Phenyl Trimethylsilyl Sulfide-Mediated Controlled Ring-Opening Polymerization of α‑Amino Acid <i>N</i>‑Carboxyanhydrides

We describe here the first example of trimethylsilyl sulfide (<i>S</i>-TMS) mediated controlled ring-opening polymerization (ROP) of α-amino acid <i>N</i>-carboxyanhydrides (NCAs). We show that phenyl trimethylsilyl sulfide (PhS-TMS), an inexpensive and commercially available compound, mediates rapid ROP of a broad scope of NCA monomers, produces functional poly­(amino acids) (PAAs) with controllable molecular weights (MWs), narrow polydispersity index (PDI), and an in situ generated phenyl thioester group at the <i>C</i>-terminus (PAA-SPhs). PhS-TMS offers more rapid chain initiation than previously reported hexamethyldisilazane (HMDS) initiator, ensuring a living polymerization with better control. Mechanistic studies suggest that a reactive trimethylsilyl carbamate (TMSC) was generated during the chain initiation and continued to regulate the chain propagation through a TMS transfer process. Considering the versatility of NCAs, and the potential of leveraging the <i>C</i>-terminal phenyl thioester for native chemical ligation (NCL), we believe this method may offer a powerful platform enabling the rapid generation of functional PAAs and their <i>C</i>-terminal conjugates for numerous biological applications

A Concise Approach to Site-Specific Topological Protein–Poly(amino acid) Conjugates Enabled by <i>in Situ</i>-Generated Functionalities

Controlling the topology of polymer-modified proteins has attracted growing interest. However, one of the main challenges in this field is the lack of efficient and site-specific methods for installing multiple bioorthogonal functionalities on substrate polymers. We report here an orchestrating strategy that provides easy access to various topological protein–poly­(amino acid) (PAA) conjugates in high yields. This method features the <i>in situ</i> installation of two “chemical handles”, including a thioester for native chemical ligation and a polyglycine nucleophile for sortase A-mediated ligation, at both ends of substrate PAAs. As a result, neither pre-functionalization of initiator or monomer units, nor post-polymerization modification of the resultant polymers, is necessary. Site-specific topological conjugates, particularly circular conjugates, can be conveniently synthesized under mild conditions from the functionalized PAAs. The biomedical utility of our method is demonstrated by the rapid and efficient generation of several therapeutic interferon-α conjugates, which exhibit significantly enhanced protease resistance and thermostability. Given the versatility of both PAAs and proteins, the method offers a convenient approach to producing libraries of conjugates for biological applications

A S-Sn Lewis Pair-Mediated Ring-Opening Polymerization of α‑Amino Acid <i>N</i>‑Carboxyanhydrides: Fast Kinetics, High Molecular Weight, and Facile Bioconjugation

The rapid and controlled generation of polypeptides with ultrahigh molecular weight (MW) and well-defined chain end functionality has been a great challenge. To tackle this problem, we report here an initiation system based on a S-Sn Lewis pair, trimethylstannyl phenyl sulfide (PhS-SnMe₃), for the ring-opening polymerization (ROP) of α-amino acid <i>N</i>-carboxyanhydrides (NCAs). This initiator displays a strong solvent effect, and can yield polypeptides with high MW (>1.0 × 10⁵ g·mol^–1) and low polydispersity index within a few hours. The MWs of the obtained polypeptides are strongly dependent on the THF/DMF ratio. The polymerization follows a typical first-order kinetic character with respect to the monomer concentration in mixed THF and DMF. Moreover, a highly reactive phenyl thioester is <i>in situ</i> generated at the C-terminus of the polypeptides, which is readily accessible for native chemical ligation affording high MW and site-specific protein–polypeptide conjugates. Together, this initiator sheds light on regulating the ROP of NCAs via appropriate Lewis pair and solvent selection, and is particularly useful in preparing ultrahigh MW polypeptides within a short period of time

Salt- and pH-Triggered Helix–Coil Transition of Ionic Polypeptides under Physiology Conditions

