Water-assisted and protein-initiated fast and controlled ring-opening polymerization of proline N-carboxyanhydride

Abstract

The production of polypeptides via the ring-opening polymerization (ROP) of N-carboxyanhydride (NCA) is usually conducted under stringent anhydrous conditions. The ROP of proline NCA (ProNCA) for the synthesis of poly-L-proline (PLP) is particularly challenging due to the premature product precipitation as polyproline type I helices, leading to slow reactions up to one week, poor control of the molar mass, and laborious workup. Here, we report the unexpected water-assisted controlled ROP of ProNCA, which affords well-defined PLP as polyproline II helices in 2-5 minutes and almost quantitative yields. Experimental and theoretical studies together suggest an as-yet-unreported role of water in facilitating proton shift, which significantly lowers the energy barrier of the chain propagation. The scope of initiators can be expanded from hydrophobic amines to also encompass hydrophilic amines and thiol-bearing nucleophiles, including complex biomacromolecules such as proteins. Protein-mediated ROP of ProNCA conveniently affords various protein-PLP conjugates via a grafting-from approach. PLP modification not only preserves the biological activities of the native proteins, but also enhances their resistance to extreme conditions. Moreover, PLP modification extends the elimination half-life of asparaginase (ASNase), a first-line anticancer drug for lymphoma treatment, by 18 folds and mitigates the immunogenicity of wt ASNase by more than 250 folds. Together, this work provides a simple solution to a longstanding problem in PLP synthesis, and offers valuable guidance for the development of water-resistant ROP of other proline-like NCAs. The facile access of PLP can greatly boost the application potential of PLP-based functional materials for engineering industry enzymes and therapeutic proteins.

High Molecular Weight Polyproline as a Potential Biosourced Ice Growth Inhibitor: Synthesis, Ice Recrystallization Inhibition, and Specific Ice Face Binding

“…Alongside this the development of ROPISA has allowed for NCA monomers to polymerize within a basic aqueous environment to outpace the hydrolysis . With regard to NCA-proline the methodology reported by Hu et al was remarkable, using DFT calculations it was shown that the presence of water was promoting polymerization, and hence this method is employed here . In this method acetonitrile/water mixtures are used which leads to fast kinetics and high yields and prevents precipitation of growing polyproline chains (Figure A).…”

“…56,57 Recent advances in proline NCA polymerizations now make high molecular weight polyproline accessible. 58 Lu and co-workers have reported the water-assisted controlled ROP of ProNCA in ACN/water mixture affording well-defined polyproline II helices. 59 Considering the above, this work explores the synthesis and ice-binding protein-mimetic activity of polyproline as a step toward identifying biosourced and scalable ice recrystallization inhibitors.…”

Memory Effect in Thermoresponsive Proline‐based Polymers

“…showed that the ROP of Pro‐NCA was water‐enhanced in biphasic mixture containing acetonitrile. [27] Following the aqueous ROP methodology developed in this latest study, we first implemented the polymerization of L ‐Proline NCA at 0.3 M in a mixture containing 60 % acetonitrile and 40 % water from hexylamine at a monomer/initiator ratio of 50. After a few minutes, the reaction was complete and upon purification, the hexyl‐PLP50 1 was obtained in high yield (90 %) and characterized by 1 H NMR, MALDI and SEC analysis (fig S1–3).…”

Cooperative Covalent Polymerization of N-carboxyanhydrides: from Kinetic Studies to Efficient Synthesis of Polypeptide Materials

“…The desired polymerization of NCA outpaces various side reactions including the water-induced side reactions, 33,38,43,45 enabling the preparation of well-defined polypeptides even in the presence of an aqueous phase. 38,43,48 Additionally, the accelerated polymerization allows the efficient synthesis of polypeptides with versatile architectures, 31,32,35,42,49 high MWs, 32,34,42,46 and predictable multiblock sequences, 39,45 which is difficult, if not impossible, to obtain with conventional polymerization methods.…”