughout 600 s, indicating that excessive conformational entropy and backbone hydrophilicity hinder translocation. Flow cytometry quantified these trends (Figure F): the dual-positive (FITC⁺/Cy3⁺) fraction increased from PArg1 (∼13%) to PArg4 (∼53%), peaking for PArg2 (∼85%) and PArg3 (∼82%). Collectively, the transmembrane behavior of the polymers is regulated by the balance between conformational adaptability and membrane interface interaction strength. Chains with moderate flexibility (PArg2 and PArg3) undergo effective conformational rearrangement at the membrane interface, lowering the energy barrier for lipid insertion. Highly flexible chains (PArg4), however, exhibit high conformational entropy, making it difficult to form stable intercalated conformations. In contrast, rigid chains (PArg1) struggle to match membrane curvature, limiting their transmembrane depth. The hydrophilic–hydrophobic balance also governs the thermodynamic driving force for membrane insertion: moderate hydrophobicity favors entropy-driven intercalation, while excessive hydrophilicity reduces this driving force. Thus, differences in transmembrane depth and penetration modes among the polymers primarily arise from the balance between these two factors (Figure G).

The antibiofilm efficacy of these polymer–membrane interactions were assessed against Gram-negative (P. aeruginosa) and Gram-positive (Enterococcus faecalis, E. faecalis) strains (Figures S32–S37). Significant inhibition was observed for cationic PArg polymers, consistent with electrostatic targeting of bacterial envelopes. The efficacy trend mirrored membrane engagement: deeply penetrating PArg2 and PArg3, together with outer-membrane–active PArg4, reduced biomass to <30% of control at 4 μg/mL, whereas rigid PArg1 remained inactive (>90% control) (Figures S32 and S33). Notably, this antibiofilm activity cannot be attributed to direct antibacterial effects, as none of the PArg polymers exhibited measurable growth inhibition at the concentrations used (Figures S38 and S39). Instead, these results indicate that effective suppression of biofilm formation arises from interference with bacterial adhesion and early aggregation processes, which are critically dependent on polymer–membrane interactions. Scanning electron microscopy (SEM) visually confirmed that only treatments with translocating polymers like PArg3 prevented dense biofilm formation (Figure S40). Together, these findings define a clear structure–function relationship: Unit A acts as a targeting “switch,” while Unit B serves as a “regulator” of interaction depth, kinetics, and efficiencytogether determining the antibiofilm outcome.

2.4. Alternating Sequence–Encoded Immunomodulation Enabling Low Immunogenicity and M2Macrophage Polarization

Ionic alternating copolymers exhibited excellent biocompatibility, with 10% hemolysis concentration (HC₁₀) values for red blood cells and half maximal inhibitory concentration (IC₅₀) values for 3T3 fibroblasts and macrophages all exceeding 1024 μg/mL (Figure S41 and Table S3). Having established this safety baseline, we evaluated interactions with macrophages to assess their specific immunocompatibility. As shown in Figure A, membrane potential sensing (DiSC₃(5)) revealed that only cationic polymers with moderately flexible backbones (PArg2 and PArg3) caused significant depolarization (1.6– and 2.3–fold relative to the control), indicating substantive membrane interaction. These findings highlight that initial macrophage engagement shares a mechanistic prerequisite with bacterial interaction: cationic charge from unit A for electrostatic anchoring, paired with sufficient backbone flexibility from unit B for productive contact with macrophages.

5.

Interactions of ionic alternating copolymers with macrophages. (A) Membrane potential changes in RAW264.7 macrophages after incubation with different polymers, evaluated using DiSC₃(5)-based fluorescence assay. (B, C) Relative expression of IL–6 (B) and anti-inflammatory cytokine IL–10 (C) in RAW264.7 macrophages after 24 h exposure to polymers. Data are presented as mean ± SD (n ≥ 3). (D) Representative CLSM images of RAW264.7 macrophages after 24 h incubation with PArg3, PGly3, PPhe3, PGlu3, and PCya3 polymers, followed by immunostaining for CD86 (red) and CD206 (green). Nuclei were counterstained with DAPI (blue). (E) Flow cytometry analysis of CD206 expression in macrophages treated with PArg3 for 24 h, showing the proportion of M2–polarized macrophages. PBS-treated macrophages served as the control. All experiments were performed at a fixed polymer concentration of 256 μg/mL.