Controlling the helix–coil transition of polypeptides under physiological conditions is an attractive way toward smart functional materials. Here, we report the synthesis of a series of tertiary amine-functionalized ethylene glycol (EG_<i>x</i>)-linked polypeptide electrolytes with their secondary structures tunable under physiological conditions. The resultant polymers, denoted as P­(EG_<i>x</i>DMA-Glu) (<i>x</i> = 1, 2, and 3), show excellent aqueous solubility (>20 mg/mL) regardless of their charge states. Unlike poly-l-lysine that can form a helix only at pH above 10, P­(EG_<i>x</i>DMA-Glu) undergo a pH-dependent helix–coil switch with their transition points within the physiological range (pH ∼5.3–6.5). Meanwhile, P­(EG_<i>x</i>DMA-Glu) exhibit an unusual salt-induced helical conformation presumably owing to the unique properties of EG_<i>x</i> linkers. Together, the current work highlights the importance of fine-tuning the linker chemistry in achieving conformation-switchable polypeptides and represents a facile approach toward stimuli-responsive biopolymers for advanced biological applications

Salt- and pH-Triggered Helix–Coil Transition of Ionic Polypeptides under Physiology Conditions

Controlling the helix–coil transition of polypeptides under physiological conditions is an attractive way toward smart functional materials. Here, we report the synthesis of a series of tertiary amine-functionalized ethylene glycol (EG_<i>x</i>)-linked polypeptide electrolytes with their secondary structures tunable under physiological conditions. The resultant polymers, denoted as P­(EG_<i>x</i>DMA-Glu) (<i>x</i> = 1, 2, and 3), show excellent aqueous solubility (>20 mg/mL) regardless of their charge states. Unlike poly-l-lysine that can form a helix only at pH above 10, P­(EG_<i>x</i>DMA-Glu) undergo a pH-dependent helix–coil switch with their transition points within the physiological range (pH ∼5.3–6.5). Meanwhile, P­(EG_<i>x</i>DMA-Glu) exhibit an unusual salt-induced helical conformation presumably owing to the unique properties of EG_<i>x</i> linkers. Together, the current work highlights the importance of fine-tuning the linker chemistry in achieving conformation-switchable polypeptides and represents a facile approach toward stimuli-responsive biopolymers for advanced biological applications

Macrocyclization of Interferon–Poly(α-amino acid) Conjugates Significantly Improves the Tumor Retention, Penetration, and Antitumor Efficacy

Cyclization and polymer conjugation are two commonly used approaches for enhancing the pharmacological properties of protein drugs. However, cyclization of parental proteins often only affords a modest improvement in biochemical or cell-based <i>in vitro</i> assays. Moreover, very few studies have included a systematic pharmacological evaluation of cyclized protein-based therapeutics in live animals. On the other hand, polymer-conjugated proteins have longer circulation half-lives but usually show poor tumor penetration and suboptimal pharmacodynamics due to increased steric hindrance. We herein report the generation of a head-to-tail interferon–poly­(α-amino acid) macrocycle conjugate <i>circ</i>-P­(EG₃Glu)₂₀-IFN by combining the aforementioned two approaches. We then compared the antitumor pharmacological activity of this macrocycle conjugate against its linear counterparts, <i>N</i>-P­(EG₃Glu)₂₀-IFN, <i>C</i>-IFN-P­(EG₃Glu)₂₀, and <i>C</i>-IFN-PEG. Our results found <i>circ</i>-P­(EG₃Glu)₂₀-IFN to show considerably greater stability, binding affinity, and <i>in vitro</i> antiproliferative activity toward OVCAR3 cells than the three linear conjugates. More importantly, <i>circ</i>-P­(EG₃Glu)₂₀-IFN exhibited longer circulation half-life, remarkably higher tumor retention, and deeper tumor penetration <i>in vivo</i>. As a result, administration of the macrocyclic conjugate could effectively inhibit tumor progression and extend survival in mice bearing established xenograft human OVCAR3 or SKOV3 tumors without causing severe paraneoplastic syndromes. Taken together, our study provided until now the most relevant experimental evidence in strong support of the <i>in vivo</i> benefit of macrocyclization of protein–polymer conjugates and for its application in next-generation therapeutics