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

Abstract

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 in situ 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.

Synthesis and Applications of Protein/Peptide-Polymer Conjugates

“…In light of the advances made by the latest ring opening polymerization ([C]ROP, ROMP) and RDRP (ATRP, SET‐LRP, RAFT, NMP) protocols, such polymers are ideal candidates for post‐polymerization modification and protein/peptide modification via ‘grafting‐to,’ ‘grafting‐from,’ and ‘grafting‐through’ techniques which can be acheieved via covalent or non‐covalent intereactions. ROP of N ‐carboxyanhydrides has been developed whereby functionality for site‐specific protein ligation can be topologically installed in situ . Cationic ROP of oxazolines can be terminated using functional nucleophilies, and the resulting polymers have been ‘grafted‐to’ proteins .…”

“…ROP of N-carboxyanhydrides has been developed whereby functionality for site-specific protein ligation can be topologically installed in situ. [43] Cationic ROP of oxazolines can be terminated using functional nucleophilies, and the resulting polymers have been 'grafted-to' proteins. [44] Furthermore, side chain functionality, incorporated either in the oxazoline monomer or through post-polymerization modification has been exploited for the 'grafting-through' modification of polymer chains [45] and particles.…”

Hydroxyl‐tolerated polymerization of N‐phenoxycarbonyl α‐amino acids: A simple way to polypeptides bearing hydroxyl groups

“…Polypeptides are attractive materials with protein‐like structures for numerous applications in biochemical and biomedicine due to their excellent biocompatibility with blood and tissue, biodegradability, and biorecognition properties . Amino acids (AAs) with various side groups qualify polypeptides with various properties, fascinating researchers with great interests . For example, polylysine and poly(glutamic acid), bearing amino groups and carboxyl groups, respectively, are widely used in stimuli‐responsive biomaterials .…”

Topology Engineering of Proteins in Vivo Using Genetically Encoded, Mechanically Interlocking SpyX Modules for Enhanced Stability

“…12 Among them, cyclic topology is the most commonly encountered nonlinear protein topology in nature and has also been extensively studied to date. 18−20 Various technologies have been developed to prepare cyclic proteins, both in vivo (21) and in vitro , 22 including intein technology, 23 SpyTag-SpyCatcher chemistry, 24,25 sortase/butlase-mediated ligation, 26−30 etc. Typical benefits from backbone cyclization include the dramatically improved stability against both thermal denaturation and proteolytic digestion.…”