“Dual Self-Promoted Ring-Opening Polymerization towards Cationic Polypeptoids with Stable Helices”

Today, we share significant research led by Zhengbiao Zhang's team, published in Angewandte Chemie International Edition. This study successfully synthesized cationic polypeptoid mimics bearing bulky chiral side chains via an innovative dual self-promoted ring-opening polymerization strategy. Contrary to conventional understanding, these cationic polypeptoids not only did not disrupt the helical structure but instead formed exceptionally stable, polyproline type I-like helices. This work challenges the traditional paradigm that "cationic side chains inevitably destabilize helices" and opens new avenues for designing advanced functional polymers combining low toxicity and high cellular uptake efficiency.

01 Research Background

Cationic helices are key structural motifs ubiquitous in living systems, playing central roles in biological processes such as membrane interactions, protein translocation, and immune defense. Natural cationic helices are primarily α-helices, composed of peptide segments containing charged amino acid residues like lysine and arginine. The stability of α-helices relies on intramolecular N–H···O hydrogen bonds along the backbone and hydrophobic interactions among side chains.

However, in biomimetic synthesis, introducing charged residues into polypeptide chains typically disrupts the crucial hydrogen bonds that maintain the α-helix due to electrostatic repulsion between side chains, leading to structural instability. For instance, poly-L-lysine, one of the most common cationic polypeptides, adopts a random coil conformation when its side chains are positively charged. As helical polymers often exhibit superior performance compared to random coils in applications like cell penetration, gene delivery, and antimicrobial materials, stabilizing the helical structure of cationic polymers is not only a fundamental scientific challenge but also a practical strategy for optimizing their biomedical application performance.

Polypeptoids, structural analogues of peptides, have their side chains attached to the backbone nitrogen atoms rather than the α-carbons. This slight change removes the chiral center and hydrogen bond donor from the backbone, preventing the formation of strong intramolecular hydrogen bonds typical of peptides. Furthermore, the similar energy barriers for cis/trans isomerization of the tertiary amide bonds result in an inherently flexible backbone, making it difficult to form rigid secondary structures. Current strategies to induce helicity in polypeptoids (e.g., introducing bulky side chains) mostly rely on solid-phase synthesis, which suffers from tedious steps, low yields, and limited degrees of polymerization. Moreover, no cationic polypeptoids reported to date have been able to form helical structures.

Figure 1. Cationic α-helical polypeptides and PPI-like helical Polypeptoids

02 Innovative Highlights

Discovery of a Novel "Dual Self-Promoted" Ring-Opening Polymerization Mechanism:​ For N-substituted N-carboxyanhydride monomers bearing side chains with significant steric hindrance, this study unexpectedly discovered their rapid polymerization exhibiting living characteristics. Mechanistic studies revealed this stems from a dual self-promotion mechanism: firstly, the tertiary amine group on the side chain acts as a "proton shuttle," facilitating proton transfer via intermolecular hydrogen bonding to promote polymerization; secondly, the formed polymer chains rapidly adopt a rigid helical structure, which in turn accelerates the chain-end attack on monomers. These two effects act synergistically to overcome the substantial steric hindrance, enabling highly efficient polymerization.

Overturning Conventional Wisdom: Cationic Side Chains Stabilize Helical Structures. Contrary to the destabilizing effect of cationic side chains on helices in traditional polypeptides, this study demonstrates that a series of cationic peptidomimetics synthesized via quaternization reactions not only maintain but significantly enhance their helical stability. As the first report confirming that cationic side chains can stabilize helical conformations in peptidomimetics, this work fundamentally challenges the established paradigm.

Construction of a Stable Helical Platform with Excellent Biocompatibility: The cationic helical polypeptoids synthesized based on the above mechanism exhibit significantly lower cytotoxicity and faster cellular uptake kinetics compared to traditional polylysine. This provides a versatile material platform for constructing a new generation of safe cell-penetrating carriers or antimicrobial materials.

03 Results and Discussion

3.1 Efficient Monomer Synthesis and Controlled Polymerization

The study first efficiently synthesized a series of NNCA monomers bearing chiral tertiary amine side chains with high yield and easy purification. Using benzylamine as the initiator and in the presence of triethylamine, these monomers underwent controlled ring-opening polymerization in chloroform. The polymer molecular weight could be precisely controlled by adjusting the monomer-to-initiator ratio, yielding polymers with narrow molecular weight distributions. The polymerization exhibited living characteristics, with molecular weight increasing linearly with conversion, and successful chain extension was demonstrated.

