Open-vessel polymerization of N-carboxyanhydride (NCA) for polypeptide synthesis

Synthetic polypeptides, also known as poly(α-amino acids), have the same polyamide backbone structures as natural proteins and peptides. As an important class of biomaterials, polypeptides have been widely used because of their biocompatibility, bioactivity and biodegradability. Ring-opening polymerization of N-carboxyanhydride (NCA) is a classical and widely used method for the synthesis of polypeptides. The dominantly used primary amine-initiated NCA polymerization can yield well-defined polymers and complex macromolecular architectures, but the reaction is slow and sensitive to moisture, making it necessary to use anhydrous solvents and a glovebox. One solution is to use lithium hexamethyldisilazide (LiHMDS) as the initiator, as described in this protocol. LiHMDS-initiated NCA polymerization is less sensitive to moisture and can be carried out in an open vessel outside the glovebox. It is also very fast; the reaction can be complete within 5 min to produce 30-mer polypeptides. In this protocol, poly(γ-benzyl-L-glutamate) is prepared as an example, but the protocol can easily be adapted to the synthesis of other polypeptides by generating NCAs from different amino acids, making it particularly suitable for the efficient parallel synthesis of polypeptide libraries. We provide detailed procedures for NCA synthesis and purification, the method of polymer end-group modification and measurement of polymerization kinetics and reactivity ratio. The procedure for synthesis of monomers and polymerization to form polypeptides requires <1 d. The superfast and open-vessel NCA polymerization method described here will probably enable a wide range of applications in the synthesis and functional study of polypeptide biomaterials.