However, the downstream immune consequences of this engagement revealed a more nuanced structure–activity landscape. Cytokine analysis showed that none of the copolymers significantly induced interleukin-6 (IL-6) production (Figure B). Since IL-6 is typically regulated by pattern recognition receptors like Toll-like receptors and the nuclear factor-kappa B (NF-κB) pathway, these results suggest that the polymers did not trigger typical pro-inflammatory cascades. , Meanwhile, PArg, PGly, and PCya series copolymers mildly upregulated the anti-inflammatory cytokine IL-10 (Figure C). Overall, while these polymers electrostatically interact with cell membranes, the immune response appears to be more influenced by their structural features and modulation of immune recognition and signaling, rather than by membrane perturbation alone.

To further assess functional outcomes, macrophage polarization was examined using DEP3-based copolymers with consistent backbone properties (Figure D). Immunofluorescence revealed negligible CD86 expression, excluding M1 polarization, whereas CD206 was strongly upregulated by PArg3, moderately by PGly3 and PCya3, and weakly by PPhe3 and PGlu3. Flow cytometry confirmed that PArg3 increased CD206⁺ macrophages from 1.89% (control) to 31.7% (Figure E). Notably, membrane depolarization and M2 polarization were not directly correlated: PArg3 induced both strong depolarization and M2 activation, implying regulation by cationic features and backbone flexibility. In contrast, zwitterionic PGly3 and anionic PCya3 elicited moderate M2 phenotypes without depolarization, indicating nonperturbative signaling. Overall, ionic alternating copolymers exhibit low pro-inflammatory activation yet tunable immunomodulatory potential. The decoupling of membrane perturbation from polarization outcome suggests that cationic character is not indispensabledistinct Unit A side chains can independently direct immune signaling, underscoring the potential of sequence-defined polymers for precision immune engineering.

2.5. Alternating Sequence–Defined Thermal Programming: Orthogonal Control of Decomposition and Glass Transition

To clarify how alternating backbones influence thermal resistance, we first determined the maximum decomposition temperatures (T _d) of the copolymers by thermogravimetric analysis (TGA) (Figures A and S42). Polymers bearing the alkyl-chain DEP2 backbone showed the greatest stability, with PArg2 reaching 400 °C, in line with the high bond dissociation energies of C–C linkages. By contrast, incorporation of cycloaliphatic rings (DEP1) or ether chains (DEP3 and DEP4) reduced T _d substantially, underscoring that thermal stability is dictated primarily by the intrinsic chemistry of Unit B, while Unit A side chains exert little influence.

6.

Thermal behavior of ionic alternating copolymers. (A) Thermal decomposition temperatures (T _d) and (B) glass transition temperatures (T _g) of ionic alternating copolymers, determined by TGA and DSC, respectively. Radar plots summarizing the dependence of T _d (C) and T _g (D) on alternating units A and B. Data are presented as mean ± SD (n = 3). The “>” indicates the T _g is higher than 220 °C.

Building on this decomposition profile, we next asked how the alternating units control chain dynamics as reflected in glass transition temperatures (T _g) (Figures B and S43–S47). Most polymers (PArg, PGly, PPhe, and PGlu) showed T _g values below 150 °C, consistent with side chains that impose only limited restrictions on motion. This result is due to the minimal spatial restrictions of the side chains on chain mobility and the relatively weak interchain interactions, which do not effectively restrict chain segment relaxation. In contrast, the PCya series shows significantly different thermal behavior. The strong polar sulfonic acid groups in the side chains create ion-dipole and dipole–dipole interactions, forming a denser interaction network that inhibits chain motion and increases T _g above 220 °C. On the other hand, polymers with the DEP4 structure, due to low chain regularity from long ether linkages, exhibit poor chain packing and have a relatively low T _g between −15 and −25 °C. Backbones of intermediate rigidity (DEP1, DEP2, and DEP3) yielded progressively higher T _g, revealing that T _g is jointly tuned by Unit A–mediated interchain interactions and Unit B–mediated flexibility.