Figure 2. Synthesis and characterization of tertiary amine-pendent peptoid helices.

3.2 Elucidation of the Dual Self-Promoted Polymerization Mechanism

Despite monomer L-M1 having the greatest steric hindrance, its polymerization rate was the fastest. Circular dichroism spectroscopy indicated that its polymer L-P1 formed a PPI-like helical structure, whereas polymers derived from monomers with less hindered side chains adopted random coils. This suggests that the rigid helical structure can, in turn, promote polymerization. Further kinetic comparisons revealed that the chiral tertiary amine side chain of the monomer significantly promotes polymerization. DFT calculations supported the side-chain facilitation mechanism: calculations indicated a preference for polymerization via an eight-membered ring transition state where the growing chain-end secondary amine forms a hydrogen bond with the monomer's side-chain tertiary amine, which acts as a proton shuttle facilitating a concerted nucleophilic addition-elimination process, although a five-membered ring pathway cannot be entirely ruled out. Thus, "helix-induced acceleration" and "side chain-mediated proton transfer" constitute the dual self-promotion mechanism.

Figure 3. Dual self-promoted ROP of l-M1 involving side chain-mediated acceleration and helix-induced acceleration.

3.3 Exceptionally Stable Helical Structure and Its Stabilization Mechanism

The helical structure of L-P1 exhibited remarkable stability, with its CD signal remaining almost unchanged across a temperature range of 20–90°C, a pH range of 1.7–7.0, and in the presence of high concentrations of denaturants (guanidine hydrochloride, urea). This contrasts sharply with the fragility of traditional peptide helices reliant on hydrogen bonding. This extraordinary stability originates from the bulky, chiral side chains forcing the backbone to adopt the cis-amide conformation. Both NMR analysis and DFT calculations confirmed that C–H···O hydrogen bonding between the side chain and the backbone carbonyl oxygen, along with the steric bulk of the side chain itself, are key to stabilizing the cis conformation and, consequently, the entire helical structure.

Figure 4. DFT calculations of model chain propagation reaction between the tertiary amine-pendant l-M1 and the secondary amine 1.

3.4 Helicity Enhancement via Quaternization and Preliminary Biological Performance

Quaternization of L-P1 yielded a series of structurally diverse cationic polypeptoids. Surprisingly, the helicity of the polymers increased significantly after quaternization, with CD signal intensity enhancing several-fold. DFT calculations suggested that quaternization introduces additional C–H···O hydrogen bonds and increases steric hindrance, further stabilizing the cis-amide conformation and thereby enhancing helicity.

Regarding biological performance, these cationic helical polypeptoids exhibited much lower cytotoxicity than traditional polylysine. More importantly, the helical L-P1 demonstrated very fast cellular uptake kinetics, reaching uptake equilibrium within 2 hours, whereas random-coil polylysine required about 12 hours. This indicates the helical structure facilitates cell penetration. Although the quaternized derivative also showed rapid uptake, its total uptake was lower, indicating a need for fine balance among charge density, hydrophobicity, and uptake efficiency.

04 Conclusion and Future Perspectives

This study represents a conceptual breakthrough. It is the first report demonstrating that cationic side chains can stabilize, rather than disrupt, the helical structure of polypeptoids, elucidating the underlying mechanism. Furthermore, the discovered dual self-promoted polymerization mechanism provides a new method for synthesizing difficult monomers with high stereoregularity. In summary, the profound significance of this work lies in providing a highly customizable platform. By adjusting the side chain structure, the helical rigidity, charge density, and amphiphilicity of cationic polypeptoids can be finely tuned to optimize their interactions with biological systems. This holds direct application potential for developing a new generation of low-toxicity, high-efficiency gene delivery vectors and antimicrobial agents. Moreover, the design principle of "ion-dipole interactions stabilizing helices" revealed here offers important guidance for the broader design of biomimetic functional polymeric materials. Future exploration of this platform in areas like drug delivery, antimicrobial/antiviral applications, and even biomimetic catalysis appears highly promising.

Original Article: 

Gan K, Zuckermann RN, Zhao N, ,et al. Dual Self-Promoted Ring-Opening Polymerization towards Cationic Polypeptoids with Stable Helices. Angew Chem Int Ed Engl. 2025 Nov 10:e21129. 

https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.202521129#