Notably, no distinct endothermic melting peaks were detected in the DSC profiles of any sample, confirming that all copolymers are amorphous. This lack of crystallinity arises from the strong polarity and steric disparity between alternating units, which frustrates periodic chain packing and prevents three-dimensional ordering. Taken together, these results establish an orthogonal design principle for thermal properties. As shown in Figure B and D, the decomposition stability (T _d) reflects the chemical strength of the backbone (Unit B), whereas the glass transition (T _g) is independently programmed by the balance of side-chain interactions (Unit A) and backbone flexibility (Unit B). This decoupled control spans an exceptionally broad range (−20 °C to >220 °C), providing a rational framework for tuning stability and dynamics in application-oriented design.

3. Conclusions

In summary, this study presents a self-catalyzed aqueous polymerization strategy that enables the precise synthesis of charge-programmable alternating copolymers from amino acid salts and diepoxides. This modular approach allows the sequence-defined incorporation of cationic, zwitterionic, and anionic units without external catalysts or protecting groups. The resulting polymers exhibit programmable biointerfacial behaviors: cationic sequences promote bacterial membrane anchoring, while backbone flexibility governs translocation kinetics. In macrophages, all copolymers display low immunogenicity, with specific variants effectively inducing M2-type polarization. Thermally, they combine high decomposition stability with orthogonally tunable glass-transition temperatures. This work establishes a direct molecular link between alternating sequence design and predictable multifunctional performance, providing a versatile platform for creating next-generation biointerface materials with precisely tailored charge, conformation, and thermal properties.

4. Methods

4.1. Synthesis of Model Compounds

A representative procedure is as follows. Sodium glycinate (GlyS, 1.0 g, 10.0 mmol) was dissolved in 2 mL of deionized water in a round-bottom flask equipped with a magnetic stir bar. Glycidyl methyl ether (GME, 1.78 g, 20.0 mmol) was added dropwise at room temperature. The mixture was stirred for 24 h, after which the reaction mixture was directly lyophilized to yield the bis-adduct compound 1 as a white fluffy solid (>98% yield).

4.2. Synthesis of Amino Acid–Based Alternating Copolymers

All amino acid–based polymers were synthesized by reacting amino acid salts with corresponding diepoxide monomers in water under ambient conditions. Taking PGly3 as a representative example, sodium glycinate (GlyS, 1.0 g, 10.0 mmol) was dissolved in 2 mL of deionized water, followed by the dropwise addition of 1,4-butanediol diglycidyl ether (DEP3, 2.0 g, 10.0 mmol, 1.0 equiv) under magnetic stirring at room temperature. The reaction mixture was stirred for 24 h and then lyophilized. The resulting solid was redissolved in water and purified by dialysis (molecular weight cutoff 3500 Da) against deionized water for 48 h to remove unreacted monomers and low-molecular-weight oligomers. The purified solution was finally lyophilized to afford PGly3 as a white fibrous solid (2.8 g, 92% yield).

4.3. Computational Details

All quantum chemical calculations were carried out using the Gaussian 16 software package (Revision C.01). Geometry optimizations and vibrational frequency analyses were performed at the B3LYP/6–31+G­(d) level with Grimme’s DFT–D3 dispersion correction (BJ damping) and the SMD implicit solvation model (H₂O). Thermochemical corrections were obtained from frequency calculations at 298.15 K. Single-point energies were refined using the M06–2X/6–311+G­(d,p) level of theory with the same dispersion and solvation settings. All transition states were verified by intrinsic reaction coordinate (IRC) analyses to confirm connectivity between reactants and products. Molecular structures and energy diagrams were visualized with CYLview 20.

Glossary

Abbreviations

GlyS

glycine sodium salt

GME

glycidyl methyl ether

LC–MS

liquid chromatography–mass spectrometry

DFT

density functional theory

MALDI–TOF MS

matrix–assisted laser desorption/ionization time–of–flight mass spectrometry

P. aeruginosa

Pseudomonas aeruginosa

DiSC₃(5)

3,3′-dipropylthiadicarbocyanine iodide

NPN

N-phenyl-1-naphthylamine

CLSM

confocal laser scanning microscopy

E. faecalis

Enterococcus faecalis

HC₁₀

10% hemolysis concentration

IC₅₀

half maximal inhibitory concentration

T _d

decomposition temperatures

TGA

thermogravimetric analysis

T _g

glass transition temperatures.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.5c01566.

• Materials, characterizations, experimental and computational details (PDF)

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

National Natural Science Foundation of China (U25A20259, 52473132); State Key Laboratory of New Textile Materials and Advanced Processing Technologies, No. FZ20230044.

The authors declare no competing financial interest.